CA2276548C - Improved methods for the preparation of endotoxin-binding proteins - Google Patents

Abstract

Disclosed are improvements in methods for the isolation of endotoxin-binding proteins which are secreted by transfected host cells in appropriate cell culture media.
In its preferred embodiments, the invention comprises addition of a cation exchange material to the media as the means of increasing the yield of recombinant endotoxin-binding proteins, such as bactericidal/permeability-increasing protein and lipopolysaccharide-binding protein.

Description

r CA 02276548 1999-08-09 IHPROVED HETHODS FOR T$E PREPARATION OF
ENDOTOYIN-BINDING PROTEINS
LDOF TgE INVENTION
The present invention generally relates to improved procedures for the preparation of endotoxin-binding proteins by recombinant methods and more to particularly to processes for the large scale production of recombinar~t endotoxin-binding proteins, such as bactericidal/ permeability-increasing (BPI) protein, lipopolysaccharide-binding protein (LHP)) high density lipoprotein, L.imulus anti-LpS factor, 15 tachyplesin, and structurally related proteins using genetically transformed host cells grown in culture, including culture in fermentors. -HIsrCICGROUND OF THE INVENTION
Endotoxin, or lipopolysaccharide, is a 20 component of the cell wall of Gram-negative bacteria and is implicated in the manifestation of acute bacterial infections. Numerous proteins have been reported which bind to the principal form of endotoxir~, lipopolysaccharide ("LPS"). Examples of 2 5 s a c h L P S - b i n d i n g p r o t a i n s a r a bactericidal/permeability-increasing protein ('~BPI

r CA 02276548 1999-08-09 .. 2 _ proteirx") l.ipopolysaccharide-binding protein ("LBP"), high density lipoprotein, and tachyplesin [Nal~amuxa, et al., J. Biol) Chem., 263: 16709-16713 (1988)l.
Certain o~ these proteins share significant structural homology. For example) both sPI and LBP
possess a positively-charged amino terminal region of approximately 25 kDa which is the portion of each molecule which binds to the lipid A portion of LPS. See SChumanzi, et ai., Science, 249:1429-19:31 (1990).
Binding of BPI pxotein to membrane-bound LPS
increases the ezwelope permeability o~ susceptible Gram negative bacteria. Ooi, et al., J. Biol. Chem., 262:14891 (~.987j. BPI protezn also binds to soluble LPS
anal human BPT protein has been isolated from 7.5 polymarphonucJ.ear neutrophils ( "PMNS" ) by acid extraction combined with either ion exchange chromatography or E.
coli a~fin,~.ty chromatography. Elsbach, et al. J. giol.
Chem., 254:11000 (1979); Weiss et al_, Blood, 69:652 (1987). The bolo-BPI protein, isolated from human pMNs has potent bactericidal activity against a broad spectrum of Gram-negative bacteria. ElsbaCh, et al., J. Biol.
Chem., 254:1.1000 {1979). This antibacterial activity appears to be associated with the amino= ~.ermir~.al region of the isolated human holo-BPI protein. In contrast, the carboxyl terminal region of the isolated human BPI
protein displays only slightly detectable anti-bacterial activity. Ooi, et al., J. Exp. Med., 174:549 (1991).
Human DNA encoding BPI has been cloned and the amino acid sequence of the encoded protein has been elucidated [See, Gray et al., J'. Biol. Chem., 264:9505 (1989), hereinafter referred to as "Gray"; U.S. Letters Patent No. 5,198,541 dated March 30, 1993] allowing for the large scale production of recombinant BP'I and biologically active (e. g., amino and carboxyl terminal) fragments thereof. Xnitial attempts to purify recombinant BPI and HPI-related proteins fxom the medium of transfected cells util~.zing traditional protein purification methods provided low yields. Pulse-chase 5 experiments using 3sS labelled methionine and performed on cell cultures of traz~sfected Chinese Hamster Ovary (CHO) cells expressiza.g a recombinant product comprising the amino terminal 199 amino acids of the mature BPI protein (hereinafter rHPX(1-199)] indicated that the recombinant BPI fragment disappeared from the media during 3.5 hours to 7 hours of chase. Preliminary experimental. procedures aimed at determining the basis for this low product yield indicated that the protein product displ-ays significant "sticaciness" and, In fact, adheres to itself, to other Z5 media components (including host cells), and to plastic and glass culture vessels. However, the precise reasons) fox protein loss have yet to be elucidated.

