Indian Patents
Title of Invention

PURIFICATION OF POLYPEPTIDES
Abstract A method for purifying a desired heterologous polypeptide from microbial fermentation broth or homogenate in which it is produced and solubilized comprising adding to the broth or homogenate an effective amount of a solution of a solution of ethacridine lactate to precipitate host cell impurities under conditions wherein the majority of the polypeptide remains soluble, and separating the desired polypeptide from the the broth or homogenate, wherein the temperature of the broth or homogenate after addition of the ethacridine lactate is from room temperature to 700c and the polypeptide is an antibody.
Full Text

Attorney Docket No: P1941R1
PURIFICATION OF POLYPEPTIDES
Background of the Invention
1. Field of the Invention
This invention relates to a process for purifying polypeptides of interest from microbial
fermentation broth or homogenate. More particularly, a precipitation agent is introduced to the broth or
homogenate to effect, for example, protein, DNA, and cell debris removal.
2. Description of Related Art
The advent of recombinant technology now allows for the production of high levels of proteins
within suitably transformed host cells. As a result, there is increased demand for fast, robust, and efficient
purification methods to recover the recombinantly produced proteins. Generally, proteins are produced by
culturing cells, such as mammalian, insect, fungal, and bacterial cell lines, engineered to produce the protein
of interest by insertion of a recombinant plasmid containing the gene for that protein. Since the cell lines
used are living organisms, they must be fed with a complex growth medium, containing sugars, amino acids,
and growth factors, usually supplied from preparations of animal serum. Separation of the desired protein
from the mixture of compounds fed to the cells and from the by-products of the cells themselves to a purity
sufficient for use as a human therapeutic poses a formidable challenge.
Procedures for purification of proteins from cell debris initially depend on the site of expression of
the protein. Some proteins can be secreted directly from the cell into the surrounding growth media; others
are made intracellularly. For polypeptides produced in mammalian cells, the purification scheme is
significantly easier than for polypeptides produced in other types of host cells. Mammalian cells export the
polypeptides so that they can be collected from the growth media, where they are present in relatively pure
form. However, if the polypeptide is produced in a non-mammalian cell, e.g., a microorganism such as
fungi or E. coli, the polypeptide will be recovered inside the cell or in the periplasmic space (Kipriyanov and
Little. Molecular Biotechnology, 12: 173-201 (1999); Skerra and Pluckthun, Science, 240: 1038-1040
(1988)). Hence, it is necessary to release the protein from the cells to the extracellular medium by extraction
such as cell lysis. Such disruption releases the entire contents of the cell into the homogenate, and in
addition produces subcellular fragments that are difficult to remove due to their small size. These are
generally removed by differential centrifugation or by filtration.
Cell lysis is typically accomplished using mechanical disruption techniques such as homogenization
or head milling. While the protein of interest is generally effectively liberated, such techniques have several
disadvantages (Engler, Protein Purification Process Engineering, Harrison eds., 37-55 (1994)). Temperature
increases, which often occur during processing, may result in inactivation of the protein. Moreover, the
resulting suspension contains a broad spectrum of contaminating proteins, nucleic acids, and
polysaccharides. Nucleic acids and polysaccharides increase solution viscosity, potentially complicating
subsequent processing by centrifugation, cross-flow filtration, or chromatography. Complex associations of
these contaminants with the protein of interest can complicate the purification process and result in
unacceptably low yields.

