WO1993025229A1

WO1993025229A1 - A method for purifying recombinant tap

Info

Publication number: WO1993025229A1
Application number: PCT/US1993/005283
Authority: WO
Inventors: E. Dale Lehman; Daniel Freymeyer
Original assignee: Merck & Co., Inc.
Priority date: 1992-06-12
Filing date: 1993-06-02
Publication date: 1993-12-23
Also published as: AU4404793A

Abstract

The invention is a method for purifying recombinantly produced TAP, a 60 amino acid anticoagulant protein derived from ticks. S. cerevisiae ATCC 20984 yeast cells are cultured in yeast cell culture media suitable for expressing TAP. The cells are separated from expressed protein by crossflow ultrafiltration and diafiltration to clarify the broth. Preferably, pH is thereafter adjusted to about 3.5. The TAP is then concentrated and partially purified by adsorption on to a POROS cation-exchange resin, preferably a POROS II HS/P sulfopropyl cation-exchange resin. The broth is preferably pumped onto the resin using a peristaltic pump. The protein is eluted from the resin with a concentrated salt buffer, preferably 1 M NaCl. The TAP in the eluate is then purified to greater than 96 % homogeneity with a step gradient generated with a preparative HPLC pump. The protein is then desalted and concentrated on a reverse phase chromatography column, preferably a POROS R/M reverse phase chromatography column, and thereafter lyophilized.

Description

TITLE OF THE INVENTION

A METHOD FOR PURIFYING RECOMBINANT TAP

BACKGROUND OF THE INVENTION

European Publication 419,099 describes a 60 amino acid protein ("TAP-l ", or tick anticoagulant protein, referred to hereinafter as "TAP") which specifically inhibits coagulation Factor Xa, a process for purifying the protein from Ornithodorous moubata tick extracts, and a method for recombinantly producing the protein using transformed yeast cells.

The method for recombinantly producing TAP involved preparing synthetic TAP gene, and using the gene to construct pKH4«TAP and transform DMY6, forming S. cerevisiae MY 2030 9718 P 281-3 (deposited with American Type Culture Collection as ATCC No. 20984).

Lehman et al., Journal of Chromatography. volume 574 (1992), pp. 225-235, describes large-scale purification and characterization of recombinant tick anticoagulant peptide. Fermentation broth obtained from ATCC 20984 yeast cells was clarified, diafiltered, and pumped onto Sepharose columns. Protein bound to the columns was eluted. Active protein fractions were concentrated by preparative reversed-phase HPLC. The product from HPLC was subjected to volume reduction under vacuum to remove organic solvents, lyophilized, decolorized and re-lyophilized. The entire process required about 217 hours and resulted in a yield, as calculated based on TAP present in unclarified fermentation broth and TAP present in re-lyophilized product, of 32%.

Protein purification can be carried out using column chromatography, including chromatography employing silica-based media and media strengthened mechanically. These systems, when run at high linear flow rates, suffer from diminished dynamic capacity and diminished resolution.

Perfusion chromatography (Afeyan et al. Bio/Technology 8:203-206; Afeyan et al. J. Chromatography 544:267-279; Regnier Nature 350:634-635; Afeyan et al., LC-GC, vol. 9, no. 12, pp. 824-832 (1991); Fulton et al., BioTechniques, vol. 12, no. 5, (1992); and Afeyan et al., United States Patent 5,019,270) chromatographic media are composed of a poly(styrenedivinylbenzene) matrix. The media are particles, which include first and second interconnected sets of pores, are designed so that, at achievable high fluid flow rates, convective flow occurs in both pore sets, and the convective flow rate exceeds the rate of solute diffusion in the second pore set.

The media is transected by 6,000-8,000 A tliroughpores which permit convective flow within the particle. The throughpores are lined with 500-1500 A diffusive pores containing functional groups that make a certain portion of the internal surface available for solute binding as a result of convective flow of solvent through the particle. The net result is that the media display augmented dynamic capacity and higher resolution at substantially greater linear flow rates while exhibiting significantly less backpressure.

SUMMARY OF THE INVENTION

The invention is a method for recombinantly producing TAP, a 60 amino acid anticoagulant protein derived from the Ornithodorous moubata tick. S_.. cerevisiae ATCC 20984 yeast cells are cultured in yeast cell culture media suitable for expressing TAP. The cells are separated from expressed protein by crossflow ultrafiltration and diafiltration. Preferably, pH is thereafter adjusted to about 3.5. The TAP is then concentrated and partially purified by a chromatographic step in which TAP is bound to perfusion chromatography chromatographic media, preferably a POROS cation- exchange resin, more preferably a POROS II HS/P sulfopropyl cation- exchange resin. The broth is preferably pumped onto the resin using a peri-staltic pump. The protein is eluted from the resin with a concentrated salt buffer, preferably 1 M NaCl. The eluate is purified to greater than 96% homogeneity with a step gradient generated with a preparative HPLC pump. The protein is then desalted and concentrated on a reverse phase chromatography column, preferably a POROS R/M reverse phase chromatography column, and thereafter lyophilized.