r CA 02276548 1999-08-09 Like 8PT protein, LBP binds to the lipid A
portion of LPS. The bolo-LBP protein is a 60 kD protein secreted by the liver and has been reported to be responsible for delivering LPS to macrophages. Ooi; et al., J. Exp. Med., 7.7~: 649-65 (1991). Unlike BPT
protein, LBP generally enhances the inflammatory response generated by LPS. For example, LBP stimulates LPS-induced tumor necx~osls factor ( ~rTNFr~ ) production.
Of interest to the present invention is the use of ion exchange materials in the isolation and purificatioz~ of proteins and related substances. For example, Centocox Inc. PCT application No. W089/05157 published June 15, 1989, reports the purification and isolation of recombinant immunoglobulins by passing the cell culture medium over a chromatography column, wherein the immunoglobulin is adsorbed onto axi exchange material.
The immundglobulin is then eluted by raising the salt concentration in the column. As another example, published PCT application No. w0 90/087.59 by Robins, et a3.., reports removal of DNA from protein. preparations by incubation in the presence of an anion exchange material.
Wang, Ann. N.Y. Acad. Sci., 413:313-321 (1983) presents the results of ~~hybridrr ferment=ation-extraction procedures for the production and isolation of a model antibiotic, cycloheximide, from fermentation cultures using non-ionic resins and noted that for one resin (XAD-4, Rohm and Haas, Philadelphia, PA) the product was absorbed on the resin surface making it ~~conceivable~~ to harvest the product from the resin.
Due to the utility of endatoxin binding proteins such as BPI protein and LBP as regulators of bacterial infection and the sequelae thereof, there exists a need in the art for improved methods for the isolation of such proteins from cell culture media.
$RIEF SUl~ARY OF THE IN~E
The present invention provides improved methods which facilitate the isolation of endvtoxih-binding proteins, and especially lipid A binding proteins, in high yields.
15_ The improved methods generally comprise the incorporation of a particulate ration exchange material into cell culture medium containing host cells which have been genetically trahsfected with DNA
encoding the endotoxin-binding protein. Such proteins 2o which are secreted into the cell culture medium by said host cells are reversibly bound to said ration exchange material. The canon exchange material with bound protein is then separated from the cell culture medium. Finally, the desired endotoxin-binding 25 protein is then isolated from the ration exchange material.
The improved methods comprise the incorporation of a particulate ration exchange material (preferably S-Sepharose~"particles) into a cell culture medium containing host cells (preferably CHO--Kl or CHO-DG44 Cells) which have been genetically transformed with DNA for expression of endotoxin-binding proteins or fragments thereof. The protein secreted into the cell medium by said host cells .is reversibly bound to said cation exchange material.
The cation exchange material with bound protein is then separated from the cell culture medium. Finally, 1D the protein is isolated from the cation exchange material.
A presently preferred cativn exchange material for practice of the invention is S-Sepharose alzd presently preferred isolation procedures comprise sequentially contacting the canon exchange laaterial with a gradient or steps of increasing ionic strength.
In a preferred embodiment of the invention, the improved methods are applied to the production of recombinant BPI products, including but not limited 2.0 to, bactericidal/permeability-increasing protein and biologically active fragments thereof as well as gpI-related products such as fusion proteins comprising, at their amino terminal, the BfI protein or a biologically active fragment thereof and, at their carboxy teL'~riinal, at least one Constant domain of an immunoglohulxn heavy chain region or an allelic ~ariatlt thereof. Proteins of interest are secreted by genetically transformed host cells which are grown and maintained in a culture medium suitable for growth of host cells and secretion of the protein products.
Also, in another embodiment of the invention, the present improved methods are applied to the isolation of lipopolysaccharide-binding protein and amino-terminal fragments thereof.
The foregoing brief summary illustrates the preferred embodiments of the invention. Numerous aspects and advantages of the present invention will become 1o apparent to the skilled artisan upon reading the following detailed description thereof.
pESCRIPTION OF THE DRAWING
FIGS. lA, 1B, 1C and 1D depict the results of comparative experiments using methods according tv the invention and traditional chromatography methods to isolate the rBPI(1-199) protein.
FIG. 2 depicts the results of the stepwise elution of rBPI(1-99) from S-Sepharose.
FIG. 3 depicts results of Western blot analysis of products prepared according to the invention.
FIG. 4 is a Western blot depicting rBPI-Ig fusion products prepared according to the methods of the present invention.
.FIG. 5 depicts the results of a Cvomassie-stained gel depicting isolation of LBP prepared according to methods of the present invention.

DETAILED DESCRIPTION OF THE INVENTIC~I~i The following detailed description illustrates practice of methods of the invention in the context of recombinant production of three particular endotoxin-binding proteins, an amino-terminal portion of recombinant BPI protein ("rBPI
protein"}) an amino-terminal portion of recombinant LBP ("rLBP"), and rBPI-immunoglobulir~ fusion proteins ("rHPI-Ig fusions") from animal cell cultures. While the practise of the invention is exemplified herein by certain specific endotoxin-binding proteins, it is apparent to the skilled artisan that, due to their general structural and functional similarities, any endotoxin-binding protein may be isolated using methods of the invention. Such proteins include, but are not limited to, polymyxin S, high density lipoprotein, Limulus anti-LPS factor, and tachyplesin.
More specifically, example 1 demonstrates that addition of the ration exchange material, S
Sepharose, to a cell culture medium results in increE~sed yields of rBPI protein. Example 2 provides further results which demonstrate that introduction of the ration exchange material to cell cultures produces increased yields of rBPI protein. Example 3 illustrates practice of the improved methods in the isolation of rSPi-immunoglobulin fusion proteins and Example 4 de7adonstrates the use of a ration exchange material in the isolation of LBP.