Attorney Docket No: P1941R1
As such, more selective means of releasing intracellular proteins facilitates further downstream
processing. Several techniques have been reported to permeabilize cells and/or to extract intracellular
proteins. These methods include the use of solvents, detergents, chaotropic agents, antibiotics, enzymes, and
chelating agents to enhance cell permeability and/or promote extraction. Additions of certain compounds,
such as glycine, to the fermentation medium during culture growth have also been reported to promote
release of certain intracellular enzymes. Finally, techniques such as freeze-thaw treatment or osmotic shock
have also been shown to release subsets of intracellular proteins.
However, these techniques are not necessarily applicable to all intracellular microbial proteins, and
all have limited application for large-scale processing, and/or other disadvantages. For example, while
solvents such as toluene and chloroform promote release of intracellular proteins, these substances are
known to be toxic and/or carcinogenic (Windholtz et al., The Merck Index 10th Edition: 300 and 1364
(1983)). Ionic detergents, such as SDS, often irreversibly denature isolated proteins. Although non-ionic
detergents are not normally denaturing, the recovered proteins are often associated with detergent micelles
that can require additional processing to yield detergent-free protein. Chaotropic agents, such as urea and
guanidine hydrochloride, can be denaturing at the concentrations required for complete release, and their
effectiveness may be dependent on the growth phase of the culture. The use of lysozyme, which provides
for a relatively gentle means of protein release, is limited because of its relatively high cost and because of
the subsequent need to purify the protein of interest from the enzyme reagent. In addition, chelating agents,
often used to enhance the effectiveness of other permeabilizing/release techniques such as lysozyme or
toluene extraction, suffer from the disadvantage of non-specific release of host proteins.
Other methods for protein release also have disadvantages. For example, osmotic shock, in which
cells are suspended in a high osmolarity medium, recovered, and subsequently placed in a low osmolarity
buffer, requires additional processing steps with respect to other extraction alternatives (Moir et al,
Separation Processes in Biotechnology. Asenjo eds: 67-94 (1990)) or necessitates the handling of large
liquid volumes at low temperatures. This renders the method unattractive for large-scale processing.
Freeze-thaw treatment also releases intracellular proteins, although relatively low yields often result
in multiple cycles or additional processing requirements. In addition, cell paste freezing is an added non-
trivial processing requirement compared with other extraction alternatives.
Finally, reagents, such as glycine, have been added during fermentation to promote protein release
to the extracellular medium (Aristidou et al.. Biotechnology Letters 15: 331-336(1993)). While partial
release of several intracellular proteins has been reported, this approach requires direct coupling of
fermentation and release strategies and subsequent separation of the protein of interest from a potentially
complex extracellular broth.
Once the polypeptide of interest is released from the host cell, purification thereof from other cell
components is required. Unfortunately, most extraction approaches, such as cell lysis, not only expose the
protein to potential degradation by host cell proteases, but also make isolation of the protein from other
elements of the resulting suspension more difficult. For example, the presence of negatively charged
molecules, such as DNA, RNA, phospholipids, and lipopolysaccharides (LPS), often requires the use of
anion-exchange chromatography (Sassenfeld, TIBTECH. 8: 88-93 (1990); Spears, Biotechnology, vol. 3-
Bioprocessing, Rehm eds:40-51 (1993)) and/or precipitation with polycations, such as protamine sulfate
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the process selected to facilitate the subsequent isolation of soluble, correctly folded and assembled
antibody, substantially free of other antibody-related material.
U.S. Pat. No. 5,760,189 discloses a method for releasing a thioredoxin-like fusion protein from E.
coli. including negatively charged non-proteiriaceous material, into a solution by adding chelator to the
solution, and precipitating the negatively charged non-proteinaceous material from the solution by adding a
divalent cation/alcohol solution to the solution to form a first soluble fraction containing the protein and a
first insoluble fraction containing unwanted contaminants. Optionally, the temperature prior to the addition
of chelator may be substantially cooler than after the addition of chelator. The divalent cation includes, for
example, magnesium, manganese, and calcium, alone or in combination.
US Pat. No. 5,714,583 discloses methods for the purification of factor IX in a solution comprising
the steps of applying the solution containing factor IX to an anion-exchange resin, washing the anion-
exchange resin with a solution having a conductivity that is less than required to elute factor IX from the
resin, eluting the anion-exchange resin with a first eluant to form a first eluate, applying the eluate to a
heparin or heparin-like (e.g., negatively charged matrix) restn, eluting the heparin or+ieparin-like resin with
a second eluant to form a second eluate, applying the second eluate to an hydroxyapatite resin, and then
eluting the hydroxyapatite resin with a third eluant to form a third eluate containing the purified factor IX.
US Pat. No. 6,322,997 discloses a method for recovering a polypeptide comprising exposing a
composition comprising a polypeptide to a reagent that binds to, or modifies, the polypeptide, wherein the
reagent is immobilized on a solid phase; and then passing the composition through a filter bearing a charge
that is opposite to the charge of the reagent in the composition, so as to remove leached reagent from the
composition.
US Pat. No. 6,214,984 discloses low-pH hydrophobic interaction chromatography (LPHIC) for
antibody purification. In particular, the patent provides a process for purifying an antibody from a
contaminant that comprises loading a mixture containing the antibody and the contaminant on a hydrophobic
interaction chromatography column and eluting the antibody from the column with a buffer having a pH of
about 2.5-4.5. Usually, the mixture loaded onto the column is at about the same pH as the elution buffer.
US Pat. No. 6,121,428 provides a method for recovering a polypeptide comprising exposing a
composition comprising a polypeptide to a reagent that binds to, or modifies, the polypeptide, wherein the
reagent is immobilized on a solid phase; and then passing the composition through a filter bearing a charge
that is opposite to the charge of the reagent in the composition, so as to remove leached reagent from the
composition.
US Pat. No. 5,641,870 provides a process for purifying an antibody is provided, wherein a mixture
containing the antibody and contaminant is subjected to LPHIC optionally at low salt concentration. The
antibody is eluted from the column in the fraction that does not bind thereto. In the extraction step, frozen
cell pellets are re-suspended at room temperature in 20 mM MES buffer, pH 6.0 containing 5 mM EDTA
and 20 mM 4,4'-DTP previously dissolved in ethanol (3 liters of buffer/kg of cell pellet). The suspended
cells are disrupted by two passages through a Mantin Gaulin homogenizer at 5500 to 6500 PSI. The
homogenate is adjusted to 0.25% (v/v) with polyethyleneimine (PEI) and diluted with an equal volume of 2-
8°C. purified water. The diluted homogenate is then centrifuged. The antibody fragment is found in the
supernatant.
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Historically, immunoglobulin G (IgG) has been purified from human serum and plasma (Putnam,
cd.. The Plasma Proteins. vol. 1 (Academic Press, 1975)). The purification process has often contained one
or more precipitation steps. The most commonly used precipitation scheme for recovering IgG is the Colin
iractionation (Cohn et al., J. Amer. Chem. Soc. 72: 465 (1950)). However, other precipitation techniques
have been reported (Niederauer and Glatz, Advances in Biochemical Engineering Biotechnology, v. 47
(Springer-Verlag Berlin Heidelberg, 1992): Steinberg and Hershberger, Biochim. et Biophys. Acta. 342:
195-206 (1974)). The pioneering work of purifying IgG from plasma using 6,9-diamino-2-ethoxyacridine
lactate (USAN name and herein called ethacridine lactate and also known by the names ETHODINIM or
RIVANOL™), a highly aromatic cationic dye, is reported by Horsjsi and Smetana, Acta Med. Scand., 155:
65 (1956). The following decade produced a number of publications showing the capability of 6,9-diamino-
2-ethoxyacridine lactate to purify IgG and other proteins (Miller, Nature. 184: 450 (1959); Steinbuch and
Niewiarowski, Nature. 186: 87 (1960); Neurath and Brunner, Experientia. 25: 668 (1969)) from biological
materials, e.g., plasma and growth media. Use of ethacridine lactate to recover antibodies and other proteins
from other sources has been reported. See Tchernov et al., J. Biotechnol.. 69: 69-73 (1999); SU 944580
published 28 July 1982; Franek and Dolnikova, Biotech-Forum-Eur. 7: 468-470 (1990); EP 250288
published 23 Dec. 1987; DE3604947 published 20 August 1987; Rothwell et al., Anal. Biochem.. 149: 197-
201 (1985); Lutsik and Antonyuk, Biokhimiva. 47: 1710-1715 (1982); and Aizenman et al., Mikrobiol-Zh.,
44:69-72(1982).
The primary step of recovering polypeptides from microorganisms is most often concerned with
removing solid material, e.g., cells and cellular debris. It is important to recognize the need to separate the
desired product from components present in conditioned medium with which it specifically interacts. Where
the protein of interest is positively charged, it will tend to bind to any negatively charged molecules present,
thereby making purification of the protein by traditional methods very difficult. Additional removal of
contaminating soluble protein from crude microbial extracts, e.g., E. coli homogenate. during this step would
simplify subsequent chromatography steps. Such additional removal would be especially valuable for
industrial-scale production, resulting in decreased chromatography column size and production times.
Summary of the Invention
The invention involving purification is as claimed.
Specifically, in one aspect, the invention provides a method for purifying a desired heterologous
polypeptide from microbial fermentation broth or homogenate in which it is produced and solubilized
comprising adding to the broth or homogenate an effective amount of a solution of 6,9-diamino-2-
ethoxyacridine lactate (ethacridine lactate) to precipitate host cell impurities under conditions wherein the
majority of the polypeptide remains soluble, and separating the desired polypeptide from the broth or
homogenate.
In another aspect, the invention provides a microbial cells fermentation broth or homogenate
comprising ethacridine lactate and a polypeptide heterologous to the cells.
Addition of ethacridine lactate as a precipitation agent unexpectedly results in a dramatic removal
of host debris including host proteins. In this process, the majority of host proteins will be recovered in a
precipitate together with the cell debris, and the polypeptide is recovered in the clarified supernatant. The
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Attorney Docket No: P1941R1
improved purity of the clarified extract when using ethacridine lactate results in reduction in the volume of
chromatographic media or resin required for the columns, thereby reducing the scale needed for subsequent
purification. It also results in elimination of some chromatographic step(s), which improves processing time
and cost. In addition, the process herein results in a stable feedstock and can be operated at a neutral pH.
Brief Description of the Drawings
Figure 1 is a schematic representation of the construction of the antiCD18 F(ab')2(-leucine zipper)
plasmids pS1130 (single promoter) and pxCD18-7T3 (dual-promoter).
Figure 2 depicts the inserted nucleic acid sequence (designated as Anti-CD 18-7T3.DNA; SEQ ID
NO:1) of the dual-promoter construct pxCD18-7T3.
Figures 3 A and 3B depict the amino acid sequences (designated in combination as Anti-CD 18-
7T3.Protein) encoded by the two translational units within the construct pxCD18-7T3 (SEQ ID NOS:2 and
3). designated as STII + Anti-CD18 light chain (Fig. 3A) and STII + Anti-CD18 heavy chain (Fig. 3B),
respectively. N-terminal STII secretion signal sequences are underlined.
Figure 4 is a schematic of the anti-Tissue Factor IgGl plasmids paTF130 (phoA/phoA promoters)
and pxTF-7T3FL (phoA/tacII-promoters).
Figure 5 depicts the inserted nucleic acid sequence (designated as Anti-TF-7T3FL.DNA; SEQ ID
NO:4) of the phoA/tacII-promoter construct pxTF-7T3FL.
Figures 6A and 6B depict the amino acid sequences (designated in combination as Anti-TF-
7T3FL.Protein) encoded by the two translational units within the construct pxTF-7T3FL (SEQ ID NOS:5
and 6) designated as STII + Anti-TF light chain (Fig. 6A) and STII + Anti-TF heavy chain (Fig. 6B),
respectively. N-terminal STII secretion signal sequences are underlined.
Figure 7 depicts the chemical structure of ethacridine lactate.
Figures 8A-8C show a non-reduced SDS-PAGE Coomassie-blue-stained gel analysis of three
supernatants after precipitation with ethacridine lactate. The precipitation was performed at different pH
values, as indicated at each lane. The lanes indicated with an X are the clarified supernatant of the
respective E. coli homogenate, i.e., anti-CD18 F(ab')2, anti-TF F(ab')2, and full-length anti-TF (Figs. 8A,
8B, and 8C, respectively). The homogenates were diluted 4-fold with a 0.8% ethacridine lactate solution,
i.e.. a final concentration of 0.6% ethacridine lactate in each experiment. All samples were volume
compensated before loading on to the gel. Hence, the intensity of the bands should be comparable to the
extract (X) if a 100% recovery is obtained. The arrows indicate product band.
Figures 9A-9C show a non-reduced SDS-PAGE Coomassie-blue-stained gel analysis of
supernatants after precipitation with ethacridine lactate. The precipitation was performed with different
ethacridine lactate concentrations, as indicated at each lane. The lanes indicated with an X are the clarified
supernatant of the respective K coli homogenate, i.e., anti-CD 18F(ab')2, anti-TF F(ab')2, and full-length anti-
TF (Figs. 9A, 9B. and 9C, respectively). The pH of the anti-CD18 F(ab')2, anti-TF F(ab')2, and full-length
anti-TF was 8.5, 7.5, and 6.0. respectively. The conductivity in the samples was 3.2 + 0.2 mS/cm. All
samples were volume compensated before loading on to the gel. Hence, the intensity of the bands should be
comparable to the extract (X) if a 100% recovery is obtained. The arrows indicate product band.
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Attorney Docket No: P1941R1
Figures 10A and 10B show a non-reduced SDS-PAGE Coomassie-blue-stained gel analysis of two
supernatants after dilution with water or with ethacridine lactate, respectively. The precipitation was
performed at different conductivity levels. E. coli homogenate containing anti-CD18 F(ab')2 was used for
this study. The homogenate was diluted 4-fold with either water (Fig. 10A) or a 0.8% ethacridine lactate
solution, i.e., a final ethacridine lactate concentration of 0.6% in each experiment (Fig. 10B), and the pH was
adjusted to 8.3. To alter the conductivity, NaCl was added to the samples in different concentrations of 0-
400 mM (as indicated in the figures). The arrows indicate product band.
Figure 11 shows a graph of solubility of ethacridine lactate at increasing sodium chloride
concentrations. The samples were incubated for three hours at room temperature before the concentration of
soluble ethacridine lactate was determined. The open keys symbolize the 1.2% ethacridine lactate solution
and the closed keys the 0.6% solution. The solid line is the 0.6% ethacridine lactate solution at pH 6.0, the
broken line is the 1.2% ethacridine lactate solution at pH 6, the dotted line is the 0.6% ethacridine lactate
solution at pH 9, and the broken line with dots is the 1.2% ethacridine lactate solution at pH 9.
Figures 12A-12C show a non-reduced SDS-PAGE Coomassie-blue-stained gel analysis of three
supernatants after precipitation with ethacridine lactate. The precipitation was performed at elevated
temperatures. The lanes indicated with an X are the clarified supernatant of the respective E. coli
homogenate, i.e.. anti-CDI8 (F(ab')2), anti-TF (F(ab')2), and full-length anti-TF (Figs. 12A, 12B, and 12C.
respectively). The homogenate was diluted 4-fold to a final ethacridine lactate concentration of 0.6% and
the pH was adjusted to 8.5, 7.5, and 6.0 for anti-CD18 (F(ab')2), anti-TF (F(ab')2), and full-length anti-TF.
respectively. The temperature and time for incubation are indicated in the figures. The arrows indicate
product band.
Figure 13 shows a graph of turbidity as a function of time for three different supernatants.
Supernatants from anti-CD 18 homogenate treated with 0.6% ethacridine lactate are symbolized by solid
circles (4°C) or open circles (21°C), and 0.2% PEI-treated sample is shown as solid squares (4°C) and open
squares (21°C). A supernatant recovered from a clarified anti-CD18 homogenate that had been diluted with
water before concentration is shown as solid triangles (4°C) and open triangles (21 °C). In all cases the anti-
CD 18 homogenate had been diluted 4-fold and the pH was 7.2.
Detailed Description of the Preferred Embodiments
Definitions
The expression "microbial fermentation broth or homogenate" refers to broth, paste, or extract,
preferably resuspended. obtained from microorganisms, including yeast, fungi, and prokaryotes such as
bacteria, that are cultured and consuming nutrients, no matter what culturing vessel is utilized, for example,
a shake flask or fermentor. Preferably, the broth or homogenate is from yeast or prokaryotes. More
preferably, the broth or homogenate is from bacteria. Homogenate is preferred herein. In some cases if the
solution has very high conductivity it may be preferred to harvest the cells and re-suspend them, but
otherwise, it is preferred to use the homogenate as it is directly from the fermentor. The components of the
broth or homogenate include cell debris, host cell protein, DNA, RNA, etc. Thus, the addition of the lactate
herein leads to selective precipitation of host cell proteins, etc., giving better purification power than not
using the lactate.
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Attorney Docket No: P1941R1
The expression "under conditions wherein the majority of the polypeptide remains soluble" refers to
addition of ethacridine lactate to the broth or homogenate in amounts and at a temperature and conductivity
level that prevent the majority of the target polypeptide from precipitating from the broth or homogenate.
Preferably, such conditions prevent more than about 60% of the polypeptide from precipitating, more
preferably more than about 70%. still more preferably more than about 75%, even more preferably more
than about 80%, even still more preferably more than about 85%, and still more preferably more than about
85%, even still more preferably more than about 90%, and most preferably more than about 95%. This
degree of solubility is measured by an appropriate assay, such as, for example, RP-HPLC, affinity
chromatography, ELI SAs, RIAs, and a combination of SDS-PAGE and high-performance affinity
chromatography (HPAC). The choice of assay depends on such factors as the type of host cell used and
polypeptide being produced.
The "bacteria" for purposes herein include eubacteria and archaebacteria. Preferred of these are
eubacteria, including gram-positive and gram-negative bacteria. More preferred are gram-negative bacteria.
One preferred type of bacteria is Enterobacteriaceae. Examples of bacteria belonging to Enterobacteriaceae
include Escherichia, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, Serratia, and Shigella. Other
types of suitable bacteria include Azotobacler, Pseudomonas, Rhizobia, Vitreoscilta, and Paracoccus. E.
co/i is preferred herein. Suitable E. coli hosts include E. coli W3110 (ATCC 27,325), E. coli 294 (ATCC
31,446), E. coli B, and E. coli X1776 (ATCC 31,537). These examples are illustrative rather than limiting,
and W3110 is preferred. Mutant cells of any of the above-mentioned bacteria may also be employed. It is,
of course, necessary to select the appropriate bacteria taking into consideration replicability of the replicon
in the cells of a bacterium. For example, E. coli, Serratia, or Salmonella species can be suitably used as the
host when well-known plasmids such as pBR322, pBR325, pACYC177, or pKN4lO are used to supply the
replicon. See further below regarding examples of suitable bacterial host cells.
As used herein, the expressions "cell," "cell line," "strain," and "cell culture" are used
interchangeably and all such designations include progeny. Thus, the words "transformants" and
"transformed cells" include the primary subject cell and cultures derived therefrom without regard for the
number of transfers. It is also understood that all progeny may not be precisely identical in DNA content,
due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity
as screened for in the originally transformed cell are included. Where distinct designations are intended, it
will be clear from the context.
As used herein, "'polypeptide" refers generally to peptides and proteins from any cell source having
more than about ten amino acids. "Heterologous" polypeptides are those polypeptides foreign to the host
cell being utilized, such as a human protein produced by E. coli. While the heterologous polypeptide may be
prokaryotic or eukaryotic. preferably it is eukaryotic, more preferably mammalian, and most preferably
human. Preferably, it is a recombinantly produced, or recombinant polypeptide.
The polypeptide is produced and solubilized in the fermentation broth or homogenate, meaning that
it is made in such broth or homogenate and is either already in a soluble fraction resulting from production,
or is in an insoluble fraction or form or phase that is treated or contacted with a solubilizing agent such as a
chaotrope (e.g., urea or guanidine) or detergent (such as sodium dodecyl sulfate (SDS)), with or without a
reducing agent (such as dithiothreitol or beta-mcrcaptoethanol) so as to be solubilized. "'Soluble,"
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"solubilized." "solubilization," "dissolved." or "dissolution" in the sense used herein means that the
polypeptide is in the supernatant rather than in the solids fraction after centrifugation. Precipitation or
degree of solubility can be determined, for example, by the appropriate assays as noted above.
Examples of mammalian polypeptides include molecules such as, e.g., renin, a growth hormone,
including human growth hormone; bovine growth hormone; growth hormone releasing factor; parathyroid
hormone; thyroid stimulating hormone; lipoproteins; 1-antitrypsin; insulin A-chain; insulin B-chain;
proinsulin; thrombopoietin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting
factors such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor; anti-clotting factors such as
Protein C; atrial naturietic factor; lung surfactant; a plasminogen activator, such as urokinase or human urine
or tissue-type plasminogen activator (t-PA) and variants thereof such as RETEVASE™ and TNKASE™;
bombesin; thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and -beta; antibodies to ErbB2
domain(s) such as 2C4 (WO 01/00245; hybridoma ATCC HB-12697), which binds to a region in the
extracellular domain of ErbB2 (e.g., any one or more residues in the region from about residue 22 to about
residue 584 of ErbB2, inclusive), enkephalinase; a serum albumin such as human serum albumin;
Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-
associated peptide; a microbial protein, such as beta-lactamase; DNase; inhibin; activin; vascular endothelial
growth factor (VEGF); receptors for hormones or growth factors; integrin; protein A or D; rheumatoid
factors; a neurotrophic factor such as brain-derived rieurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6
(NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF; cardiotrophins (cardiac hypertrophy
factor) such as cardiotrophin-1 (CT-1); platelet-derived growth factor (PDGF); fibroblast growth factor such
as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factor (TGF) such as TGF-alpha
and TGF-beta, including TGF- 1, TGF- 2, TGF- 3, TGF- 4, or TGF- 5; insulin-like growth factor-I and -II
(1GF-I and IGF-II); des( l-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins; CD proteins
such as CD-3. CD-4. CD-8. and CD-19; erythropoietin; osteoinductive factors; immunotoxins; a bone
morphogenetic protein (BMP); an interferon such as interferon-alpha, -beta, and -gamma; serum albumin,
such as human serum albumin (HSA) or bovine serum albumin (BSA); colony stimulating factors (CSFs),
e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; anti-HER-2 antibody; Apo2
ligand; superoxide dismutase; T-cell receptors; surface membrane proteins; decay accelerating factor; viral
antigen such as, for example, a portion of the AIDS envelope; transport proteins; homing receptors;
addressins; regulator) proteins; antibodies; and fragments of any of the above-listed polypeptides.
Preferred polypeptides herein include human serum albumin (HSA), 2C4, tissue factor, anti-tissue
factor. anti-CD20, anli-HER-2, heregulin, anti-lgE, anti-CDl la, anti-CD18, VEGF and receptors and
antibodies thereto such as rhuFab V2 and AVASTIN™, growth hormone and its variants, such as hGH,
growth hormone receptors, growth hormone releasing protein (GHRP), LIV-1 (EP 1,263,780), TRAIL,
tumor necrosis factor (TNF) and antibodies thereto, TNF receptor and related antibodies, TNF-receptor-IgG.
TNF receptor associated factors (TRAFs) and inhibitors thereof, Factor VIII, Factor VIII B domain,
interferons such as interferon-gamma, transforming growth factors (TGFs) such as TGF-beta, anti-TGF such
as anti-TGF-beta. activin. inhibin, anti-activin. anti-inhibin, tissue-plasminogen activators and their variants
such as t-PA. RETliPLASEIM. and TNKase, anti-Fas antibodies, Apo-2 ligand; Apo-2 ligand inhibitor; Apo-
2 receptor, Apo-3. apoptotic factors, Ced-4, DcR3, death receptor and agonist antibodies (DR4, DR5),
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lymphotoxin (LT). prolactin, prolactin receptor. SOB proteins, WISP (wnt-induced secreted proteins),
neurotoxin-3 (NT-3). nerve growth factor (NGF) and anti-NGF, DNase, hepatitis antigen, herpes simplex
antigen, leptin. insulin-like growth factors (IGFs) such as IGF-1 and IGF-2 and their binding proteins and
receptors such as IGFBP-l-IGFBP-6, insulin, fibroblast growth factors (FGFs) such as FGF-17, Toll protein.
TIE ligands. CD40 and anti-CD40, immunoadhesins, subtilisin, hepatocyte growth factor (HGF),
thrombopoietin (TPO). interleukins such as IL-2, IL-12, IL-17, IL-22, IL-8, IL-9, and antibodies thereto, and
prostrate-specific cancer antigen (PSCA).
Examples of antibodies that bind HER2 include 4D5, 7C2, 7F3 and 2C4, as well as humanized
variants thereof, including huMAb4D5-l, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5,
huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 as described in Table 3 of U.S. Patent 5,821,337; and
humanized 2C4 mutant nos. 560, 561, 562, 568, 569, 570, 571, 574, or 56869 as described in WO01/00245.
7C2 and 7F3 and humanized variants thereof are described in WO98/17797.
Examples of antibodies that bind the CD20 antigen include: "C2B8" which is now called
"Rituximab" ("RITUXAN®") (US Patent No. 5,736,137); the yttrium-[90]-Jabeled 2B8 murine antibody
designated -'Y2B8" (US Patent No. 5,736,137); murine IgG2a "Bl" optionally labeled with I31I to generate
the "131I-B1" antibody (BEXXAR™) (US Patent No. 5,595,721); murine monoclonal antibody "1F5" (Press
el a/., Blood. 69(2): 584-591 (1987)); "chimeric 2H7" antibody (US Patent No. 5,677,180); and monoclonal
antibodies L27. G28-2. 93-1B3, B-Cl or NU-B2 available from the International Leukocyte Typing
Workshop (Valentine el al. In: Leukocyte Typing III (McMichael, Ed., p. 440, Oxford University Press
(1987)).
More preferred polypeptides are 2C4, anti-tissue factor, anti-CD20, anti-HER-2, heregulin, anti-
IgE, anti-CDl la, anti-CD18, anti-VEGF such as rhuFab V2, hGH, GHRP, LIV-1, TRAIL, antibodies to
TNF and TNF receptor and related antibodies, inhibitors of TRAF, TNF-receptor-IgG, Factor VIII, Factor
VIII B domain, interferon-gamma, TGF-beta and anti-TGF-beta, activin, inhibin, anti-activin, anti-inhibin, t-
PA. TNKase, anti-Fas antibodies. Apo-2 ligand; Apo-2 ligand inhibitor; Apo-2 receptor, Apo-3, DcR3, death
receptor and agonist antibodies (DR4, DR5), lymphotoxin (LT), prolactin, prolactin receptor, WISP, anti-
NGF. NGF, NT-3. anti-IL-8. anti-IL-9. IL-17. IL-22, DNase, GHRP, hepatitis antigen, herpes simplex
antigen. leptin. IGF-1 and IGFBP1-6, insulin. FGF-17, Toll protein, TIE ligands, CD40, immunoadhesins,
subtilisin, HGF. and TPO.
Still more preferred polypeptides are 2C4, anti-tissue factor, anti-CD20. anti-HER-2, anti-IgE.anti-
CDI la, anti-CDl 8. anti-VEGF such as rhuFab V2, hGH. LIV-I, TRAIL, antibodies to TNF and TNF
receptor and related antibodies, TNF-receptor-IgG. Factor VIII, Factor VIII B domain, interferon-gamma,
TGF-beta, activin, inhibin, anti-activin, anti-inhibin, t-PA, TNKase, Apo-2 ligand; Apo-2 ligand inhibitor;
Apo-2 receptor, Apo-3. DcR3, death receptor and agonist antibodies (DR4, DR5), WISP, inhibitors of
TRAF, anti-NGF, NGF. NT-3, anti-IL-8, anti-IL-9, IL-17, IL-22, anti-TGFs, DNase, GHRP, hepatitis
antigen, herpes simplex antigen, leptin, IGF-1 and IGFBPI-6, insulin, FGF-17, Toll protein, TIE ligands,
anti-CD40, HGF, and TPO.
Particularly preferred polypeptides are recombinant polypeptides, more preferably antibodies,
which include monoclonal antibodies and humanized antibodies. Such antibodies may be full-length
antibodies or antibody fragments. More preferably, these antibodies are human or humanized antibodies.
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Attorney Docket No: P1941R1
These include, e.g., the particularly preferred polypeptides 2C4, anti-tissue factor Fab'2 and full-length, anti-
CD20, anti-HER-2. anti-IgE, anti-CDl la, anti-CD18 Fab'2 and full-length, anti-VEGF full-length and
rhuFab V2. LIV-1, DR4. DR5. and TRAIL.
Still more preferably, the antibody is an anti-lgE. anti-CDl8, anti-VEGF, anti-tissue factor, 2C4.
anti-Her-2. anti-CD20. anti-CD40, or anti-CDl la antibody. Antibody fragments encompassed within the
definition of polypeptide preferably comprise a light chain, more preferably a kappa light chain. Such
preferred fragments include, for example, a Fab, Fab', F(ab')2, or F(ab')2-leucine zipper (LZ) fusion, and
most preferably are F(ab')2 The most preferred antibodies are anti-CDl8 F(ab')2, anti-tissue factor F(ab')2,
full-length anti-tissue factor antibody, and anti-VEGF antibody.
The term "antibody" herein is used in the broadest sense and specifically covers intact monoclonal
antibodies, polyclonal antibodies, muitispecific antibodies (e.g. bispecific antibodies) formed from at least
two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity.
The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of
substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical
except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to
polyclonal antibody preparations that include different antibodies directed against different determinants
(epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to
their specificity, the monoclonal antibodies are advantageous in that they may be synthesized
uncontaminated by other antibodies. The modifier "monoclonal" indicates the character of the antibody as
being obtained from a substantially homogeneous population of antibodies, and is not to be construed as
requiring production of the antibody by any particular method. For example, the monoclonal antibodies to
be used in accordance with the present invention may be made by the hybridoma method first described by
Koehler et al., Nature, 256: 495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S.
Patent No. 4,816.567). The "monoclonal antibodies" may also be isolated from phage antibody libraries
using the techniques described in Clackson el al, Nature, 352: 624-628 (1991) and Marks et al., J. Mol.
BioL 222: 581-597 (1991), for example.
The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of
the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies
derived from a particular species or belonging to a particular antibody class or subclass, while the remainder
of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from
another species or belonging to another antibody class or subclass, as well as fragments of such antibodies,
so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al, Proc.
Natl. Acad. Sci. USA. 81_: 6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized"
antibodies comprising variable-domain antigen-binding sequences derived from a non-human primate (e.g.
Old World Monkey, Ape etc) and human constant-region sequences.
"Antibody fragments" comprise a portion of an intact antibody, preferably comprising the antigen-
binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv
fragments; diabodics; linear antibodies; single-chain antibody molecules; and muitispecific antibodies
formed from antibody fragment(s).
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Attorney Docket No: P1941R1
An "intact" antibody is one that comprises an antigen-binding variable region as well as a light
chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3. The constant domains
may be native sequence constant domains (e.g. human native-sequence constant domains) or amino acid
sequence variant thereof. Preferably, the intact antibody has one or more effector functions.
Antibody "effector functions" refer to those biological activities attributable to the Fc region (a
native-sequence Fc region or Fc region with amino acid sequence variation) of an antibody. Examples of
antibody effector functions include Clq binding, complement dependent cytotoxicity, Fc receptor binding,
antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down- regulation of cell-surface
receptors (e.g. B cell receptor; BCR), etc.
Depending on the amino acid sequence of the constant domain of their heavy chains, intact
antibodies can be assigned to different "classes". There are five classes of immunoglobulins: IgA, IgD, IgE,
IgG, and IgM, having heavy chains designated V, *, „ (, and µ, respectively. The ( and V classes are further
divided into subclasses on the basis of relatively minor differences in CH sequence and function, e.g.,
humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. The subunit structures
and three-dimensional configurations of different classes of immunoglobulins are well known.
"Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in
which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g. Natural Killer (NK) cells,
neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the
target cell. The primary cells for mediating ADCC, NK cells, express FcRIII only, whereas monocytes
express FcRI, FcRII and FcRIII. FcR expression on hematopoietic cells in summarized is Table 3 on page
464 of Ravetch and Kinet, Annu. Rev. Immunol.. 9: 457-492 (1991). To assess ADCC activity of a
molecule of interest, an in vitro ADCC assay, such as that described in US Patent No. 5,500,362 or
5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear
cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule
of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. Proc.Natl.
Acad. Sci. USA, 95: 652-656 (1998).
"Human effector cells" are leukocytes that express one or more FcRs and perform effector
functions. Preferably, the cells express at least FcRIII and perform ADCC effector function. Examples of
human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer
(NK) cells, monocytes, cytotoxic T cells, and neutrophils, with PBMCs and NK cells being preferred. The
effector cells may be isolated from a native source thereof, e.g. from blood or PBMCs as described herein.
"Native antibodies" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed
of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy
chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains
of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain
disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant
domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end.
The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the
light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid
residues are believed to form an interface between the light-chain and heavy-chain variable domains.
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Attorney Docket No: P1941R1
The term "variable" refers to the faet that certain portions of the variable domains differ extensively
in sequence among antibodies and are used in the binding and specificity of each particular antibody for its
particular antigen. However, the variability is not evenly distributed throughout the variable domains of
antibodies. It is concentrated in three segments called hypervariable regions both in the light-chain and the
heavy-chain variable domains. The more highly conserved portions of variable domains are called the
framework regions (FRs). The variable domains of native heavy and light chains each comprise four FRs,
largely adopting a beta-sheet configuration, connected by three hypervariable regions, which form loops
connecting, and in some cases forming, part of the beta-sheet structure. The hypervariable regions in each
chain are held together in close proximity by the FRs and, with the hypervariable regions from the other
chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat el al.. Sequences of
Proteins of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda,
MD. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but
exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular
cytotoxicity (ADCC).
The term "hypervariable region" when used herein refers to the amino acid residues of an antibody
that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues
from a "complementarity-determining region'"or "CDR" (e.g. residues 24-34 (LI), 50-56 (L2) and 89-97
(L3) in the light-chain variable domain and 31-35 (HI), 50-65 (H2) and 95-102 (H3) in the heavy-chain
variable domain; Kabat et al., Sequences of Proteins of Immunological Interest. 5th Ed. Public Health
Service, National Institutes of Health, Bethesda, MD. (1991)) and/or those residues from a "hypervariable
loop" (e.g. residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light-chain variable domain and 26-32
(HI), 53-55 (H2) and 96-101 (H3) in the heavy-chain variable domain; Chothia and Lesk, J. Mol. Biol.. 196:
901-917 (1987)). "Framework Region" or "FR" residues are those variable domain residues other than the
hypervariable region residues as herein defined.
Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab"
fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its
ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites
and is still capable of cross-linking antigen.
"Fv" is the minimum antibody fragment that contains a complete antigen-recognition and antigen-
binding site. This region consists of a dimer ol one heavy-chain and one light-chain variable domain in~
tight, non-covalent association. It is in this configuration that the three hypervariable regions of each
variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively,
the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single
variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has
the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
The Fab fragment also contains the constant domain of the light chain and the first constant domain
(CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the
carboxyl terminus of the heavy-chain CH 1 domain including one or more cysteines from the antibody hinge
region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains
bear at least one free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab'
13