-g-Egampla t isolation of recombinant gpI products Methods of the present invention were used to isolate a recombinant HPI protein which is the expression product of DNA encoding the 31-residue signal sequence and the first 199 amino acids of the N-terminus of the mature human BPI, as set out in SEQ
ID NOS: 1 and 2 and is desj~gnated "rBPI(1-199)"
herein. The DisA sequence employed differs from the BPI-encoding DNA sequence reported in Gray, et al., supra, in that the vaiine at position 151 of the rSPI(1-199) expression product is specified by GTG
rather than by GTC as in Gray et~al. and rBPI(1-199) encodes giutamic acid (specifi.ed by GAG) at position 185 rather than lysine (specified by AAG) as reported at that position in Gray et al. Recombinant production of rBPI(1-199) protein is_ reported in Gazzano-Santoro, et al., Infect~an and Immunity, 60:
4754-4761 (1992), wherein the protein is referred to as "rBPI-23".
The host cells employed in this example were CHO-K1 cells transformed with a DNA vector including DNA encoding the initial 199 amino terminal amino acids of human BPI preceded by its endogenous 31 residue secretory signal sequence. The desired expression product, rBPI(1-199), was a biologically active fragment of the human BPI protein comprising the initial 199 amino terminal residues from whi,eh the signal sequence residues were removed in the course of pQSt-translational secretory processing by the host cells.
Two roller bottles containing the transfected CHI host cells in Hams' F12 medium supplemented with 5% fetal bovine serum were prepared and the cells were grown to confluence (approximately 3 days). Once confluency was reached, the Hams Fl2 medium was removed and replaced with 500 ml of HH-CHo serum free medium (Irvine Scientific, Irvine, Ca.).
In the first roller bottle, approximately 8 gm (wet weight) of sterilized S-Sepharose (Pharmacia, fast flow, ~1~-0512-O1, Uppsula, Sweden) was added to one of the roller bottles for 3 days. The S-Sepharose was then isolated in order tv generate a first column.
Growth medium and S-Sepharose resin were removed from roller bottles, pooled and left for at least 15 minutes to allow the S-Sepharose to=_settle to the bottom of the container. The bulk of the medium, clear of resin, was removed by decanting and then filtered through a device, such as a fritted disc, to permit the removal of cells and the retention of the S-Sepharose. Following the decanting of the medium, the S-Sepharose was suspended in an Acetate buffer comprising 20 mM sodium acetate/acetic acid at pH 4.0 Containing 0.1 M NaCl, stirred gently, and allowed to settle for 10 minutes. The buffer was then decanted and the S-Sepharose was transferred in a small volume to an appropriately-sized liquid chromatography column (1 x 10 cm, Econocolumn~ BioRad, Richmond) CA).

..11-The second roller battle contained cells grown under the conditions stated above but in the absence of S-Sepharose. The u~edium from this second roller bottle was removed from the roller bottle. CHO
cells were removed by centrifugation and the clatrified medium was adjusted to contain 20 mM sodium acetate/acetic acid, pH 4Ø The medium was diluted to a conductivity of 10-1S mS/cm and was then loaded onto a second, traditional S-Sepharose column which had bean equilibrated in 2o mM sodium acetate/acetic acid pH 4.0 (Acetate buffer) containing 0.1 M NaCl in order to maximize binding of the xBp=(1-199) protein.
Both the first and the second S-Sepharose columns were washed with 0.1 M NaCl-Acetate buffer until the AZ80 absorbance of the eluate w2~s equal to that of the 0.1 M NaCI-Acetate buffer alone. The protein bound to each column was then eluted in a single step with 1.0 M NaCl-Acetate buffer.
The eluates from both columns ~ were subjected to an ELISA assay wherein samples from the eluates ,.~
were bound to Immulon-2 flat bottom multiwell plates (Dynetech Labs) in the presence of PBS overnight at 4~C. The plates were then washed with 0.05% Tween-20'~
~n PBS, and then incubated with a 1:1000 dilution of rabbit anti-rBPI(1-199) antisera in PBS containing 0.05% Tween-2o for one hour at room temperature.
After incubation) the plate was again washed With 0.05% Tween-?.0 in PBS and the ELISA was developed using the TMB reagent (Pierce Rockford, IL) according to the manufactures instructions and read at 450 nm in an EL309 micxoplate reader (Biotek Instruments, Winoosaci Vt. ) .
The ELISA results revealed that the eluate from the S-sepharose column derived from the cell culture medium produced 3-8 fold stronger reactivity compared to the eluate from the S-Sepharose column to which medium had been added.
Example 2 provides fuxther results, demonstrating that culturing S-Sepharose together with transfected CHO cells increased the yield of rBPI(1 199) protein produced by the transfected cells.
Exasap 1 a 2 puantitative Anal~rsis of Isolation or rBPI(1-1991 In order to more quantitatively demonstrate that the yield of the rHPZ(1-1.99) protein product obtained from the CHO cell cultures in=Example 1 was greater when a cation exchange material was added to the cell culture medium, stained gel and Western analyses were carried out on the eluate samples described above.
Protein samples obtained from the 1.0 M
NaCI-Acetate buffer eluates described in Example 1 were separated by SDS-polyacryl.amide gel electrophoresis (SDS-PAGE). The samp7.es of rBPZ(1-199) were first adjusted to contain less than O_5 M
NaCl and were then precipitated by the addition of ice-cold acetone to a final concentration of 75%. The resulting protein precipitate was then pelleted by centrifugation at greater than 10,000 rpm far 5 to to minutes. The supernatant was removed and the precipitate was suspended in a gel sample buffer containing a M Urea, 2% sDS, 60 mM Tris Hcl at pH 6.8.
The suspended samples and appropriate protein molecular weight standards (BioRad, Richmond, CA and BRL, Bethesda, MD) were heated to 95 C for 3-5 minutes and then loaded onto uniform percentage or gradient percentage polyacrylamide gels (BioRad) and separated using a mini Protean~II gel electrophoresis apparatus (HioRad). Following eJ.ectrophoresis the gels were used directly for Coomassie staining (0.5% Coomassie Brilliant slue-R, 25% isopropanol, ~o% methanol, lo%
acetic acid) or were used for electrotransfer. The proteins which were separated by SDS-PAGE were electrotransferred along with appropriate prestain standard protein (BioRad) onto either nitrocellulose (8A85, Schleicher and Schuell, Keene, NH) or PVDF
(Immobilon-P;~~Millipore, Bedford, MA) membranes. The transfer was achieved in 10% CAPS (cyclohexylamino-1-propane-sulgonic acid), 10% methanol, pH 1.1.5 for 20 minutes at 0.5 amps. The resulting blots were processed using a 1:1000 dilution of rabbit anti-human BPr (holoprotein) antisera and the Western Lite Chemiluminescent Detection Systems (Tropix System, Bedford, MA) according to the manufacturer's instructions. Gelatin (BioRad) at 0.25% was used in place of Tropix I-Blocky and the membranes were not - . - - CA 02276548 1999-08-09 dried following electrotransfer. The processed membranes were exposed to Cronex ~4 film (Dupont) Wilmington, DE).
The results of the stained gel and Western analysis are shown in Figs. 1A-1D, wherein Fig. lA and 1B
respectively present coomassie stain and Western blot analysis of the flow through (FT), 0.1 M NaCl and l.0 M
NaCl e~.uates of columns formed from S-Sephdrose beads incubated with culture medium. Figs. iC and 1D
correspondingly present Coomassie stain and Western blot analysis of eluates from "traditional" columns of S-Sepharose. The arrow in Figs. lA and 1C indicates the region correspot'~ding the molecular weight of the rBPI ( 1-199) protein product. The yield of rHPI(1-199) protein was estimated to be at.least 10-fold higher when the cationic exchange xesin, 5-Sephaxose, had been added to the culture medium during cell growth.
Subsequent Experiments involved the isolation of rBPI(1-199) from 20 to 40 g of s~Sepharose obtained from 3 to 5 roller bottles. The bound samples were eluted with increasing concentrations of NaCl in Acetate buffer. As Shown in Fig. 2, the rHPI(1-199) product, visualized by Coomassie blue staining, is seen as a 23 kd protein in the 0.8, 0.9, 1.0, and 1.5 M NaCI-Acetate buffer eluates from the S-Sepharose columns. Lrittle or no rHPI(1-199) protein was observed in the 0.2 M to 0.7 M NaCl-Acetate buffer eluates. The xesults of the Western Hlot (Figure 3) indicated that the strongest detectable rBPx(1--199) protein signal was obtained in the 0.8 M to 1.0 M NaCI-Acetate buffer eluates of the S-Sepharose columns.
The 1.5 M NaCl eluate froaa the S-Sepharose column also contained protein which was identified as rBPI(1-199) (See Figure 2) right lade). The 1.5 M
NaCl-Acetate buffer eluate from the S-Sepharose column contained protein having molecular weights of approximately 40 kDa and gxeater than 66 kDa (Fig. 2) which, upon treatment with dithiozhreitol, could be reduced to a single band of approximately 23 kDa. The reduced protein was cross-reactive with anti-BPI
antisera and had the N-terminal sequence of correctly processed rHPI(i-199). The 40 kDa and greater than 66 kDa proteins appear to be disulfide-linked multimers of rBPI(1-199).
The aforementioned results indicate that the addition of a cation exchange material to the cell culture medium improved recovery of rBPI(1-199) protein. Zn order to determine optimum concentration of S-Sepharose, 1.25 g to 10 g quantities of S-Sepharose were added to roller bottles containing 5D0 ml culture medium 2ind transfeeted CHO cells and allowed to incubate as described above. The medium containing the Gation exchange material was then poured into columns as described above. The columns were washed with 0.1 M NaC1-Acetate buffer, then with 0.7 M NaCl-Acetate buffer arid the rBPI{1-199) sample was eluted with 1.0 M NaC1-Acetate buffer. The yield of rBPI(1~199) was determined by chromatography on C4 reverse phase HPLC and was essentially constant for 2.5 g, 5.0 g, arid 10 g quantities of S-Sepharose. 'fhe yield was decreased by approximately 50% in roller bottles containing only 1.25 g S-Sepharose per roller bottle.
Example 3 provides results demonstrating that increased yield of rBPI fusion proteins is obtained using methods according to the present invention.
lQ EXAMPLE
Isolatio of rBPZ-Ig k'usion Proteins Host cells employed in this example are CHO-DG44 ce~.ls transfected with a DNA vector comprising DNA
encoding the initial 1.99 amino acids of BPI protein fused to at least one constant region of an immunoglobulin heavy chain_ Transfected CHO-DG44 cells were grown in roller bottles. For eack~ roller bottle) a T150 flask (containin.g 50 ml ~-MEM without nucleosides and 10%
dialyzed fetal bovine serum) was inoculated with transfected Cells and the cells were grown to confluence (approximately 3-4 days). The --cells were then trypsiniaed and transferred into a 90ocm2 roller bottle containing 500 ml Ham's F12 media and 10% fetal bovine serum and grown to confluence for approximately 3 days. Once confluency was reached, the Ham's F12 medium was removed arid replaced with 500 ml HB-CHO serum-free medium (Irvine Scientific, 3rvine, CA) .
S-Sepharose beads, which had first been washed with Dulbecco's phosphate buffered saline (PBS) l0 and autoclaved for 20 minutes at 120°C were added aseptically to the roller bottles. The cells were then incubated at 37~ C for 3 days) at which time the beads and growth medium were removed and left undisturbed for at least 15 minutes. The bulk of the medium, clear of resin, was removed from by decanting and filtered through a device) such as a fritted disc, which allows removal of the cells and retention of the 5-Sepharose. Following the decanting of the medium, the S-Sepharose was suspended in an acetate buffer comprising 20 mM sodium acetate/acetic acid, 0.1 M
NaCI at pH 4.0, stirred gently, and allowed to settle for 10 minutes. The buffer was then decanted and the S-Sepharose was transferred in a small volume to an appropriately-sized liquid chromatography column. An Econocolumn (2.5 x 10 cm, BioRad, Richmond, CA) was used for a 20 to 40 g pooled sample of S-Sepharose collected from 3 to 5 roller bottles. The packed S-Sepharose column Was washed wi~.h o.i M NaCI-acetate buffer until the A280 absorbance of the eluate was equal to that of the O.l M NaCl-acetate buffer alone, with 0.5 M NaCl-acetate buffer, with 1.0 M NaCl-acetate buffer and again with 1.5 M NaCl-acetate buffer.
Additional CHO-DG44 cells were prepared as above except that S-Sepharose beads were not added to the cu3ture medium. Instead, an attempt was made to purify the rBPI fusion expression product utilizing two different protein A coiumns. A first sample of HB-CHO medium (see above) was filtered through a 0.45 ycm filter in order to separate the CHO-DG4a cells from the zest of the medium. The sample was than adjusted ", .
to pH 8.0 and placed on a 1?roSepA ~(Biopracessing) column. A second preparation was placed on an AvidGel""
(Bioproeessing) column. The elution og bath columns was, performed with 25 mM citrate buffer at pH 5 . 5 . No rBPI fusion product was recovered from=-either protein A Column. Nor was any product visualized from the ProSepA column following reduction (Figure 4, lane 1).
However, when the ProSepA and Avidgel columns were stripped with 100 mM citrate buffer at pH 3. D, rBPI
fusion prote~.n was detected as shown respectively in lanes 2 and 3 of Figure 4. Lanes 4-6 of Figure a represent the 0.5 M, 1.0 M, and 1.5 M eluates from the S-Sepharose column. Of the eluates from the s-Sepharose column, the 2.5 M eluate contained material corresponding to a fusion diner of approximately 100 kD.