Attorney Docket No: P1941R1
fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also
known.
The "light chains" of antibodies from any vertebrate species can be assigned to one of two clearly
distinct types, called kappa (6) and lambda (8), based on the amino acid sequences of their constant domains.
"Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody,
wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further-
comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired
structure for antigen binding. For a review of scFv, see Pliickthun in The Pharmacology of Monoclonal
Antibodies, vol. 113, Rosenburg and Moore eds. (Springer-Verlag, New York, 1994), pp. 269-315. Anti-
ErbB2 antibody scFv fragments are described in WO93/16185; U.S. Patent No. 5,571,894; and U.S. Patent
No. 5,587,458.
The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which
fragments comprise a variable heavy domain (VH) connected to a variable light domain (VL) in the same
polypeptide chain (VH - VL). By using a linker that is too short to allow pairing between the two domains on
the same chain, the domains are forced to pair with the complementary domains of another chain and create
two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161;
and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).
"Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain
minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are
human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient
are replaced by residues from a hypervariable region of a non-human species (donor antibody), such as
mouse, rat. rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some
instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding
non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the
recipient antibody or in the donor antibody. These modifications are made to further refine antibody
performance. In general, the humanized antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to
those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human
immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see
Jones et al.. Nature, 321: 522-525 (1986); Riechmann el al.. Nature, 332: 323-329 (1988); and Presta. Curr.
Op. Struct. BioL 2: 593-596 (1992).
An "isolated" antibody is one that has been identified and separated and/or recovered from a
component of its natural environment. Contaminant components of its natural environment are materials
that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes,
hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody
will be purified (I) to greater than 95% by weight of antibody as determined by the Lowry method, and most
preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or
internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE
14

Attorney Docket No: P1941R1
under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Isolated
antibody includes the antibody in situ within recombinant cells, since at least one component of the
antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared
by at least one purification step.
A "leucine zipper" is a peptide (often about 20-40 amino acid residues long) having several
repeating amino acids, in which every seventh amino acid is a leucine residue. Such leucine zipper
sequences form amphipathic alpha-helices, with the leucine residues lined up on the hydrophobic side for
dimer formation. Examples of leucine zippers herein include the Fos-Jun leucine zipper (O'Shea-et a/..
Science, 245: 646 (1989)), which may be used for forming heterodimers (e.g.. bispecific antibodies); the
GCN4 leucine zipper from yeast (Landschulz el a/., Science, 240: 1759-1764 (1988)) which may be used for
forming homodimers (e.g.. monospecific antibodies); and leucine zippers found in other DNA-binding
proteins, such as C/EBP and c-myc, as well as variants of any of these.
The expression "control sequences" refers to DNA sequences necessary for the expression of an
operably linked coding sequence in a particular host organism. The control sequences that are suitable for
bacteria include a promoter, optionally an operator sequence, and a ribosome-binding site.
Nucleic acid is "operably linked" when it is placed into a functional relationship with another
nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA
for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a
promoter is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome
binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of
a secretory leader, contiguous and in reading phase. Linking is accomplished, for example, by ligation at
convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are
used in accordance with conventional practice.
The term "recovery" of a polypeptide generally means obtaining the polypeptide free from the cells
in which it was produced.
"Host cell impurities" means contaminating host proteins and other biomolecular impurities such as
DNA and cell debris in the fermentation broth or homogenate.
Modes for Carrying Out the Invention
The invention provides in one aspect a method for purifying a desired heterologous polypeptide
from a microbial fermentation broth or homogenate in which it is produced and solubilized. The polypeptide
may already be produced in a soluble fraction, or it may be insoluble (e.g., produced in an insoluble fraction,
phase, or form) and therefore contacted or treated so as to dissolve the polypeptide. If the polypeptide is
produced in an insoluble state, it is solubilized by exposure to or contact with a solubilizing agent (as noted
above) before the ethacridine lactate is added, for example, adding such agent to a fraction containing the
insoluble polypeptide. It is preferred that the polypeptide is already in the soluble fraction. The method
herein involves adding to the broth or homogenate an effective amount of a solution of ethacridine lactate to
precipitate host cell impurities contained in the broth or homogenate. Such addition takes place under
conditions wherein the majority of the polypeptide remains soluble. In the next step the desired polypeptide
is separated from the broth or homogenate. including cell debris, host cell protein, DNA, RNA, etc.
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Attorney Docket No: P1941R1
Because most host proteins precipitated by the ethacridine lactate have a negative charge while the
target polypeptide has a positive surface charge, it is preferred that the target polypeptide have a higher.p1
than the average p1 of host proteins contained in the host cell impurities, so that it can be recovered in the
supernatant from the precipitated host proteins. Such average p1 can be determined by a 2-D gel of the host
proteins, wherein, for example, the band of p1 ranges from about 7.5 to 5.0, the average being 6.25.
Alternatively, isoelectrofocusing alone (which is the first dimension on a 2-D gel) can be used for this
determination, as well as chromatofocusing, and calculations by amino acid composition. The more
preferred polypeptides are those having a p1 of at least about 7, and preferably about 7-10.
The preferred polypeptides to be employed are set forth above.
The concentration of ethacridine lactate employed is dependent, for example, on the amount of
negative charges in solution, which are on the surface of most host cell impurities present in the solution.
Hence, the ethacridine lactate concentration depends at least on the amount of host cell impurities such as
the DNA and host protein concentration in the solution. The higher the concentration of host protein and
DNA in the homogenate, the higher the amount of ethacridine lactate required. Hence, the more negatively
charged components available for the ethacridine lactate to complex with and thus precipitate, the higher
amount of ethacridine lactate needed to maximize precipitation. The preferred concentration of ethacridine
lactate is generally more than about 0.1% weight/volume. More preferred is an ethacridine lactate
concentration of about 0.1-5%, still more preferred about 0.4-5%, and most preferably about 0.6-5%
weight/volume.
In general, the lower the conductivity of the solution when performing the precipitation, the more
efficient the purification of the polypeptide from the cell debris and DNA. The conductivity can be
controlled, for example, by the amount of salt in the homogenate or broth or by diluting the homogenate or
broth with water or other suitable solvent. It is preferred that the conductivity of the broth or homogenate
after addition of the ethacridine lactate is less than about 16 milliSiemens (mS), more preferably about 1-15
mS, still more preferably about 1-10 mS, and most preferably about 1-5 mS.
The conductivity of the solution during the precipitation will depend at least in part on the type of
salt present therein. Halides (e.g., chlorides or bromides) are not preferred anions for the salts, but if they
are present, they are preferably at a concentration of less than about 100 mM in solution before adding the
ethacridine lactate, and below about 50 mM once the ethacridine lactate is added. Some exemplary salts to
employ herein include buffer salts, TR1S, MES, MOPS, acetate, and citrate. The concentration of salts
present must not be above an amount that would precipitate the ethacridine lactate. The exact amount is
dependent mainly on the type of salt and the stoichiometry between the salt and ethacridine lactate, and the
limit is at the low end of the stoichiometry, i.e., the low end means more salt relative to the ethacridine
lactate.
The pH of the broth or homogenate after addition of the ethacridine lactate depends, for example,
on the p1 of the polypeptide, the amount of negative surface charges on the polypeptide, the amount of host
cell impurities in the solution, and the concentration of ethacridine lactate. The pH is preferably not higher
than the p1 of the polypeptide. Generally, the pH range is about 4-10; however, for efficient host cell
impurity precipitation, the pH of the broth or homogenate after addition of the ethacridine lactate is
preferably no greater than about 9, since the ethacridine lactate becomes less charged above this pH. with the
16

Attorney Docket No: P1941R1
preferred range of about 4-9. More preferably the pH of the broth or homogenate after addition of the
ethacridine lactate is about 5-9. and still more preferably, about 6-9. The more negative surface charges on
the polypeptide. the lower the pH within this range, with a preferred range for such polypeptides of about pH
6-7.
The broth or homogenate after addition of the ethacridine lactate optionally is incubated at an
elevated temperature for a period of time. Whether to raise the temperature and for how long depends on
many factors, including the type of polypeptide of interest, what, if any, modifications to the polypeptide of
interest occur if exposed to elevated temperature during this period in the process, etc. For example, for the
anti-tissue factor F(ab')2 purifications, elevated temperatures are preferred, whereas for full-length antibody,
it is preferred to have no heat or have the temperature no higher than about 25°C. With these factors in
mind, in general, the temperature of the broth or homogenate after addition of the ethacridine lactate ranges
from about room temperature to about 70°C, more preferably, from about room temperature to about 65°C
held for about 1-60 minutes. If the temperature should be elevated, one preferred range is from about 50 to
65°C held for about 1-60 minutes.
In another aspect, the invention provides a composition of matter that is a fermentation broth or
homogenate from microbial cells comprising ethacridine lactate and heterologous polypeptide. Preferably,
the polypeptide is dissolved in such broth or homogenate. The cells, polypeptide, concentration, and
conditions for the broth or homogenate are as indicated above. The dissolution degree of the polypeptide
can be determined by an appropriate assay such as the assays as noted above. Culturing parameters are used
and polypeptide production is conducted in a conventional manner, such as those procedures described
below.
A. Selection of Nucleic Acid and Modifications Thereof
While the polypeptide herein, such as an antibody, may be produced from any source (e.g., peptic
cleavage of intact antibodies), preferably it is made recombinantly. The nucleic acid encoding the
polypeptide of interest is suitably RNA, cDNA, or genomic DNA from any source, provided it encodes the
polypeptide(s) of interest. Methods are well known for selecting the appropriate nucleic acid for expression
of heterologous polypeptides (including varianis thereof) in microbial hosts. Selection of appropriate
nucleic acid to prepare non-antibody polypeptides in microbial cell culture is well known in the art.
If monoclonal antibodies are being produced, DNA encoding the monoclonal antibodies is readily
isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable
of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma
cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression
vectors, which are then transformed into the microbial host cells herein to obtain the synthesis of monoclonal
antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of DNA
encoding the antibody include Skerra et al., Curr. Opinion in Immunol.. 5: 256-262 (1993) and Pluckthun,
Immunal._Reys;. 130: 151-188 (1992).
Methods for humanizing non-human antibodies have been described in the art. Preferably, a
humanized antibody has one or more ami no acid residues introduced into it from a source that is non-human.
These non-human amino acid residues are often referred to as "import" residues, which are typically taken
from an "import" variable domain. Humanization can be essentially performed following the method of
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Attorney Docket No: P1941R1
Winter and co-workers (Jones et al., Nature. 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327
(1988): Verhoeyen et al.. Science. 239: 1534-1536(1988)), by substituting hypervariable region sequences
for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are
chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially less than an intact human variable
domain has been substituted by the corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some hypervariable region residues and
possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
The choice of human variable domains, both light and heavy, to be used in making the humanized
antibodies is very important to reduce antigenicity. According to the so-called "best-fit" method, the
sequence of the variable domain of a rodent antibody is screened against the entire library of known human
variable-domain sequences. The human sequence that is closest to that of the rodent is then accepted as the
human framework region (FR) for the humanized antibody (Sims et al., J. Immunol.. 151: 2296 (1993)-;-
Chothia et al., J. Mol. Biol.. 196: 901 (1987)). Another method uses a particular framework region derived
from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The
same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.
USA. 89: 4285 (1992); Presta et al., J. Immunol.. 151: 2623 (1993)).
It is further important that antibodies be humanized with retention of high affinity for the antigen
and other favorable biological properties. To achieve this goal, according to a preferred method, humanized
antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized
products using three-dimensional models of the parental and humanized sequences. Three-dimensional
immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer
programs are available that illustrate and display probable three-dimensional conformational structures of
selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely
role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of
residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR
residues can be selected and combined from the recipient and import sequences so that the desired antibody
characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable
region residues are directly and most substantially involved in influencing antigen binding.
Various forms of the humanized antibody or affinity-matured antibody are contemplated. For
example, the humanized antibody or affinity-matured antibody may be an antibody fragment, such as a Fab.
that is optionally conjugated with one or more targeting agent(s) in order to generate an immunoconjugate.
Alternatively, the humanized antibody or affinity-matured antibody may be an intact antibody, such as an
intact IgGl antibody.
Fab'-SH fragments can be directly recovered from £ colt and chemically coupled to form F(ab')2
fragments (Carter et al., Bio/Technology. 10: 163-167 (1992)). According to another approach, F(ab')2
fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of
antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice
is a single-chain Fv fragment (scFv) (WO 93/16185; U.S. Pat. Nos. 5.571,894 and 5,587,458). The antibody
fragment may also be a "linear antibody", e.g., as described in U.S. Pat. No. 5,641,870. Such linear antibody
fragments may be monospecific or bispecific.
18

Attorney Docket No: P1941R1
Bispecific antibodies are antibodies that have binding specificities for at least two different
epitopes. Exemplary bispecific antibodies may bind to two different epitopes of the Dkk-1 protein.
Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific
antibodies).
According to a different approach, antibody variable domains with the desired binding specificities
(antibody-antigen combining sites) are fused to immunoglobulin constant-domain sequences. The fusion
preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge,
CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the
site necessary for light-chain binding, present in at least one of the fusions. DNAs encoding the
immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are co-transfected into a suitable bacterial host organism. This provides for
great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when
unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is,
however, possible to insert the coding sequences for two or all three polypeptide chains in one expression
vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when
the ratios are of no particular significance.
In a preferred embodiment of this approach, the bispecific antibodies are composed of a hybrid
immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy
chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this
asymmetric structure facilitates the separation of the desired bispecific compound from unwanted
immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of
the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690.
For further details of generating bispecific antibodies see, for example, Suresh el at.. Methods in
Enzymology. 121: 210(1986).
According to another approach described in U.S. Pat. No. 5,731,168, the interface between a pair of
antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from
recombinant cell culture. The preferred interface comprises at least a part of the CH3 domain of an antibody
constant domain. In this method, one or more small amino acid side chains from the interface of the first
antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory
"cavities" of identical or similar size to the large side chain(s) are created on the interface of the second"
antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine).
This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products
such as homodimers.
Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the
antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for
example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for
treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies
may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in
the art, and are disclosed, e.g., in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
19

Attorney Docket No: P1941RI
Techniques for generating bispecific antibodies from antibody fragments have also been described
in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al.
Science. 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to
generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent
sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab'
fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an
equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific
antibodies produced can be used as agents for the selective immobilization of enzymes.
Additionally. Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to
form bispecific antibodies (Shalaby et al, J. Exp. Med.. 175: 217-225 (1992)).
Various techniques for making and isolating bispecific antibody fragments directly from
recombinant cell culture have also been described. For example, bispecific antibodies have been produced
using leucine zippers (Kostelny et al, J. Immunol.. 148: 1547-1553 (1992)). The leucine zipper peptides
from the Fos and Jun proteins are linked to the Fab' portions of two different antibodies by gene fusion. The
antibody homodimers are reduced at the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The
"diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA. 90: 6444-6448 (1993) has
provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a
heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker that is too
short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of
one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby
forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of
single-chain Fv (sFv) dimers has also been reported (Gruber et al, J. Immunol.. 152: 5368 (1994)).
Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can
be prepared (Tutt et al., J. Immunol.. 147: 60 (1991)).
Nucleic acid molecules encoding polypeptide variants are prepared by a variety of methods known
in the art. These methods include, but are not limited to, isolation from a natural source (in the case of
naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-
directed) mutagenesis. PCR mutagenesis, or cassette mutagenesis of an earlier prepared variant or a non-
variant version of the polypeptide.
It may be desirable to modify the antibody of the invention with respect to effector function, e.g.. so
as to enhance Fc receptor binding. This may be achieved by introducing one or more amino acid
substitutions into an Fc region of the antibody. Alternatively or additionally, cysteine residue(s) may be
introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region.
To increase the serum half-life of the antibody, one may incorporate a salvage receptor binding
epitope into the antibody (especially an antibody fragment) as described in U.S. Pat. 5,739,277, for example.
As used herein, the term "salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG
molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the
IgG molecule.
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Attorney Docket No: P1941R1
Other modifications of the antibody are contemplated herein. For example, the antibody may be
linked to one of a variety of non-proteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol,
polyoxyalkylenes. or copolymers of polyethylene glycol and polypropylene glycol.
B. Insertion of Nucleic Acid Into a Replicable Vector
The heterologous nucleic acid (e.g., cDNA or genomic DNA) is suitably inserted into a replicable
vector for expression in the microorganism under the control of a suitable promoter. Many vectors are
available for this purpose, and selection of the appropriate vector will depend mainly on the size of the
nucleic acid to be inserted into the vector and the particular host cell to be transformed with the vector. Each
vector contains various components depending on the particular host cell with which it is compatible.
Depending on the particular type of host, the vector components generally include, but are not limited to,
one or more of the following: a signal sequence, an origin of replication, one or more marker genes, a
promoter, and a transcription termination sequence.
In general, plasmid vectors containing replicon and control sequences that are derived from species
compatible with the host cell are used in connection with microbial hosts. The vector ordinarily carries a
replication site, as well as marking sequences that are capable of providing phenotypic selection in
transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E.
coli species (see, e.g.. Bolivar et al., Gene. 2: 95 (1977)). pBR322 contains genes for ampicillin and
tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR322
plasmid, or other bacterial plasmid or phage, also generally contains, or is modified to contain, promoters
that can be used by the host for expression of the selectable marker genes.
(i) Signal Sequence Component
The DNA encoding the polypeptide of interest herein may be expressed not only directly, but also
as a fusion with another polypeptide, preferably a signal sequence or other polypeptide having a specific
cleavage site at the N-terminus of the mature polypeptide. In general, the signal sequence may be a
component of the vector, or it may be a part of the polypeptide DNA that is inserted into the vector. The
heterologous signal sequence selected should be one that is recognized and processed (i.e., cleaved by a
signal peptidase) by the host cell.
For prokaryotic host cells that do not recognize and process the native or a eukaryotic polypeptide
signal sequence, the signal sequence may be substituted by a prokaryotic signal sequence selected, for
example, from the group consisting of the lamB. ompF, alkaline phosphatase, penicillinase, Ipp, or heat-
stable enterotoxin II leaders. For yeast secretion the signal sequence may be, e.g., the yeast invertase leader,
alpha factor leader (including Saccharomyces and Klttyveromyces -factor leaders, the latter described in U.S.
Patent No. 5,010,182), or acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179
published 4 April 1990), or the signal described in WO 90/13646 published 15 November 1990.
(ii) Origin of Replication Component
Expression vectors contain a nucleic acid sequence that enables the vector to replicate in one or
more selected host cells. Such sequences are well known for a variety of microbes. The origin of replication
from the plasmid pBR322 is suitable for most Gram-negative bacteria such as E. coli.
(iii) Selection Gene Component
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Attorney Docket No: P1941R1
Expression vectors generally contain a selection gene, also termed a selectable marker. This gene
encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture
medium. Host cells not transformed with the vector containing the selection gene will not survive in the
culture medium. This selectable marker is separate from the genetic markers as utilized and defined by this
invention. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins,
e.g.. ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies other than
those caused by the presence of the genetic marker(s), or (c) supply critical nutrients not available from
complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
One example of a selection scheme utilizes a drug to arrest growth of a host cell. In this case, those
cells that are successfully transformed with the nucleic acid of interest produce a polypeptide conferring
drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs
neomycin (Southern et al, J. Molec. Appl. Genet.. I: 327 (1982)), mycophenolic acid (Mulligan et al..
Science, 209: 1422(1980)) or hvgronrycin (Sugden et al. Mol. Cell. Biol.. 5: 410-413 (1985)). The three
examples given above employ bacterial genes under eukaryotic control to convey resistance to the
appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid), or hygromycin, respectively.
(iv) Promoter Component
The expression vector for producing the polypeptide of interest contains a suitable promoter that is
recognized by the host organism and is operably linked to the nucleic acid encoding the polypeptide of"
interest. Promoters suitable for use with prokaryotic hosts include the beta-lactamase and lactose promoter
systems (Chang et al., Nature. 275: 615 (1978); Goeddel et al., Nature. 281: 544 (1979)), the arabinose
promoter system (Guzman et al., J. Bacteriol.. 174: 7716-7728 (1992)), alkaline phosphatase, a tryptophan
(trp) promoter system (Goeddel, Nucleic Acids Res.. 8: 4057 (1980) and EP 36,776) and hybrid promoters
such as the tac promoter (deBoer et al, Proc. Natl. Acad. Sci. USA. 80: 21-25 (1983)). However, other
known bacterial promoters are suitable. Their nucleotide sequences have been published, thereby enabling a
skilled worker operably to ligate them to DNA encoding the polypeptide of interest (Siebenlist et al, Cell.
20: 269 (1980)) using linkers or adaptors to supply any required restriction sites.
Promoters for use in bacterial systems also generally contain a Shine-Dalgarno (S.D.) sequence
operably linked to the DNA encoding the polypeptide of interest. The promoter can be removed from the
bacterial source DNA by restriction enzyme digestion and inserted into the vector containing the desired
DNA.
Promoters suitable for use in yeast are well known in the art. Examples of suitable promoting
sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase (Hitzeman et al., L
Biol. Chem., 255: 2073 (1980)) or other glycolytic enzymes (Hess el al., J. Adv. Enzyme Reg.. 7: 149
(1968); Holland, Biochemistry, JJ7: 4900 (1978)), such as enolase, glyceraIdehyde-3-phosphate
dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and
glucokinase.
Other yeast promoters, which are inducible promoters having the additional advantage of
transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism.
22

Attorney Docket No: P1941R1
metallothionein, gIyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and
galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP
73,657.
(v) Construction and Analysis of Vectors
Construction of suitable vectors containing one or more of the above-listed components employs
standard ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and re-ligated in the
form desired to generate the plasmids required.
For analysis to confirm correct sequences in plasmids constructed, the ligation mixtures are used to
transform E. coli KI2 strain 294 (ATCC 31,446) or other strains, and successful transformants are selected
by ampicillin or tetracycline resistance where appropriate. Plasmids from the transformants are prepared,
analyzed by restriction endonuclease digestion, and/or sequenced by the method of Sanger et al, Proc. Natl.
Acad. Sci. USA, 74: 5463-5467 (1977) or Messing et al., Nucleic Acids Res.. 9: 309 (1981), or by the
method of Maxam et al.. Methods in Enzymology, 65: 499 (1980).
C. Selection and Transformation of Host Cells
Suitable host cells for cloning or expressing the DNA in the vectors herein are any microbial cells,
including prokaryotes and fungal cells, including yeast. Suitable prokaryotes for this purpose include
bacteria as defined above, preferably eubacteria, such as Gram-negative or Gram-positive organisms.
Examples include Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella,
Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, andShigella, as well
as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710
published Apr. 12. 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. One preferred E. coli
cloning host is E. coli 294 (ATCC 31, 446), although other strains such as E. coli B, E. coli X1776 (ATCC
31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.
Mutant cells of any of the above-mentioned strains may also be employed as the starting hosts that are then
further mutated to contain at least the minimum genotype required herein.
E. coli strain W3110 is a preferred parental E. coli host because it is a common host strain for
recombinant DNA product fermentations. Examples of starting E. coli hosts to be used as parent hosts,
along with their genotypes, are included in the table below:



Also suitable are the intermediates in making strain 36F8, i.e., T1BA (U.S. Pat. No. 5,304,472) and 35E7 (a
spontaneous temperature-resistant colony isolate growing better than 27B4). An additional suitable strain is
the E. coli strain having the mutant periplasmic protease(s) disclosed in U.S. Pat. No. 4,946,783 issued
August 7, 1990.
The above strains may be produced by chromosomal integration of the parental strain or other
techniques, including those set forth in the Examples below.
Full-length antibodies may be made in E. coli in accordance with the teachings of WO 02/061090
published August 8, 2002.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable
expression hosts for polypeptide-encoding vectors. Saccharomyces cerevisiae is a commonly used lower
eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Beach and Nurse, Nature.
290: 140 (1981); EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Patent No. 4,943.529;
Fleer el al., Biotechnology, 9: 968-975 (1991)) such as, e.g., K. lactis (MW98-8C, CBS683, CBS4574;
Louvencourt et a/., J. Bacteriol.. 737 (1983)), K.fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.
wickeramii (ATCC 24,178), K. wallii (ATCC 56,500), K. drosophi/arum (ATCC 36,906; Van den Berg el
a/.. Biotechnology. 8: 135 (1990)), A'. thermotolerans. and K. marxianus; yarrowia (EP 402,226): Pichia
pastoris (EP 183,070; Sreekrishna el at., J. Basic Microbiol., 28: 265-278 (1988)); Candida; Trichoderma
reesia (EP 244,234); Neurospora crassa (Case et at., Proc. Natl. Acad. Sci. USA. 76: 5259-5263 (1979));
Sclwanniomyces such as Schwanniomyces occidentals (EP 394,538 published 31 October 1990); and
filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10
24