, CA 02276548 1999-08-09 _19-Example 4 provides results demonstrating that increased product y~.eld of LBP is also obtaiz~ed when cells transfected with DNA encoding LB12 are incubated with a cation exck~ange resin.

isolation Of Lipopolysacch ride-Binding Protein The DNA sequence obtained for the 25 kD amino term3.nal of LBP is shown iz~ SEQ ID NO: 3. That Sequence differs in two regions from the reported sequence of Schumann, et al., Science, 249: 1429_1431 (1990) (SEQ ZD
NO: 4) . Those differez~Ces lead to amino acid differences at pos~.tions 129-232 and at position 149 (an asparagine residue at position 1~k8 is encoded by GAT in Schumanrz, supra and by SAC in SEQ ZD NO: 3). See also, Zncyte 25 Pharmaceuticals Tnc. PCT Appla.cation 93/06228 published April 1, 1.993.
Host cells employed in this example are CHO-DG44 cells transfected with a DNA vector comprising DNA
encoding the initial 197 amino acids of LBP, the expression product.
Transfected DG44 cells were=-grown in roller bottles. For each roller bottle, a T150 flask (containing 50 ml a-ME~I without nucleosides and 100 dialyzed fetal bovine serum) was inoculated with transfected cells and the cells were grown to cox~.fluence (approximately 3-4 days). The cells were then trypsinized and transferred into a 900cmz roller bottle containing 500 ml Ham's F12 media axed 10% fetal bovine serum and grown to confluence for approximately 3 days. Once confluency was reached, the Hams F1z medium was removed and replaced with 500 ml HB-CHO
serum-free medium (Irvine Scientific, Irvirie, CA).
S-Sepharose beads) which had first been washed with PBS and autoclaved for 20 minutes at 120 C were added aseptically to the roller bottles. The cells were then incubated at 37 C for 3 days, at which time the beads and growth medium were removed and left undisturbed for at least 15 minutes. The bulk of the medium, clear of resin, was removed from by decanting and filtered through a device, such as a fritted disc, which allows removal of the cells and retention of the s-Sepharose. Following the decanting of the medium, the S-Sepharose was suspended in an acetate buffer comprising 20 mM sodium acetate/acetic acid, 0.1 M
NaCI at pH 4.0, stirred gently, and allowed to settle for 10 minutes. The buffer was then decanted and the S-Sepharose was transferred in a small volume to an appropriately--sized liguid chromatography column. An 2o Econocolumn (2.5 x 10 cm, BioRad, Richmond, CA) was used for a 2o to 4o g pooled sample of S-Sepharose collected from 3 to 5 roller bottles. The packed 5-Sepharose column was washed with 0.1 M NaCl-acetate buffer until the A280 absorbance of the eluate was equal to that of the 0.1 M NaCl-acetate buffer alone, with o.7 M NaCl-acetate buffer and again with 1 . o M
NaCl-acetate buffer.
Yield of rLBP from cell eult~ares in which S-Seprarose beads were added is shown in Figure 5 which is a Coomassie-stained ge7~ of the 0.7 M (lanes 1 and 3) and 1.0 M (lanes 2 and 4) eluates described above.
As shown in the figure, a significant amount of LBP
eluted at 1.0 M. That S-Sepharose is able to facilitate LBP production from cell cultures is unexpected based on its calculated pI {6.6). In the culture medium used above, in which the pH was approximately 7.0, LBP would be expected, based on its pI, to be uncharged oz- slightly negatively charged and thus unreactive with a cation exchange resin, such as s--sepharose.
Numerous modifications and variations in the practice of the invention are expected to occur to those skilled in the art upon consideration of the foregoing description of the presently preferred embodiments thereof. For example, the concentration of cation exchange material used in the-invention may be varied according to the number and type (i.e., efficiency of production of the recombinant product) of cells used. As another example, while cation exchange materials other than S~Sepharose (e. g., Biorex 7oTM and SP (sulfopropyl) type materials such as Sp-Sephadex as well as CM (carboxymethyl) type materials such as CM Sepharose and CM Sephadex) can be employed in processes of the invention, S-Sepharose was preferred as being most readily handled, subjected to sterilization processing, and the like. As still another example, whl.le the above illustrative examples address recombinant production of endotoxin binding . CA 02276548 1999-08-09 proteins in roller bottles, processes of the invention are readily "scaled up" to production izx fermentors.
Typical fermentation conditions for such processes as applied to production of rBPI(1-199) include use of a 60OL working volume iri a 75OL Chemap'" (woodbury, N.Y.) fern~.entor wherein CHO-KJ. cells transfected with plasmid pING4502 [see, Gazzano et al., supxa7 are grown, in ExCell~' 301 medium (,HRH Scientific) supplemented with 0.05% FBS and 0.01 Antifoam (U Carferm Adjuvant~" 27, Union Carbide) and Z°s SP Sepharose "big beads" (1.00-300 micron diameter, pharmacia) is added. Finally, the precise elution proffiles of recombinant endotoxin-binding proteins isolated according to the invention are expected to vary depending on the precise identity of the protein 25 involved. .~s one example, rBPI(1-199) is readily a.solated from resiza. beads foJ.low~ing a 0 , 7 M NaCl-Acetate buffer wash. However, yields of cysteine replacement analogs of BPI protein such as described in co-owned, co-pending Canadian patent application No. 2,155,00 are enhanced by isolation following a 0.6 M NaCI-Acetate buffer wash. Consequently, the only limitations which.
should be placed upon, the scope of the present invention are those which appear in the appended=claims.