Attorney Docket No: P1941R1
January 1991), and Aspergillus hosts sucli as A. nidulans (Ballance el al., Biochcm. Biophys. Res.
Commun., .112: 284-289 (1983); Tilbum et al.. Gene. 26: 205-221 (1983): Yd Ion el al.. Proc, Natl, Acad.
Sci,_USA- 81: 1470-1474 (1984)) and A. niger (Kelly and Hynes, EMBOJ., 4: 475-479 (1985)).
Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on
methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces,
Torulopsis. and Rhodotorula. A list of specific species that are exemplary of this class of yeasts may be
found in C. Anthony. The Biochemistry of Methylotrophs. 269 (1982).
The nucleic acid encoding the polypeptide is inserted into the host cells. Preferably, this is
accomplished by transforming the host cells with the above-described expression vectors and culturing in
conventional nutrient media modified as appropriate for inducing the various promoters.
Depending on the host cell used, transformation is done using standard techniques appropriate to
such cells. The calcium treatment employing calcium chloride, as described in section 1.82 of Sambrook et
al, Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), is
generally used for prokaryotic cells or other cells that contain substantial cell-wall barriers. Another method
for transformation employs polyethylene glycol/DMSO, as described in Chung and Miller, Nucleic Acids
Res_. 16: 3580 (1988). Transformations into yeast are typically carried out according to the method of Van
Solingen el al.. J.Bact, 130:946 (1977) and Hsiao el al., Proc. Natl. Acad. Sci. (USA). 76:3829 (1979).
However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation.
bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene, polyornithine, may also be used.
P. Culturing the Host Cells
Prokaryotic cells used to produce the polypeptides of the invention are grown in media known in
the art and suitable for culture of the selected host cells, including the media generally described by
Sambrook et al., supra. Media that are suitable for bacteria include, but are not limited to, AP5 medium,
nutrient broth, Luria-Bertani (LB) broth, Neidhardt's minimal medium, and C.R.A.P. minimal or complete
medium, plus necessary nutrient supplements. In preferred embodiments, the media also contains a selection
agent, chosen based on the construction of the expression vector, to selectively permit growth of prokaryotic
cells containing the expression vector. For example, ampiciHin is added to media for growth of cells
expressing ampicillin resistant gene. Any necessary supplements besides carbon, nitrogen, and inorganic
phosphate sources may also be included at appropriate concentrations introduced alone or as a mixture with
another supplement or medium such as a complex nitrogen source. Optionally the culture medium may
contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine.
thioglycollate, dithioerythritol, and dithiothreitol.
Examples of suitable media are given in U.S. Pat. Nos. 5,304,472 and 5,342,763. C.R.A.P.
phosphate-limiting media consists of 3.57g (NH4)2(SO4), 0.71g NaCitrate-2H2O, l.07g K.CI, 5.36g Yeast
Extract (certified), 5.36g HycaseSF™-Sheffield, adjusted pH with KOH to 7.3, qs to 872 ml with SQ H2O
and autoclaved; cooled to 55° C and supplemented with 110 ml 1 M MOPS pH 7.3, 11 ml 50% glucose, 7 ml
1M MgSO4). Carbenicillin may then be added to the induction culture at a concentration of 50ug/ml
The prokaryotic host cells are cultured at suitable temperatures. For E. coli growth, for example,
the preferred temperature ranges from about 20°C to about 39°C, more preferably from about 25°C to about
37°C, even more preferably at about 30°C.
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Attorney Docket No: P1941R1
Where the alkaline phosphatase promoter is employed, E. coli cells used to produce the polypeptide
of interest of this invention are cultured in suitable media in which the alkaline phosphatase promoter can be
partially or completely induced as described generally, e.g., in Sambrook el al.. supra. The culturing need
never take place in the absence of inorganic phosphate or at phosphate starvation levels. At first, the
medium contains inorganic phosphate in an amount above the level of induction of protein synthesis and
sufficient for the growth of the bacterium. As the cells grow and utilize phosphate, they decrease the level of
phosphate in the medium, thereby causing induction of synthesis of the polypeptide.
If the promoter is an inducible promoter, for induction to occur, typically the cells are cultured until
a certain optical density is achieved, e.g., a A55O of about 200 using a high cell density process, at which
point induction is initiated (e.g., by addition of an inducer, by depletion of a medium component, etc.), to
induce expression of the gene encoding the polypeptide of interest.
Any necessary supplements may also be included at appropriate concentrations that would be
known to those skilled in the art, introduced alone or as a mixture with another supplement or medium such
as a complex nitrogen source. The pH of the medium may be any pH from about 5-9, depending mainly on
the host organism. For E. coli, the pH is preferably from about 6.8 to about 7.4, and more preferably about
7.0.
One selective media that can be used for culturing yeast is a synthetic complete dextrose agar
lacking uracil (SCD-Ura) prepared as described in Kaiser et al., Methods in Yeast Genetics (Cold Spring
Harbor Press, Cold Spring Harbor, NY, 1994), p. 208-210.
E. Detecting Expression
Gene expression may be measured in a sample directly, for example, by conventional Southern
blotting, northern blotting to quantitate the transcription of mRNA (Thomas, Proc. Natl. Acad. Sci. USA. 77:
5201-5205 (1980)), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled
probe, based on the sequences of the polypeptide. Various labels may be employed, most commonly
radioisotopes, particularly 32P. However, other techniques may also be employed, such as using biotin-
modified nucleotides for introduction into a polynucleotide. The biotin then serves as the site for binding to
avidin or antibodies, which may be labeled with a wide variety of labels, such as radionuclides, fluorescers.
enzymes, or the like. Alternatively, assays or gels may be employed for detection of protein.
F. Purification of Polvpeptides
When using recombinant techniques, the polypeptide herein is produced intracellularly or in the
periplasmic space. If the polypeptide is produced intracellularly, as a first step, the particulate debris, either
host cells or lysed cells (e.g. resulting from homogenization), is removed, for example, by centrifugation or
ultrafiltration, to produce a cell broth or homogenale.
Then, in accordance with this invention, the cellular impurities as defined above are removed from
the homogenate or broth by precipitation using the ethacridine lactate under the conditions as set forth
above, and the resulting mixture is treated so that the polypeptide of interest, in soluble form, is recovered.
The separation of the target polypeptide from the broth or homogenate may be accomplished by any-
suitable means, including those well known in the art such as centrifugation and filtration. Preferably, the
separation is performed using centrifugation or tangential flow filtration, for example, using a filter of about
300 kiloDaltons to 1 micron.
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Attorney Docket No: P1941R1
After the polypeptide is separated from the broth or homogenate. it may be purified by any known
means, including chromatography or filtration such as ultrafiltration/diafiltration or tangential flow filtration.
In particular, the following procedures, individually or in combination, are exemplary of suitable purification
procedures, with the specific method(s) used being dependent on the type of polypeptide: immobilized metal
affinity chromatography (IMAC), aqueous two-phase separation (ATPS), fractionation on immunoaffinity or
ion-exchange columns: ethanol precipitation; reversed-phase HPLC; hydrophobic-interaction
chromatography (HIC) chromatography on silica; chromatography on an ion-exchange resin such as S-
SEPHAROSE™ and DEAE; chromatofocusing; SDS-PAGE; ammonium-sulfate precipitation;
ultraflltration/diafiltration, tangential flow filtration, and gel filtration using, for example, SEPHADEX™G-
75.
For example, as part of the overall recovery process for the protein, the polypeptide may be
exposed to an immobilized reagent that binds to or modifies the polypeptide. Thus, the polypeptide may be
subjected to affinity chromatography wherein an immobilized reagent that binds specifically to the
polypeptide, such as an antibody, captures the antibody and impurities pass through the affinity
chromatography column. The polypeptide can be subsequently eluted from the column by changing the
conditions such that the polypeptide no longer binds to the immobilized reagent. The immobilized reagent
may also be an enzyme such as a protease that modifies the polypeptide (Sahni et al., Anal. Biochem.. 193:
178-185 (1991) and Voyksner etal., Anal. Biochem.. 188: 72-81 (1990)).
Another type of purification process is filtration. Filtration of fine particle size contaminants from
fluids has been accomplished by the use of various porous filter media through which a contaminated
composition is passed such that the filter retains the contaminant. Retention of the contaminant may occur
by mechanical straining or electrokinetic particle capture and adsorption. In mechanical straining, a particle
is retained by physical entrapment when it attempts to pass through a pore smaller than itself. In the case of
electrokinetic capture mechanisms, the particle collides with a surface within the porous filter and is retained
on the surface by short-range attractive forces. To achieve electrokinetic capture, charge-modifying systems
can be used to alter the surface charge characteristics of a filter (see, e.g., WO 90/11814). For example,
where the contaminant to be removed is anionic, a cationic charge modifier can be used to alter the charge
characteristics of the filter such that the contaminant is retained by the filter.
Monoclonal antibodies may be suitably separated from the precipitants by conventional antibody
purification procedures such as, for example, gel filtration or electrophoresis, dialysis, HIC, affinity
chromatography, e.g. protein-A SEPHAROSE™, protein-G, antigen-affinity or anti-IgG affinity
chromatography, homogenization, clarification by filtration of centrifugation, precipitation, e.g. by treatment
with ammonium sulfate. polyethylene glycol or caprylic acid, ion-exchange chromatography, e.g. using
resins such as hydroxyapatite, e.g., resins containing calcium-phosphate such as ceramic-hydroxyapatite and
BIOGEL HT™, and anion-exchange resins including those having a positively charged moiety (at neutral
pH). such as diethylaminoethane (DEAE). polyethyleneimine (PEI), and quaternary aminoethane (QAE). for
example, Q-SEPHAROSE FAST FLOW™ resin (Pharmacia), DEAE-SEPHAROSE FAST FLOW™ resin,
DEAE-TOYOPEARL™ resin, QAE-TOYOPEARL™ resin, POROS-Q™ resin, FRACTOGEL-DMAE™
resin, FRACTOGEL EMD-TMAE™ resin, MATREX CELLUFINE DEAE™, and the like. Methods for
27

Attorney Docket No: P1941R1
isolating and purifying antibodies are further described in Antibodies: A Laboratory Manual"; Harlow and
Lane. eds. (Cold Spring Harbor Laboratories, New York: 1988).
In one specific embodiment, the recovery step involves exposing the solubilized polypeptide to a
solid phase to which is immobilized a reagent that binds to, or modifies, the polypeptide. In one
embodiment, the solid phase is packed in a column and the immobilized reagent captures the polypeptide. In
another embodiment, the reagent chemically and/or physically modifies the polypeptide and is immobilized
on the solid phase that is. e.g., packed in a column, and the composition is passed through the column. For
example, the polypeptide may comprise a precursor domain that the immobilized reagent removes as part of
the recovery process, e.g., the precursor polypeptide is an antibody with a leucine zipper dimerization
domain, which is removed by immobilized pepsin in the recovery process.
In this embodiment, the composition comprising the polypeptide and leached reagent (and
optionally one or more further contaminants) is then passed through a filter bearing a charge that is opposite
to the charge of the reagent at the pH of the composition, so as to remove leached reagent from the
composition. The filter may be positively charged to remove contaminants that are negatively charged at the
pH of the composition, such as acidic proteases, protein A, protein G or other reagents that can leach from
affinity columns. Alternatively, the filter may be negatively charged to remove contaminants that are
positively charged at the pH of the composition, such as basic proteases. Preferably, the charge
characteristics of the polypeptide of interest in the composition passed through the filter are such that the
polypeptide is not significantly retained by the filter and passes therethrough. The filter may be placed "in
line" with the effluent treated as in the previous step (i.e., the effluent flows directly though the filter). This
can be achieved by connecting the filter directly to the column effluent port, before the effluent is collected
into a pool tank. The filter may be regenerated using techniques applicable to the type of filter used.
HIC has also been used for purifying antibody fragments. See, for example, Inouye el al., Protein
Engineering, pp. 6, 8 and 1018-1019(1993); Inouye el al., Animal Cell Technology: Basic & Applied
Aspects. 5: 609-616 (1993); Inouye el al.. Journal of Biochemical and Biophysical Methods. 26: 27-39
(1993); Morimoto el al.. Journal of Biochemical and Biophysical Methods. 24: 107-117 (1992); and Reae/
al., Journal of Cell. Biochem., Suppl. 0, Abstract No. Xl-206 (17 Part A), p. 50 (1993). HIC columns
normally comprise a base matrix (e.g., cross-linked agarose or synthetic copolymer material) to which
hydrophobic ligands (e.g.. alkyl or aryl groups) are coupled. Many HIC columns are available
commercially. Examples include, but arc not limited to, Phenyl SEPHAROSE 6 FAST FLOW™ column
with low or high substitution (Pharmacia LKB Biotechnology, AB, Sweden); Phenyl SEPHAROSE™ High
Performance column (Pharmacia LKB Biotechnology, AB, Sweden); Octyl SEPHAROSE™ High
Performance column (Pharmacia LKB Biotechnology, AB, Sweden); FRACTOGEL™ EMD Propyl or
FRACTOGEL™ EMD Phenyl columns (E. Merck, Germany); MACRO-PREP™ Methyl or MACRO-
PREP™ t-Butyl Supports (Bio-Rad, California); WP Hl-Propyl (C 3 )™ column (J. T. Baker, New Jersey);
and TOYOPEARL™ ether, phenyl or butyl columns (TosoHaas, PA).
Examples of batch hydrophobic chromatography matrices are well known in the art and include C18
alkyl chains linked to a support matrix such as SEPHAROSE , agarose, or silica, e.g., butyl, phenyl, or
octyl SEPHAROSE™. or polymers such as cellulose or polystyrene. US Pat. No. 6,214,984 describes use of
low-pH hydrophobic interaction chromatography (LPH1C) for antibody and antibody fragment purification.
28

Attorney Docket No: P1941R1
This method is particularly useful for purifying antibody fragments, especially correctly folded and disulflde
linked antibody fragments (e.g.. Fab fragments) from contaminating antibody fragments that are not
correctly folded and/or disulfide linked. Prior to LPH1C, the antibody composition prepared from the cells
is preferably subjected to at least one purification step, with examples including hydroxyapatite
chromatography. gel electrophoresis, dialysis, and affinity chromatography. The suitability of protein A as
an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the
antibody. Protein A can be used to purify antibodies that are based on certain human heavy chains
(Lindmark et a/., i. Immunol. Meth., 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes
and for one human isotype (Guss el al., EMBOJ.. 5: 1567-1575 (1986)). The matrix to which the affinity
ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such
as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing
times than can be achieved with agarose. Where the antibody comprises a CH 3 domain, the BAKERBOND
ABX™ resin (J. T. Baker, Phillipsburg, N.I.) is useful for purification. Other techniques for protein
purification such as fractionation on an ion-exchange column, ethanol precipitation, reverse-phase HPLC,
chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on an anion- or
cation-exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium
sulfate precipitation are also available depending on the antibody to be recovered.
G. Uses of Polypeptides
The polypeptide thus recovered may be formulated in a pharmaceutically acceptable carrier and is
used for various diagnostic, therapeutic, or other uses known for such molecules. For example, antibodies
described herein can be used in immunoassays, such as enzyme immunoassays.
Therapeutic uses for the polypeptides purified using the method described herein are also
contemplated. For example, a growth factor or hormone can be used to enhance growth as desired, and an
antibody can be used for redirected cytotoxicity (e.g., to kill tumor cells), as a vaccine adjuvant, for
delivering thrombolytic agents to clots, for delivering immunotoxins to tumor cells, for converting enzyme
activated prodrugs at a target site (e.g., a tumor), for treating infectious diseases, or for targeting immune
complexes to cell surface receptors.
Therapeutic formulations of the polypeptide are prepared for storage by mixing the polypeptide
having the desired degree of purity with optional physiologically acceptable carriers, excipients, or
stabilizers (Remington's Pharmaceutical Sciences. 16th edition, Osol, A., Ed., (1980)), in the form of ""
lyophilized cake or aqueous solutions.
Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides;
proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine;
monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating
agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium;
and/or nonionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).
29

Attorney Docket No: P1941R1
The polypeptide also may be entrapped in microcapsules prepared, for example, by coacervation
techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules
and poly-(methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example,
liposomes. albumin microspheres. microemulsions, nano-particles and nanocapsules), or in macroemulsions.
Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.
The polypeptide to be used for in vivo administration must be sterile. This is readily accomplished
by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitutiort.The
antibody ordinarily will be stored in lyophilized form or in solution.
Therapeutic polypeptide compositions generally are placed into a container having a sterile access
port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection
needle.
The route of polypeptide administration is in accord with known methods, e.g., injection or infusion
by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, or intralesional routes,
or by sustained-release systems as noted below. The polypeptide is administered continuously by infusion or
by bolus injection.
Suitable examples of sustained-release preparations include semipermeable matrices of solid
hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g.,
films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-
hydroxyethyl-methacrylate) as described by Langer el al., J. Biomed. Mater. Res., 15: 167-277 (1981) and
Langer, Chem. Tech.. 12: 98-105 (1982) or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919, EP
58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman el al., Biopolymers. 22:
547-556 (1983)), non-degradable ethylene-vinyl acetate (Langer el al., supra), degradable lactic acid-
glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-
glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid (EP 133,988).
While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of
molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When, for example,
encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of
exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in
immunogenicity. Rational strategies can be devised for antibody stabilization depending on the mechanism
involved. For example, if the aggregation mechanism is discovered to be intermolecular S--S bond formation
through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues,
lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing
specific polymer matrix compositions.
Sustained-release polypeptide compositions also include liposomally entrapped polypeptides.—
Liposomes containing the antibody are prepared by methods known per se: DE 3,218,121; Epstein el al.,
Proc. Natl. Acad. Sci. USA, 82: 3688-3692 (1985); Hwang el al., Proc. Natl. Acad. Sci. USA. 77: 4030-4034
(1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patent application 83-118008;
U.S. Pat. Nos. 4,485.045 and 4,544,545; and EP 102,324. Ordinarily the liposomes are of the small (about
200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. % cholesterol,
the selected proportion being adjusted for the most effective therapy with polypeptide.
30

Attorney Docket No: P1941R1
An effective amount of polypeptide to be employed therapeutically will depend, for example, upon
the therapeutic objectives, the route of administration, and the condition of the patient. Accordingly, it will
be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain
the most beneficial therapeutic effect. A typical daily dosage might range from about 1 µg/kg to up to 10
mg/kg or more, depending on the factors mentioned above. Typically, the clinician will administer
polypeptide until a dosage is reached that achieves the desired effect. The progress of this therapy is easily
monitored by conventional assays.
The invention will be more fully understood by reference to the following examples. They should
not, however, be construed as limiting the scope of the invention. All literature and patent citations herein
are incorporated by reference.
EXAMPLE 1
Materials and Methods
A. Plasmids. Transformation. Fermentation
1. Production of rhuFab'2 (xCD 18)
a. Plasmid Construction
The control plasmid, pSl 130, was designed for the dicistronic expression of anti-CD18 F(ab')2 and
it was based on the vector described by Carter el al., Bio/Technology. JO: 163-167 (1992). This design
places transcription of the genes for both light chain and the heavy chain fragment with a C-termina! leucine
zipper under the control of a single phoA promoter. Transcription ends with a Xlo transcriptional terminator
located downstream of the coding sequence for the heavy chain-leucine zipper (Scholtissek and Grosse,
Nucleic Acids Res.. 15(7): 3185 (1987)). The heat stable enterotoxin II signal sequence (STII) precedes the
coding sequence for each chain and directs the secretion of the polypeptide into the periplasm (Lee et al.
Infect. Immun., 42: 264-268 (1983); Picken et al., Infect. Immun., 42: 269-275 (1983)). Leucine zipper was
attached to the C-terminal end of heavy chain fragment to promote the dimerization of the two Fab' arms.
The dual-promoter plasmid containing two separate translational units, pxCD18-7T3, temporally
separates the transcription of light chain from the transcription of heavy chain. As in pSl 130, light chain
remains under the control of the phoA promoter. However, in pxCD18-7T3, a Ata transcriptional terminator
follows the light chain coding sequence. Downstream of this terminator, the lacll promoter was added to
control the transcription of the heavy chain fragment/C-terminal leucine zipper (DeBoer et al., Proc. Natl.
Acad. Sci. USA. 80: 21-25 (1983)). A second At,, transcriptional terminator follows this coding sequence.
Silent codon variants of the STII signal sequence were used to direct the secretion of both chains (Simmons
and Yansura, Nature Biotechnology. .14: 629-634 (1996)).
A schematic comparison of the single promoter control plasmid vs. the dual-promoter plasmid is
depicted in Figure 1. The expression cassette sequence of pxCD18-7T3 is provided in Figure 2 (SEQ ID
NO: 1), and the amino acid sequences (SEQ ID NOS:2 and 3) from the two translational units are shown in
Figures 3A (light chain) and 3B (heavy chain), respectively.
b. Fermentation
The host strain used in fermentation was a derivative of E. coli W3110, designated 59A7. The
complete genotype of 59A7 is W3110 AflniA phoAE15 A(argF-lac)169 deoCdegP41 ilvG2096(Vaf) A/wr
31

Attorney Docket No: P1941R1
sprW148R The 59A7 host cells were iransformed with pxCD18-7T3 plasmid and successful transformants
were selected and grown in culture. An additional plasmid, pMS42l, was co-transformed along with
pxCD18-7T3. This additional plasmid, pMS421, is a pSClOl-based plasmid which provides laclq to
improve control of the tac/I promoter, and which also confers spectinomycin and streptomycin resistance.
For each 10-liter fermentation, a single vial containing 1.5 ml of culture in 10-15% DMSO was
thawed into a 1-L shake flask containing 500 ml of LB medium supplemented with 0.5 ml of tetracycline
solution (5 mg/ml) and 2.5 ml 1M sodium phosphate solution. This seed culture was grown for
approximately 16 hours at 30°C and was then used to inoculate a 10-liter fermentor.
The fermentor initially started with approximately 6.5 liters of medium containing about 4.4 g of
glucose, 100 ml of 1M magnesium sulfate, 10 ml of a trace element solution (100 ml hydrochloric acid, 27 g
ferric chloride hexahydrate, 8 g zinc sulfate heptahydrate, 7 g cobalt chloride hexahydrate, 7 g sodium
molybdate dihydrate. 8 g cupric sulfate pentahydrate, 2 g boric acid, 5 g manganese sulfate monohydrate, in
a final volume of 1 liter). 20 ml of a tetracycline solution (5 mg/ml in ethanol), 10 ml of FERMAX Adjuvant
27™ (or some equivalent anti-foam), 1 bag of HCD salts (37.5 g ammonium sulfate, 19.5 g potassium
phosphate dibasic. 9.75 g sodium phosphate monobasic dihydrate, 7.5 g sodium citrate dihydrate, 11.3 g
potassium phosphate monobasic), and 200 g of NZ Amine A (a protein hydrolysate). Fermentations were
performed at 30 °C with 10 slpm of air flow and were controlled at a pH of 7.0 ± 0.2 (although occasional
excursions beyond this range occurred in some cases). The back pressure of the fermentor and agitation rate
was varied to manipulate the oxygen transfer rate in the fermentor, and, consequently, control the cellular
respiration rate.
Following inoculation of the fermentor with the cell-containing medium from the shake flask, the
culture was grown in the fermentor to high cell densities using a computer-based algorithm to feed a
concentrated glucose solution to the fermentor. Ammonium hydroxide (58% solution) and sulfuric acid
(24% solution) were also fed to the fermentor as needed to control pH. Further additions of anti-foam were
also used in some cases to control foaming. When the culture reached a cell density of approximately 40
OD550, an additional 100 ml of 1M magnesium sulfate was added to the fermentor. Additionally, a
concentrated salt feed (consisting of approximately 10 g ammonium sulfate, 26 g dibasic potassium
phosphate, 13 g monobasic sodium phosphate dihydrate, 2 g sodium citrate dihydrate and 15 g monobasic
potassium phosphate in 1L of water) to the fermentor was started at a rate of 2.5 ml/min when the culture
reached approximately 20 OD550 and continued until approximately 1250 ml were added to the
fermentation. Fermentations were typically continued for 72-80 hours.
During the fermentation, once the dissolved oxygen setpoint for the fermentation was reached, the
concentrated glucose solution was fed based on the dissolved oxygen probe signal in order to control the
dissolved oxygen concentration at the setpoint. Consequently, in this control scheme, manipulations of
fermentor operating parameters such as the agitation rate or back pressure, which affect the oxygen transfer
capacity in the fermentation, correspondingly manipulated the oxygen uptake rate or metabolic rate of the
cells.
A mass spectrometer was used to monitor the composition of the off-gas from the fermentations
and enabled the calculation of the oxygen uptake and carbon dioxide evolution rates in the fermentations.

Attorney Docket No: P1941R1
When the culture reached a cell density of approximately 220 OD550, the agitation was decreased
from an initial rate of 1000 rpm to approximately 725 rpm over approximately 12 hours. Fifty ml of 200
mM of isopropyl p-D-thiogalactopyranoside (IPTG) was added to induce heavy chain synthesis
approximately 12 hours after the culture reached a cell density of 220 OD550.
2. Production of Anti-Tissue Factor F(ab')2
a. Plasmid Construction
A dual promoter plasmid, pxTF7T3, was created similar to the dual promoter plasmid pXCD18-
7T3 set forth above, and used to enable temporal separation of anti-Tissue Factor light-chain and heavy-
chain expression. The lacl sequence from the plasmid pMS421was also incorporated onto pxTF7T3 to
create a new dual-promoter plasmid pJVG3IL.
b. Fermentation
The host strain used in these fermentations was a derivative of E coli W3110, designated 60H4.
The complete genotype of 60H4 is: W3110 A/huAAmanA phoAAEIS A(argF-lac)169 deoC2 degP4I
lvG2096(Vaf) Aprc prc-siippressor. The 60H4 host cells were transformed with pJVG3FL and successful
transformants were selected and grown in culture.
Fermentations were run under conditions similar to those for anti-CD18 F(ab')2 as described above,
with the principal exceptions that the run length varied between approximately 72 and 114 hours, and heavy
chain was induced using IPTG from approximately 4 to 12 hours following the attainment of a culture
OD550 of 220.
3. Production of full-length anti-TF antibodies
a. Plasmid Construction
The expression cassette for the plasmid pxTF-7T3FL comprises, from 5' to 3': (1) aphoA promoter
(Kikuchi el a/., Nucleic Acids Res., 9(21): 5671-5678 (1981)); (2) trp Shine-Daigarno (Yanofsky el a/.,
Nucleic Acids Res.. 9: 6647-6668 (1981)); (3) a silent codon variant of the STII signal sequence (TIR
relative strength about 7) (Simmons and Yansura, Nature Biotechnology. J4: 629-634 (1996)); (4) coding
sequence for anti-tissue factor light chain; (5) Xto terminator (Scholtissek and Grosse, Nucleic Acids Res..
15: 3185 (1987)); (6) a tacll promoter ((DeBoer el. a/., Proc. Natl. Acad. Sci. USA. 80: 21-25 (I983)K (7) a
second trp Shine-Dalgarno; (8) a second silent codon variant of the STII signal sequence (TIR relative
strength about 3); (9) coding sequence for anti-tissue factor full-length heavy chain; and (10) a second /.to
terminator. This expression cassette was cloned into the framework of the E. coll plasmid pBR322
(Sutcliffe, Cold Spring Harbor Symp. Quant. Biol 43: 77-90 (1978)).
Thus, the vector design of pxTF-7T3FL allows for the temporally separate expression of each chain
by using two different, rather than two identical, promoters. In this plasmid, light chain is under the control
of the phoA promoter. However, the tacll promoter is used to control the transcription of heavy chain. As is
known in the art, phoA and lac// promoters are induced under substantially different conditions. A
schematic comparison of a single-promoter plasmid paTF130 and pxTF-7T3FL is depicted in Figure 4. The
nucleic acid sequence of the expression cassette of pxTF-7T3FL (SEQ ID NO:4) is provided in Figure 5. and
the polypeptide sequences it encodes (SEQ ID NOS:5 and 6) are provided in Figures 6A (light chain) and 6B
(heavy chain, respectively. The host cells below were co-transformed with pxTF-7T3FL and pJJ247.
33

Attorney Docket No: P1941RI
pJJ247 encodes a tacll promoter driving the expression of both DsbA and DsbC, with DsbA first in the
series, and its construction is described in WO02/061090.
b. .Fermentation
For small-scale expression E. coli strain 61D6, with genotype W3110 AjhuA (AtonA) plr3 laclq
lacL8 AompTA(nmpc-fepE) degP41, was used as host cells. Following transformation, selected transformant
picks were inoculated into 5 ml of Luria-Bertani medium supplemented with carbenicillin (50 :g/ml) and
kanamycin (50 :g/ml) grown at 30°C on a culture wheel overnight. A 10-liter fermentation was conducted
using the media as described in WO02/061090 and the basic fermentation conditions were also as described
in WO02/061090, except that the following modifications were made to the fermentation process: 300 mL of
1M NaPO4, pH 7.0 was added at approximately 40 hours to give a concentration of about 30 mM. 100 mL
of a 200-mM solution of IPTG was added at approximately 44 hours to give a concentration of about 2 mM.
The fermentation was harvested 80 hours after inoculation.
B. Protein Identification
The one-dimensional SDS-PAGE gel electrophoresis was carried out in a 4-12% linear acrylamide
gradient from Novex. Specifically, the system used was the NOVEX® NUPAGE™ System, consisting of
NUPAGE™ Bis-TRIS Pre-Cast Gels (for low- to mid-molecular weight proteins).
C.Chemicals
The precipitation agent ethacridine lactate was 98% pure and purchased from Sigma (St. Louis,
MO, USA). Ethacridine lactate has a molecular weight of 361.4Da. All other chemicals were of analytical
grade.
D. Precipitation
The antibody- and F(ab')2-containing E. coli materials were homogenized using a microfluidizer
from Watts Fluidair Inc. (model B12-04DJC, Kittery, MN, USA). The cells were passed three times through
the microfluidizer at 4-bar pressure. To avoid heat degradation of the proteins, the material was passed
through an ice-water bath during each pass through the microfluidizer. The total protein concentration'in the
F(ab')2 homogenates was 30 mg/ml. The full-length anti-TF homogenate, which was derived from re-
suspended paste, had a total protein concentration of 18 mg/ml. The anti-TF paste was re-suspended in 25
mM TR1S-HCI buffer, pH 7.5.
The ethacridine lactate precipitation agent was dissolved in water to the desired final concentration
(w/v).
1. pH study:
The precipitation experiments were performed with a constant ethacridine lactate concentration of
0.6% (w/v). A 0.8% ethacridine lactate solution was prepared and mixed with the E. co/i homogenate in a
3:1 ratio, e.g., 3 ml of ethacridine lactate and 1 ml of £ coli homogenate. The pH was adjusted using HCI or
NaOH depending on the pH desired.
34