SEQUENCE LISTINiG
(1) GENERAL INFORMATION:
(i) APPLICANT: Grinna, Lynn (ii) TITLE OF INVENTION: Improved Methods for the Preparation of Endotoxin-Binding Proteins (iii) NUMHER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Marshall, O'TOOle, Geretern, Murray & Borun (B) STREET: 6300 Sears Tower, ~33 South Waeker Drive (C) CITY: Chicago (p) STATE: Illinois (E) COUNTRY: USA
(F) ZIP. 60606-6402 (v) COliFUTER READABLE FORM:
(A) MEDIiJM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/HS-DOS
(D) SOFTWARE: PatentIn Release X1.0, Version ,f1.25 (vi) CURRENT APPLIGATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION: -(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: vS 07/885,501 (S) FILING DATE: 19-MAY-1992 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Meyers, Thomas C.
(8) REGISTRATION NUMBER: 36,989 (C) REFERENCE/DOCKET NUMBER: 31405 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 312/47d-6300 (B) TELEFAX: 312/474-0448 (C) TELEX: 25-3856 (2) INFORMATION FOR SEQ ID NO: l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1813 base pairs (B) TYPE: nucleic acid (C) STRANDLDNESSe slngla (D} TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(8) LOCATION: 31..1491 (ix} FEATURE:
(A} NAME/1GEY: mat peptide (8} LOCATIONS 124-_.1491 (xi) 5EQU1;NCE DESCRIPTION: SEQ ID NO:l:
CAGGCCTTGA GGTTTTGGCA
GCTCTGGAGG
ATG
AGA
GAG
AAC
ATG
GCC
AGG
G

Met Arg Glu Asn Met Ala Ar Gly -3 1 $

CCTTGCAACGCG CCG AGATGGGTG TCCCTG ATGGTGCTC GTCGCC ATA

ProCyaAanAla Pro ArqTrpVal SerLeu MetValLeu ValAla Ile GGCACCGCCGTG ACA GCGGCCGTC AACCCT GGCGTCGTG GTCAGG ATC

GlyThrAlaVal Thr AlaAlaVal AanPro GlyValVal ValArg Ile TCCCAGAAGGGC CTG GACTACGCC AGCCAG CAGGGGACG (36CGCT CTG 198 ScrGlnLyeGly Leu AspTyrAla SerGln GlnGlyThr AlaAla Leu CAGAAGGAG.CTG AAG AGGATCAAG ATTCCT GACTACTCA GACAGC TTT

GlnLyeGluLeu Lys ArgIleLye IlePro AepTyrser AepSer Phe ' AAGATCAAGCAT CTT GGGAAGGGG CATTAT AGCTTCTAC AGCATG GAC

LyeIleLyeHie Leu ClyLyeGly HieTyr Ser.phcTyr SerMet Asp b5, 50 55 ATCCGTGAATTC CAG CT'rCCCAGT TCCGAG ATAAGCATG GTGCCC AAT

IleArgGluphe Gln LeuProSer SerGln llaSerMet ValPro Aen Val GAS Leu Lya Ph~ Ser Ile Ser Asn Ala Asn Ile Lys Iie Set Gly AAA TGG AAG GCA CAA AAG AGA TTC TTA AAA ATG AGC GGC AAT 2TT GAC d38 Lys Trp Lys Ala Gln Lys Arg Phe Leu Lye Mpt Ser Gly Aon PhQ Asp _25_ LauSer IleGlu GlyMet 5erileSer AlaAep LeuLye LeuGlySer AanPro ThrSer GlyLye ProThrIle ThrCya SerSer CyaSerSer CACATC AACAGT GTCCAC GTGCACATC TCAAAG AGCAAA GTGGGGTGG

HisIle AanSer ValHis ValHisIle SerLys SerLya ValGlyTrp LeuIle GlaLQU PheHie LyaLyaIle GluSer AlaLeu ArgAsnLye ATGAAC AGCCAG GTCTGC GAGAAAGTG ACCAAT TCTGTA TCCTCCGAG 67s HetAea SerGln ValCys GluLyaVal ThrAat1&erVal SerSerGlu cTGCAA CcTTAT TTCCAG ACTCTGCCA GTAATG ACCAAA ATACATTCT 726 LeuGln ProTyr PheGln.ThrLeupro ValHat ThrLys IleAapSeer ValAla GlyIle AanTyr GlyLeuVal AlaPro ProAla ThrTttrAla GluThr LeuAap ValGln MetLyaGly GluPhe TyrSer GluAsnHia IiiaAan ProPro ProPhe AlaProPre Va1Met GluPhe ProAlaAla HiwAsp ArgHet ValTyr LeuGlyLeu SerAap TyrPhe PheAsnThr AlaGly LeuVal TyrGln GiuAiaGly ValLeu LysHet ThrLeuArg AepAsp HetIle ProLys GluserLys pheArg LeuThr ThrLysPhe PheGly ThrPhe LeuPro GluValAla LyBLye phe~Pro AsnMetLyB

PCT/US93/04752.