Attorney Docket No: P1941R1
2. Ethacridine lactate concentration study:
The homogenate was diluted 4-fold with ethacridine lactate stock solutions (1:3 as in the pH study)
and the pH was kept at a set value for each of the target proteins. For anti-CD18, the pH was 8.5 and for
anti-TF pH 7.5. The final ethacridine lactate concentrations in the precipitation systems were 0.15, 0.30,
0.45. 0.60. 0.75. and 0.9% (vv/v). As a reference a set of experiments with 0% ethacridine lactate was
performed.
3. Conductivity/dilution study:
Various concentrations of NaCI were added to anti-CD18 homogenate to evaluate the effect of
conductivity on protein precipitation. The NaCI concentrations studied were 0, 50,100, 150, 200, and 400
mM. A reference series without ethacridine lactate was also performed to determine if any protein was
precipitated due to the high salt concentration. The pH in this salt spike study was 8.5 and the anti-CD18
homogenate was diluted 4-fold. The ethacridine lactate concentration in the samples was 0.6% and 0% for
the reference experiments.
To change the conductivity of the sample the homogenates were diluted in increasing amounts, the
pH was kept constant, pH 8.5 and 7.5 for anti-CD 18 and anti-TF, respectively. All experiments had a final
ethacridine lactate concentration of 0.6% (w/v). The homogenates were diluted 2, 3,4, 5,6, and 7-fold.
4. Temperature study:
Some experiments were performed at elevated temperatures. The E. coli homogenate was diluted
4-fold and the final ethacridine lactate concentration was 0.6%. The pH was 8.5 or 7.5 for anti-CD18 and
anti-TF, respectively. The samples were incubated in a thermostated water bath at a desired temperature,
i.e., 50, 60, and 70°C. The samples were incubated for 20-120 minutes at elevated temperatures. One long-
time incubation, 16 hours, was performed at 50°C.
After mixing the precipitation agent and E. coli homogenate together and adjustment of pH, the
samples were incubated under agitating conditions for 30-60 minutes. The precipitation experiments were
performed in glass tubes, at 4-ml scale. All experiments were performed in duplicate and average values
were reported.
E. E. coli Protein Assay
Ethacridine lactate interacts with mot commonly used protein measurement assays, e.g., Bradford.
BCA. and spectrophotometric absorption measured at 280 nm. Thus, the total protein concentrations were
measured using a generic E. coli protein EL1SA. The samples were diluted in a fish gelatin-containing
buffer (0.15 M NaCI. 0.1 M NaPO4, 0.1% fish gelatin, 0.05% TWEEN 20™, 0.05% PROCL1N™ 300) to
reduce the unspecified binding to the anti-E. coli protein antibodies. The coating antibody was Goat anti-
Whole ECP. The conjugate antibody was an anti-antibody Whole ECP, attached to horseradish peroxidase.
The absorption at 405 nm was monitored using a plate reader from Molecular Devices model SPECTRA
MAX PLUS™ (Sunnyvale, CA, USA).
F. Protein G Assay
To measure the recovered F(ab')2 and antibody concentrations, a protein G affinity chromatographassay was used. An IMMUNO DETECTION™ protein G column was purchased from PerSeptive
Biosystems (Framingham. MA, USA). The column was equilibrated with phosphate-buffered saline (PBS)
and eluted with PBS that had been pH adjusted to 2.2 with HCl. To minimize the interference from
35

Attorney Docket No: P1941R1
ethacridine lactate. samples were treated on exclusion spin columns (BIO-SPIN® 6 Tris columns (Bio-Rad
Laboratories, Hercules. CA. USA)) before assay. The spin columns were used as recommended by the
vendor. A tetramethylammonium chloride (TMAC) wash step was introduced (Fahrner el at.,
Biotechnology and Genetic Engineering Reviews. 18: 302-327 (2001)) to the chromatography method to
minimize any interference of residual ethacridine lactate in the sample. The assay was performed using a
HPLC (HP 1090™ liquid chromatograph) from Hewlett Packard (Mountain View, CA, USA). The samples
were diluted with PBS. Standard curves were prepared for each of the proteins using purified protein (from
Genentech, Inc.).
G. DNA Assay
The DNA concentration in the supernatants after precipitation was measured using a Pico Green Kit
from Molecular Probes (Eugene, OR, USA). It is a fluorescence assay where the fluorescent reagent (Pico
green) binds to double-stranded DNA. The Pico green reagent is exited at 502 nm and the emission at 523
nm is recorded. The assay was performed using a fluorescence plate reader. SPECTRA MAX GENINI
XS™, from Molecular Devices (Sunnyvale, CA, USA). Ethacridine lactate interacts with the Pico green
assay and thus the precipitation agent was removed from the solution prior to analysis. Ethacridine lactate
was removed from the sample using BIO-SPIN® 6 TRIS columns, described in the protein G affinity
chromatography assay section.
H. SDS-PAGE
Supernatants obtained after ethacridine lactate precipitation were analyzed by SDS-PAGE. Non-
reduced 4-12% NUPAGE™ gels from Novex (San Diego, CA, USA) were used to visualize the purification
and recovery of anti-CD 18 and anti-TF. Pre-cast gels ere used and the running buffer was MOPS (pre-made
concentrate purchased from Novex). The gels were stained with a filtered solution of COOMASSIE
BRILLIANT BLUE R250™. The supernatants were volume compensated in regard to respective clarified
E. coli extract. In this way, the intensity of the protein bands in the E. coli extract and samples should be
identical if a 100% yield is obtained in the supernatants after precipitation with ethacridine lactate. Thus, the
gels can be used to indicate accurately the extent of purification obtained from the precipitation.
I. Ethacridine lactate Solubility
Two ethacridine lactate solutions, i.e., 0.6 and 1.2%, were studied. Each solution was divided into
two aliquots and pH adjusted to 6.0 and 9.0. respectively. To obtain a slight buffer capacity in the system,
the ethacridine lactate was dissolved in 10 mM Tris-HCl buffer. Each ethacridine lactate solution was
exposed to increasing amounts of NaCl, i.e., 0, 50. 100, 150, 200, 300, and 600 mM. The samples were
incubated for 3 hours, then centrifuged for 20 minutes at 12000 g in a microfuge (SORVALL MC12V™.
DuPont, Wilmington. DE, USA). The supernatants were assayed for ethacridine lactate by measuring the
absorption at 270 nm. The speotrophotometer used was a HP8453 UV-VIS™ from Hewlett Packard
(Wilmington, DE). now Aligent Technologies (Palo Alto, CA) and known as the AGILENT 8453 UV-
VIS1M spectrophotometer. A standard curve was derived from a solution with known ethacridine lactate
concentration.
36

Attorney Docket No: P1941R1
J. Turbidity
To measure the stability of supematants as a function of time and temperature, the turbidity was
monitored. The turbidity meter used was from HACH (model 2100N, Ames, Iowa, USA). The samples
were measured at room temperature and without dilution of the sample.
The anti-CD 18 homogenate was treated either with 0.6% ethacridine lactate, 0.2% PEI, or only
water. In all of the three samples the anti-CD 18 homogenate was diluted 4-fold and the pH was 7.2+0.2.
After centrifugation for one hour at 4000 g the supematants were recovered and divided into two aliquots.
One part of each sample was incubated at room temperature (21°C) and the other one at 4°C.
Results and Discussion
Effect of pH
The ethacridine lactate molecule is positively charged over most of the pH interval (Miller, supra;
Neurath and Brunner. supra; Franek, Methods in Enzvmology. ed. Langone, J.J., Van Vunakis, H., 121:
631 -638 (1986)). However, since pH changes affect the charge on polypeptides and there is a correlation
between the p1 of the polypeptide and the pH at which it precipitates when exposed to ethacridine lactate
(Neurath and Brunner. supra), the effect of pH on degree of purification in the method of this invention was
investigated.
Homogenates containing anti-CD18 F(ab')2, anti-TF F(ab')2, and full-length anti-TF, respectively,
were exposed to a 0.6% ethacridine lactate solution adjusted to cover the pH range 4-10. Figs. 8A-8C show
the clarified phases after ethacridine lactate treatment and centrifugation of each of these proteins,
respectively.
In Table 1. anti-TF was studied both as a full-length antibody and as a F(ab')2. E. co/i homogenates
were treated at a 1:3 ratio with a 0.8% (w/v) ethacridine lactate solution, i.e., a final ethacridine lactate
concentration of 0.6% in the sample. The pi I was adjusted with HO and NaOH respectively to obtain
desired pH. The yields and purification factors are calculated in respect to each of the clarified cell
homogenates. The DNA concentration in the recovered supematants is also reported in the table.


Attorney Docket No: P1941R1
It can be seen that for all three proteins a bell-shaped curve was obtained with respect to extent of
purification and DNA concentration over the pH range of 4 to 10. At the middle pH values, i.e., pH 5-9, an
approximately 5-fold purification was obtained. The yield ofanti-CD18 F(ab')2 and anti-TF antibody full-
length and F(ab')2 decreased with higher pH. and anti-TF antibody fulMength and F(ab')2 had a stronger pH
dependence than anti-CD18 F(ab')2. The yield of anti-CD18 F(ab')2 is decreased from 100 to 85% and of
anti-TF F(ab')2 from 93 to 18% in the pH range 4-10. Without being limited to any one theory, this could
partly be due to the lower p1 of anti-TF compared to anti-CD 18, e.g., p1 7.5 and 8.9, respectively; however,
these are theoretically calculated p1 values.
The full-length anti-TF protein has an even stronger pH dependence than the F(ab')2 version of the
protein. The purity of full-length anti-TF is highest at pH 7.0. At pH above 8 significant yield losses are
observed (Fig. 8C). For the full-length anti-TF a pH of about 7.0 is preferred, i.e., a 7.1-fold purification and
86% yield. One possible explanation for the higher losses of anti-TF compared to anti-CD18, without being
limited to any one theory, is that anti-TF has more negative surface charges than anti-CD 18 when incubated
above its p1. Analogous to this, without being limited to any one theory, the larger full-length anti-TF might
have more negative surface charges than the corresponding F(ab')2 and thus a significantly higher yield loss
is observed when increasing the pH.
However, components other than cell debris and host protein have to be removed from the target
polypeptide. One such component is DNA. The major disadvantage of having high DNA concentration
together with the target polypeptide is that the viscosity of the solution increases. This will have a negative
impact on the further downstream processing. In addition, if an anion-exchange column is used as the first
capture column, the negatively charged DNA will bind to the resin and thus reduce the protein capacity of
the column.
Hence, the DNA concentration in the supernatants after precipitation with ethacridine lactate was
determined. The results show that the DNA concentration in the supernatants after ethacridine lactate
precipitation is significantly reduced compared to the initial DNA concentration obtained in the E. coli
homogenale. However, the DNA concentration in the supernatant increased as the pH thereof decreased,
i.e., 0.1 and 0.2 µg/ml at pH 5.0 and 4.0 respectively. Without being limited to any one theory', this could be
due to the fact that the phosphates on the DNA become less negatively charged at lower pH. At very high
pH, e.g., pH 10.0. the DNA concentration is significantly increased (0.3 (ig/ml) and the protein purity also
was decreased. Without limitation to any one theory, this is partly due to the fact that the pH is above the p1
of the antibody and l'(ab')2: however, it can also partly be due to the fact that ethacridine lactate is less
charged at this pH.
Effect of ethacridine lactate concentration
The E. coli homogenates were mixed with ethacridine lactate solutions in a (1:3) ratio. The
ethacridine lactate concentration in the samples was increased in 0.15% increments from 0 to 0.9% (w/v).
This study was performed at pH 8.5, 7.5. and 6.0 for anti-CD18, anti-TF F(ab')2, and full-length antibody,
respectively.
Table 2 shows the effect of ethacridine lactate concentration on purification and yield. In Table 2.
anti-TF was studied both as a full-length antibody and as a F(ab')2. E. coli homogenates were treated at a 1:3
38

Attorney Docket No: P1941R1
ratio with different concentrations of ethacridine lactate solutions; the final ethacridine lactate concentration
in the sample is reported. The pH of the anti-CD18. anti-TF(F(ab')2), and full-length anti-TF was 8.5 .7.5
and 6.0. respectively. The DNA concentration in the recovered supernatants is also reported in the table.
Table 2
The effect on purity and yield of anti-CD18 and anti-TF when treated with increasing
ethacridine lactate concentration

* A value of 1 is the same purification as is obtained in the system not treated with ethacridine lactate.
The purity of the antibodies was shown to be strongly correlated with the concentration of
ethacridine lactate (Figs. 9A-9C and Table 2). At ethacridine lactate concentrations above about 0.6% the
effect of increased ethacridine lactate concentration on increased purification was not dramatic. However, at
lower concentrations of ethacridine lactate, i.e. when ethacridine lactate is deficient, every slight addition of
the precipitation agent resulted in a substantial additional purification of the F(ab')2.
The yield of anti-CDI8 F(ab')2 was not affected by the addition of different concentrations of
ethacridine lactate. The step recovery for both of the F(ab')2 was about 90% for all the experiments.
However, it appears easier to obtain quantitative recovery of the F(ab')2 anti-CD18 than the anti-TF F(ab')2.
Without being limited to any one theory, this could be because there are more negative surface charges on
the anti-TF F(ab')2 compared to the anti-CD18 F(ab')2 at the pH studied. The full-length anti-TF reached its
maximum purification at a lower ethacridine lactate concentration than the corresponding F(ab')2 protein.
0.3% and 0.6%, respectively. Without being limited to any one theory, this is probably due to a lower overall
protein concentration in the anti-TF full-length homogenate, i.e., 18 and 30 mg/ml for the full-length and
F(ab')2 anti-TF, respectively. The full-length anti-TF is also derived from re-suspended paste and the F(ab")2
is taken as broth directly from the fermentor. Hence, soluble culture media components present in the E. co/i
broth will not be present in the re-suspended full-length anti-TF material, which could partly explain the
differences seen, without limitation to any one theory.
The DNA concentration in the supernatants is strongly correlated with the data obtained for the
protein purification. At 0.6% or higher ethacridine lactate addition, no DNA was detected in the
39

Attorney Docket No: P1941R1
supernatants. There is also a clear trend of decreasing DNA concentration, i.e., 78 to 0 fig/ml, in the
supernatants when the ethacridine lactate concentration is increased from 0 to 0.6%.
Effect_of Conductivity
As ethacridine lactate precipitates proteins partly due to the charged characteristics of the molecule
(Neurath and Brunner. supra), the conductivity of the sample may have an effect on the purity of the
antibody and F(ab')2 after precipitation. Hence, if the sample has high salt concentration, i.e., high
conductivity, the salts could shield the proteins from the ethacridine lactate and thus reduce the purification
effect.
Anti-CD18 homogenate was subjected to two sets of experiments to separate the effect of the
conductivity from the protein concentration in the sample. To both sets of experiments NaCl was added (0-
400mM). One set of the experiments contained 0.6% ethacridine lactate and in the other set water was used.
The water-containing systems were used as controls, so that if the NaCl would give rise to any precipitation
this could be distinguished from the precipitation derived from the ethacridine lactate. In the systems without
ethacridine lactate, i.e. the water systems, no protein precipitation was observed in the 0-400mM NaCl
concentration range (Fig. 10A). The ethacridine lactate-containing experiments showed a strong increase in
the purification of anti-CDI8 when the conductivity was decreased (Fig. 10B).
Without being limited to any one theory, one reason for the improved purification of anti-CD18 at
lower conductivity could be the low shielding capacity of the charged ethacridine lactate at low salt
concentration. Analogous shielding effects have been seen when PEI is used for protein purification at
increasing salt concentration (Jendrisak, supra). However, an even more important factor is the low
solubility of ethacridine lactate at higher salt concentrations (Miller, supra; Neurath and Brunner, supra;
Franek, supra). At l00mM NaCI precipitation of ethacridine lactate was observed in the extraction system,
and as the salt concentration was increased, more of the ethacridine lactate was precipitated. Hence, less
ethacridine lactate is soluble in the system and available to precipitate proteins and other biomolecules.
When the solubility of ethacridine lactate as a function of NaCI concentration was studied, a pH
dependence was observed (Fig. 11). At pH 6 no significant difference in the solubility between the 0.6 and
1.2% ethacridine lactate solution was observed. Both solutions were soluble at 50mM NaCI but nearly fully
precipitated at l00mM. For the pH 9 solutions, where the ethacridine lactate was less charged, a significant
difference in solubility was seen between the two concentrations of ethacridine lactate. The 0.6% ethacridine
lactate solution precipitated at lower salt concentration than the more concentrated ethacridine lactate
solution (Fig. 11). Chlorides have been shown to precipitate ethacridine lactate especially efficiently
(Franek. supra).
In practice, the conductivity of a precipitation experiment will be determined by the dilution factor.
Hence, a set of experiments was performed where the impact of the dilution factor of the E. coli homogenate
was studied. The overall ethacridine lactate concentration was kept constant, i.e., 0.6% (w/v), but the
conductivity of the sample was decreased with increased dilution. The results are shown in Table 3. In
Table 3, anti-TF was studied both as a full length antibody and as a F(ab')2. The concentration of ethacridine
lactate in each experiment was 0.6% (w/v). The pH of the anti-CDI8, anti-TF(F(ab')2), and full- length anti-
TF was 8.5 ,7.5 and 6.0. respectively. The yields and purification factors are calculated in respect to each of
40

Attorney Docket No: P1941R1
the clarified cell homogenates. The DNA concentration in the recovered supernatants is also reported in the
table.
Table 3
The effect on the purity and yield of anti-CD18 and anti-TF upon dilution of the E. co/i homogenates in
different amounts

* A value of 1 is the same purification as is obtained in the system not treated with ethacridine lactate
The results showed that the purification of F(ab')2 was enhanced if the conductivity was lowered by
increasing dilution. However, at a conductivity of 3.5mS or lower the effect of conductivity was minor. For
the E. coli homogenate used in this Example a 4-fold dilution had to be performed to obtain conductivity
below 3.5mS. For the full-length anti-TF antibody a slightly lower dilution can be performed than for the
F(ab')2 homogenates. Without limitation to any one theory, this can be due to the fact that the protein
concentration in the full-length homogenate is lower. Furthermore, as the full-length material is derived from
re-suspended paste, some of the media components that might affect the precipitation have been removed
prior to the ethacridine lactate precipitation.
In these experiments the protein concentration was decreased with increased dilution (lowering of
the conductivity). Thus, even if 0.6% ethacridine lactate, which was found to be an excess concentration in
the ethacridine lactate concentration study, was added, maximum removal of host protein and DNA was not
obtained in the less diluted sample. This is due to the increased homogenate concentration, i.e., overall
protein and DNA, in these samples compared to the sample performed in the ethacridine lactate
concentration study. Hence, the overall concentration of protein, DNA, and other components will affect the
ethacridine lactate concentration or alternatively the dilution required. The yield of anti-CD 18 F(ab')2 and
full-length and F(ab')2 anti-TF increased with the decrease in conductivity.
The effect on the DNA removal was also found to be conductivity dependent. At a two-fold -
dilution, i.e.. 5.0 mS, a high DNA concentration was obtained in the supernatant, 20.7 µg/ml. However, if a
3-fold dilution was performed, i.e.. 4.0 mS. the concentration was decreased to 0.5 µg/ml, and at even higher
dilution no DNA was detected. As pointed out earlier, in this case the total amount of ethacridine lactate to
41

Attorney Docket No: P1941R1
protein and DNA increases with the higher dilutions, i.e., at lower conductivity. Thus, the effect seen in this
example is both of lowering conductivity and of increasing amounts of ethacridine lactate.
Effect of temperature
Temperature is a factor that is known to be important when performing precipitation experiments.
Thus, some elevated temperatures were studied in combination with ethacridine lactate. The effect of
incubation time at elevated temperature was also investigated.
Incubation at elevated temperature, i.e., 50-70°C, had a positive effect on the purity of the two
F(ab')2 proteins (Figs. 12A and 12B). The higher the temperature, the more efficient the purification. An
incubation at 70°C significantly improved the purity of the F(ab')2 proteins. Incubating the sample for longer
time, i.e., 40 min compared to 20 min, at 70°C did not improve the purity of the F(ab')2 (Figs. 12A and
12B), but an approximately 10% loss in yield was observed. However, when the sample was incubated
above 70°C, no F(ab')2. was recovered, which is due to temperature precipitation of F(ab')2as well as other
E. coli proteins.
To investigate the effect of the incubation time more closely, an experiment was performed where
the samples were incubated for 16 h and 30 min at 50°C. The results showed that there was no significant
purification improvement for the sample that had been incubated for 16 h compared to 30 min at this
temperature. This indicates that temperature precipitation is a rapid phenomenon, which suggests that a fast
heating to appropriate temperature would be more suitable then a long incubation time.
The full-length anti-TF was also studied at elevated temperatures. A 15-min incubation at 50°C had
a slightly positive effect on the purity of the antibody and no losses in recovered material were observed
compared to a sample incubated at room temperature (Fig. 12C). However, if the temperature was increased
to 60°C, nearly all anti-TF precipitated. The data indicate that the full-length anti-TF has lower stability at
elevated temperature than the F(ab')2 anti-TF protein.
Since an increase in temperature could give rise to modifications of the target polypeptide, the
stability of the particular polypeptide of interest at elevated temperature would need to be evaluated before
implementation.
Stability of feed stream
It is important to recover the cleanest feed streams possible. However, another important
characteristic of the feed stream is its stability over time. Hence, the stability of supernatants after
ethacridine lactate or PEI treatment and a supernatant after plain centrifugation was compared. Each of the
supernatants was incubated at two temperatures, i.e., room temperature (21°C) and 4°C. The stability was
monitored by measuring the turbidity of the respective sample.
In Fig. 13 the change in turbidity over lime is shown for the three different supernatants, i.e.,
ethacridine lactate, PEI. and non-treated clarified supernatant, respectively. It can clearly be seen that the
polyelectrolytes, i.e.. ethacridine lactate and PEI, significantly reduced the turbidity of the supernatant.
Directly after clarification the non-treated clarified supernatant had a turbidity of about 700 NTU, whereas
the PEI-treated sample only had half that turbidity, i.e., 327 NTU, and the ethacridine lactate supernatant had
a turbidity of I NTU. There was not a major difference seen, either over time or with incubation
42

Attorney Docket No: P1941R1
temperatures, when monitoring the turbidity of the PEl-treated supernatant. For the supernatant that had only
been centrifuged there was a trend of lower turbidity in the room temperature sample during the first 48
hours. However, the room temperature sample at 72 hours could not be measured due to high turbidity of the
sample. The ethacridine lactate-treated supernatant had very low turbidity that was not significantly
increased if incubated at 4°C. When the sample was incubated at 21°C the turbidity increased significantly.
i.e., from 1 to 100 NTU over 72 hours. However, 100 NTU is still less than the turbidity obtained for the
other two supernatants directly after clarification. Hence, it can be concluded that the supernatant recovered
after ethacndine lactate treatment at pH 7 is a stable feed stream.
Conclusion
Ethacridine lactate can successfully be used as precipitation agent for the primary recovery of
heterologous polypeptides from culture broth or homogenate. When ethacridine lactate is used as
precipitation agent, the ideal target polypeptide preferably has a higher pi than the average host proteins.
Hence, most proteins can be negatively charged and become precipitated by ethacridine lactate at the same
time as the target protein is positively charged and thus recovered in the supernatant.
The preferred concentration of ethacridine lactate for precipitation is highly dependent on the
concentration of host protein and DNA in the broth or homogenate. The higher the protein and DNA
concentration in the broth or homogenate, the higher is the amount of ethacridine lactate required. Hence, the
more negatively charged components available for ethacridine lactate to complex with, and thus precipitate,
the higher is the preferred amount of ethacridine lactate for precipitation. The lower the conductivity of the
solution when performing the precipitation, the more efficient is the purification of the polypeptide. The
precipitation step will give significant polypeptide purification and DNA reduction at the same time as cell
debris is removed.
For efficient host protein and DNA precipitation, the pH is generally between about 4 to 10, and
preferably no more than about pH 9, as the molecule becomes less charged above this pH. Ethacridine
lactate should more preferably be used in the about pH 5-9 range when purifying polypeptide. To improve
the purification, a short incubation at elevated temperature can be performed. However, stability at elevated
temperatures has to be determined for the particular polypeptide of interest to avoid precipitation of the
target polypeptide. Also the quality of the recovered target polypeptide has to be investigated to confirm that
no modifications of the target polypeptide take place.
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Attorney Docket No: P1941R1
WHAT IS CLAIMED IS:
1. A method tor purifying a desired heterologous polypeptide from microbial fermentation broth or
homogenate in which it is produced and solubilized comprising adding to the broth or homogenate an
effective amount of a solution of ethacridine lactate to precipitate host cell impurities under conditions
wherein the majority of the polypeptide remains soluble, and separating the desired polypeptide from
the broth or homogenate.
2. The method of claim 1 wherein the broth or homogenate is from yeast or prokaryotes.
3. The method of claim 1 or 2 wherein the broth or homogenate is from bacteria.
4. The method of any one of claims 1-3 wherein the broth or homogenate is from eubacteria.
5. The method of any one of claims 1-4 wherein the broth or homogenate is from gram-negative bacteria.
6. The method of any one of claims 1-5 wherein the broth or homogenate is from E. coli.
7. The method of any one of claims 1-6 wherein the polypeptide is separated from homogenate.
8. The method of any one of claims 1 -7 wherein the polypeptide has a higher p1 than the average p1 of host
proteins contained in the host cell impurities.
9. The method of any one of claims 1-8 wherein the polypeptide has a p10 of at least about 7.
10. The method of any one of claims 1-9 wherein the polypeptide is a recombiant polypeptide.
11. The method of any one of claims 1-10 wherein the polypeptide is an antibody.
12. The method of any one of claims 1-11 wherein the polypeptide is a humanized antibody.
13. The method of any one of claims 1-12 wherein the polypeptide is a full-length antibody.
14. The method of any one of claims 1-12 wherein the polypeptide is an antibody fragment.
15. The method of claim 14 wherein the polypeptide is an antibody fragment comprising a light chain.
16. The method of claim 15 wherein the polypeptide is an antibody fragment comprising a kappa light
chain.
17. The method of any one of claims 14-16 wherein the polypeptide is a Fab, Fab', F(ab')2, or F(ab')2-
leucine zipper fusion.
18. The method of any one of claims 14-17 wherein the polypeptide is F(ab')2.
19. The method of any one of claims 1-11 wherein the polypeptide is an anti-IgE. anti-CD18. anti-VEGF.
anti-tissue factor. 2C4. anti-Her-2. anti-CD20. anti-CD40, or anti-CD 1 la antibody or antibody
fragment.
20. The method of any one of claims 1-11 or 19 wherein the polypeptide is anti-CD 18 F(ab')2, anti-tissue
factor F(ab')2. full-length anti-tissue factor antibody, or anti-VEGF antibody.
21. The method of any one of claims 1-20 wherein the concentration of ethacridine lactate is about 0.1-5%
weight/volume.
22. The method of any one of claims 1-21 wherein the concentration of the ethacridine lactate is about 0.4-
5% weight/volume.
23. The method of any one of claims 1-22 wherein the concentration of the ethacridine lactate is about 0.6-
5% weight/volume.
24. The method of any one of claims 1-23 wherein the conductivity of the broth or homogenate after
addition of the ethucridine lactate is about 1-15 mS.
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Attorney Docket No: P1941R1
25. The method of any one of claims 1-24 wherein the pH of the broth or homogenate after addition of the
ethacridine laciaie is about 5-9.
26. The method of any one of claims 1-25 wherein the pH of the broth or homogenate after addition of the
ethacridine lactate is about 6-9.
27. The method of any one of claims 1-26 wherein the temperature of the broth or homogenate after
addition of the ethacridine lactate is from about room temperature to 70°C.
28. The method of any one of claims 1-27 wherein the temperature of the broth or homogenate after
addition of the ethacridine lactate is from about room temperature to about 65°C held for about 1-60
minutes.
29. The method of any one of claims 1-28 wherein the temperature of the broth or homogenate after
addition of the ethacridine lactate is from about 50 to 65°C held for about 1-60 minutes.
30. The method of any one of claims 1-29 wherein the separation is accomplished by centrifugation or
filtration.
31. The method of any one of claims 1-30 wherein after the polypeptide is separated from the broth or
homogenate. it is further purified by subjection to chromatography or filtration.
32. The method of any one of claims 1-31 wherein the polypeptide is produced in a soluble fraction prior to
addition of the cthyacridine lactate.
33. The method of any one of claims 1-31 wherein the polypeptide is insoluble and is dissolved by
contacting it with a solubilizing agent prior to addition of the ethacridine lactate.
34. A microbial cell fermentation broth or homogenate comprising ethacridine lactate and a polypeptide
heterologous to the cells.
35. The broth or homogenate of claim 34 that is from bacterial cells.
36. The broth or homogenate of claim 34 or 35 that is from E. coli.
37. The broth or homogenate of any one of claims 34-36 wherein the polypeptide is arecombinant
polypeptide.
38. The broth or homogenate of any one of claims 34-37 wherein the polypeptide is an antibody or antibody
fragment.
39. The broth or homogenate of any one of claims 34-38 wherein the polypeptide is F(ab')2.
40. The broth or homogenate of any one of claims 34-38 wherein the polypeptide is anti-lgE. anti-CD 18.
anti-VEGF. anti-tissue factor, 2C4. anti-Her-2, anti-CD20, anti-CD40, or anti-CDl la antibody.
41. The broth or homogenate of any one of claims 34-38 or 40 wherein the polypeptide is anti-CD 18
F(ab' )2. anti-tissue factor F(ab' )2, full-length anti-tissue factor antibody, or anti-VEGF antibody.
42. The broth or homogenate of any one of claims 34-41 wherein the concentration of the ethacridine lactate
is about 0.1-5% weight/volume.
43. The broth or homogenate of any one of claims 34-42 wherein the polypeptide is soluble in the broth or
homogenate.


A method for purifying a desired heterologous polypeptide from microbial fermentation broth or homogenate in which it is produced and solubilized is described. This method involves adding to the broth or homogenate an effective amount of a solution of 6,9-diamino-2-ethoxyacridine lactate (ethacridine lactate) to precipitate host cell impurities under conditions wherein the majority of polypeptide remains soluble, and separating the desired polypeptide from the broth or homogenate. The broth or homogenate containing the ethacridine lactate and polypeptide is also disclosed.

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Patent Number 221122
Indian Patent Application Number 01312/KOLNP/2005
PG Journal Number 25/2008
Publication Date 20-Jun-2008
Grant Date 18-Jun-2008
Date of Filing 07-Jul-2005
Name of Patentee GENENTECH, INC.
Applicant Address 1 DNA WAY, SOUTH SAN FRANCISCO, CALIFORNIA 94080-4990
Inventors:
# Inventor's Name Inventor's Address
1 PHILIP M. LESTER 15766 VIA COLUSA, SAN LORENZO, CA 94580
2 JOSEFINE PERSSON OSTERVAGEN 16A, S-169 52 SOLNA
PCT International Classification Number C12P 21/02
PCT International Application Number PCT/US2004/000499
PCT International Filing date 2004-01-08
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/439,418 2003-01-09 U.S.A.