IleGlnIle HisVal SerAla serThr ProProHis LeuSer ValGln ProThrGly LeuThr PheTyr proAla ValAapVal GlnAla pheAla GTCCTCCCC AACTCC TCCCTG GCTTCC CTCTTCCTG ATTGGC ATGCAC

ValLQUPro AsnSer SerLeu Alaser LauPheLeu I1QGly HetHie ACAACTGGT TCCATG GAGCTC AGCGCC GAGTcCAAC AGGCTT GTTGGA

ThrThrGly 5er?fetGluVal 8erAla GluSerAsn ArgLeu ValGly GluLeu38 LeuAsp ArgLeu LeuLQU GluLeuLya HiaSer AsnIle GlyProPhe ProVal GluLeu LeuGln AspilaMet AsnTyr IleVal ProIleLeu ValLeu ProArg ValAsn GluLysLeu GlnLys Glyphe CCTCTCCCG ACCCcG GCCAGA GTCCAG CTCTACAAC GTAGTG CTTCAG

laa6 ProLeuFro ThrPro AlaArg ValGln LeuTyrAsn ValVa1 LeuGln CCTCACCAG AACTTC CTGCTG TTCGGT GCAGACGTT GTCTAT AAA

ProaieGln Asnghe Leuyou pheGly AiaAspVal ValTyr Lye AGGGGTGCCG GCTGTGGGGC
GGGGCTGTCA
GCCGCACCTC

ACCGGCTGCC CAGATCTTAA CCRAGAGCCC

TTTCCCCAGG CTTGCAAACT
GAATCCTCTC

TCTTCGACTC CACGAGGAAA CATTATTCAT

AGATTCAGAA TGGAAAAGTG
ATGATCTAAA

CATGGTGTGT CTTTCAAGGG CTAAGGCTGC

ATTTTAGGGA AGAGATATTT
TTATGAGCTT.

TCGTGTTT'CR CATGAAAAAA
ATTGTAACCA

AACTTCTGGT
TTTTTTCATG
TG

2 ~ INFOR~iATZON P'OR SEQ ID NO: 2 (i) SEQUEtICE CHARACTERISTICS:

(A) LEIZGxHs 4A7 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linoar (ii) MoLECOLE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Arg Glu Asn Ket Ala Arg Gly Pro Cye Aen Ala Pro Arg Trp Val SBr Leu net Val Leu Val Aln Iie Gly Thr Ala Val Thr Ale Ala Vai Asri Pro Gly Val Val Val Arg ile Ser Gln Lye Giy Leu Asp Tyr Ala Ser Gin Gln Gly Thr Ala Ala Leu Gln Lys Glu Leu Lys Arg Ile Lye Ile Pro Aap Tyr Ser Asp Ser Phe Lys Ile Lye Hie Leu Gly Lys Gly 35 d0 45 His Tyr ser Phe Tyr Sar l3et Aap Ile Arg Glu Phe Gln Leu Pro Ser Ser Gln ile Ser Met Val Pro Aen Val Gly Leu Lya Phe Ser Ile Sar Aen Rla Asn ile Lye Ile Ser Gly Lye Trp Lyg Ala Gln Lya Arg Phg Leu Lys Met Ser Gly Aan Phe Aap Leu Ser Ile Glu Gly Met Ser Ile Ser Ale. Asp Leu Lya Leu Gly Ser Aen Pro Thr Ser Gly Lye Pro Thr Ile Thr Cya Ser Ser Cys Ser Ser His ile Asn Ser Val His Val His Ile Ser Lys Ser Lye Val Giy Trp Leu Ile Gln Leu Phe Hie Lya Lys ilQ Glu 5er Ala Leu Arg Asn Lya Met Aen Ssr Gln Val Cys Glu Lys Val Thr Asn Sar Val Ser Ser Glu Leu Gln Pro Tyr Phe Gln Thr Leu Yro Val Het Thr Lys Ile Aep Ser Val A1a Gly Ile Aen Tyr Gly Leu Vai Ala Pro Pro Ala Thr Thr Ala Glu Thr Leu Asp Val Gln Het Lye Gly Glu Phi Tyr Ser Glu Asn His Hia Asn Pro Pro Pro Pho Ala pro Pro Val Met Glu Phe Pro Ala Ala Hie Aep Arg Met Val Tyr Leu Gly Leu Ser Asp Tyr Pha phe Aan Thr Ala Gly Leu Val Tyr Gln Glu Ala 260 265 2~0 Gly Val Leu Lya t~cat Thr Leu Arg Aap Asp Met Ile pro Lys G1u Ser Lys phe Arq Leu Thr Thr Lys Pha Phe Gly Thr Phe Leu Pro clu Val Ala Lys Lys Phe Pro Aan tset Lye Ile Gin Ile His Val Ser Ala Ser Thr Pro Pro FIia Lou Ser Val Gln pro Thr G1y Leu Thr Phe Tyr Pro Ala Val Aep Val Gln Ala Phe Ala Val Leu Pro Asn Ser Ser Leu Ala Ser Leu Phe Leu Ile Gly fief His Thr Thr Gly Ser Met Glu Val Ser Ala Glu Ser Asn Rrg Lau Val Gly Glu Leu Lys Leu Asp Arg Leu Leu Leu Glu Leu Lys Iiie Ser Asn Ile Gly Pro Phe pro Val Glu Leu Leu Gln Asp Ila Met Aan T~yr Ile Val Pro Ile Leu Vai Leu Pre Arg Val Aen Glu Lya Lea Gln Lys Gly Phe pro Leu pro Thr pro Ala Arg Val Gln Leu Tyr Asn Val Val Leu Gln Pro Hie Gln Aen Phe Lau Leu Phe Gly Ala Asp Val Val Tyr Lys (2) INFOR?iATION FOR SEQ ID No:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTfi: 591 baee pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iX) FEATURE:
( A ) NAME / 1CEY : CD S
(8) LOCATION: 1..591 ( SEQtJE27CE
xi DESCRIPTION
) :
SEQ
ID
NO:

GCClIACCCCCGCTTG GTCGCC AGGATC ACCGACAAG GGACTG CAGTAT 48 AliAsn ProGlyLe5 ValA~.aArgIle Ti AspLya GlyLeu Gi Tyr r O ~

AlaAla GlnGluGly LQULeu AlaLau G1nSerGlu LeuLeu ArgIy,a ACGCTG ccTGACTTC ACCccc GACTTG AGGATCCCC cACGTC GGCCGT laa ThrLeu ProAspPhe ThrGly AspLeu ArgIlePro HisVal GlyArg GlyArg TyrGluPhe HieSer LeuAsn IleHioSer CysGlu LeuLeu HiaSer AlaLeuArg ProVal ProGly GlnGlyLau SerLeu SerIle 65 70 75 gp sQrAap serSerIla ArgVal GlnGly ArgTrpLya ValArg Lyesar TTCTTC AAACTACAG GGC.TCCTTTGAT GTCAGTGTC AAGGGC ATCAGC 336 PhePhe LyeLeuGln GlySer PheA8p Va1SerVal LyeGly IleSer ATTTcc GTCAACCTC cTCTTG GGCAGC GAGTccTcc CGGAGG cccACA 384 IleSer ValAsnGeu LeuLeu GlySer GluSerSer GlyArg ProThr 115 120 1~5 ValThr AlaSerSer CysSer SerAsp IleAlaAsp ValGlu ValAsp ATGTCG GGAGACTTG GGGTGG CTCT'IGAACCTCTTG CAC'.AAC:CAGATT 480 Met Ser Gly Asp Lau Gly Trp Leu Leu Asn Leu Phe Hia Aan Glr~ Ile Id5 150 155 160 GAG TCC AAG TTC cAG AAA GTA CTG GAG AGC AGG ATT TGC GAA ATG ATC 528 Glu ser Lys Phe Gln Lys Val Leu Glu Ser Arg Ile Cyr Glu Met Ile Gln Lye Ser Val Ser Ser Aep Leu Gln Pro Tyr Leu Gln Thr Leu pro Vnl Thr Thr 61u Ila (2) INFORMATxON FOR 5EQ ID N0:4:
(i) SEQUENCE C'NAgACTERISTICS:
(A) LENGTIi: 197 amino acids T'l~pE: a:pino acid (D) TOPOLOGY: linear ( ii ) ttOLECULE T7CgE: grotein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Ala Asn Pro Gly Leu Val Ala Arg Ile Thr Aap Lya Gly Leu Gln Tyr Ala Ala Gln Giu Gly Leu Leu Ala Leu Gln Ser Glu Leu Leu Arg Ile Thr Leu Pro Asp Phe Thr Gly Aep Leu Arg Ile Pro Hia Val Gly Arg Gly Arg Tyr Glu Phe His ser LQU Agn ile His Ser Cys Glu Leu Leu F3i~ Ser Ala Leu.Arg Pro Val Pre Gly Gln Gly Leu Ser Leu Ser Ile Ser Asp Ser Ser Ile Arg Val Gln Gly Arg Trp Lya val Arg Lya Ser Phe Phe Lye Leu Gln Gly Ser Phe Aep Val Ser Val Lya Gly Ile Ser Ile Ser Val Aan L8u Leu Leu Gly Ser Glu Ser Ser Gly Arg Pro Thr 1.15 1~0 125 Val Thr Alr SQr Ser. Cye Ser Ser Asp Ile Ala Aep Vdl G1u Val Aop ~31~
Hey ser Gly Asp Leu Gly Trp Leu Leu Aen Leu Phe His Asn Gln Ile Glu Ser Lya Pha Gln Lya Val Leu Glu Ser Arg Zle cys Glu Met Ile Gln Lys Ser Val &er Ser Aop Leu Gln Pro Tyr Leu Gln Thr Leu Arg Val Thr 2hr Olu Ile

Claims (7)

1. A hybrid fusion protein comprising, at its amino terminal, bactericidal/permeability-increasing protein (BPI) or a biologically active fragment thereof and, at its carboxy terminal, at least one constant domain of an immunoglobin heavy chain region or an allelic variant thereof.

2. The hybrid fusion protein of claim 1, wherein the biologically active fragment of BPI consists of amino acid residues 1 through 199 of BPI

3. DNA encoding the hybrid fusion protein of claim 1 or claim 2.

4. A vector comprising the DNA of claim 3.

5. A host cell transformed or transfected with the DNA of claim 3 in a manner allowing expression in the host cell of the encoded hybrid fusion protein.

6. A method of producing the hybrid fusion protein of claim 1 or claim 2, comprising the step of growing the host cell of claim 14 in a suitable culture medium and isolating the hybrid fusion protein from the host cell culture.

7. The hybrid fusion protein produced according to the method of claim 6.