TAP, which contains three disulfide bonds, has a calculated molecular mass of 6977. It is highly specific for Factor Xa (Kj = 0.58 iiM) and does not inhibit Factor Vila, kallikrein, trypsin, chymotrypsin, thrombin, urokinase, plasmin, tissue plasminogen activator, elastase, Factor XIa or S. aureus V8 protease. The inhibitor does not require calcium. Complete amino acid sequences of these proteins were determined and compared with other inhibitors of serine proteases. They have limited homology with the Kunitz-type inhibitors. However, unlike other known inhibitors of this class, which all inhibit trypsin, it inhibits Factor Xa almost exclusively.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a method for purifying recombinantly produced TAP at high production levels.

TAP, later identified as SEQ. ID NO. 1, has the following sequence:

12 NH2-Tyr-Asn-Arg-Leu-Cys-ϊle-Lys-Pro-Arg-Asp-Trp-Ile-

24 Asp-Glu-Cys-Asp-Ser-Asn-Glu-Gly-Gly-Glu-Arg-Ala-

36 Tyr-Phe-Arg-Asn-Gly-Lys-Gly-Gly-Cys-Asp-Ser-Phe-

48 T -Ile-Cys-Pro-Glu-Asp-His-Thr-Gly-Ala-Asp-Tyr-

60 Tyr-Ser-Ser-Tyr-Arg-Asp-Cys-Phe-Asn-Ala-Cys-Ile-COOH

TAP is a 60 amino acid peptide found in the saliva of the soft tick Ornithodorous moubata which specificaly inhibits blood coagulation factor Xa (Waxman et al, Science 248:593-596). To recombinantly produce TAP, a synthetic TAP gene was fused to the secretory pre-pro leader of the yeast pheromone α-mating factor (MF l) and expressed in the yeast Saccharomvces cerevisiae (ATCC 20984) under control of a galactose-inducible GALIO promoter (Neeper et ak, J. Biol Chem. 265:17746-17752. This vector was transformed into a diploid strain of S. cerevisiae DMY6 (Mata/ . adel, ura3-52, his3Δ::GAL10μ-GAL4- a3,leu2-2,ii2/Ieu 2-04, cir°/cir°) also as described in Neeper et al. A clonal isolate designated 281 -3 was selected to prepare a master seed which was stored frozen at -70°C in the presence of 20% (v/v) glycerol.

The purification method of the present invention has numerous advantages over the prior art method. Advantages achieved using Perfusion Chromatography flow-through particles to purify TAP from fermentation broth containing yeast proteins, nucleic acids, nutrients, pigments, lipids, carbohydrates, and mannans are surprising in view of the teachings by Lehman, et al. (Journal of Chromatography) concerning TAP and Afeyan et al. concerning these particles.

The process of the present invention purifies TAP in half the time required by Lehman, et al. (Journal of Chromatography). The process can be entirely operated at ambient temperature instead of reduced temperature conditions, such as 4 C. The capture and initial purification steps take less than 5 hours, compared to 29 hours using the procedure in Lehman et al. Conveniently, high resolution purification is done using the same chromatographic media as the capture step.

The process of the present invention also uses a step NaCl gradient for high resolution purification rather than a linear gradient, resulting in improved resolution and yield. Product from the high resolution purification can be collected and pooled based on the UV absorbance elution profile.

The process also removes pigments from the fermentation broth by RP-HPLC, rather than requiring an additional charcoal adsorption purification step. EXAMPLE 1

Preparation of ATCC 20984

A recombinant gene encoding the inhibitor was synthesized and constructed based on the primary amino acid sequence of TAP. The properly modified synthetic gene was inserted into a yeast expression vector that allows for secretory expression. Yeast cells were transformed with the vector containing the synthetic gene.

Because the amino acid sequence of TAP-l was identified, appropriately chosen synthetic ohgonucleotides were used to construct the gene encoding the inhibitor. Eight ohgonucleotides were synthesized, and the synthetic gene constructed by annealing and ligation.

Each oligonucleotide was purified by electrophoresis on a 15% polyacrylamide gel, isolation and electroelution. Ohgonucleotides II through VII were treated with polynucleotide kinase and annealed in complementary pairs (ID and IV) and (V and VI). Ohgonucleotides I and Vπi were annealed directly with kinased II and VII respectively. The ohgonucleotides were annealed in kinase reaction buffer by heating to 80°C for two minutes and slow cooling over the course of an hour. The four annealed oligonucleotide pairs were pooled and treated with T4 ligase. The resulting product was digested with EcoRI. The product, representing the synthetic gene, was isolated as a 200 bp fragment after electrophoresis of the mixture on a 2% agarose gel, the identified fragment excised and electroeluted.

The DNA fragment representing the synthetic gene was ligated to pJC264 (Gan, Z.-R. et al. (1989) Gene 79:159-166) which had been previously digested with EcoRI and treated with alkaline phosphatase to yield plasmid 276-2E. The ligation mixture was used to transform competent E. coli (JM109 available from Stratagene, California, U.S.A.) Ampicillin resistant cells were obtained and selected for on ampicillin plates. The correct insert sequence in resulting plasmid clones was confirmed by DNA sequence analysis. The synthetic gene was inserted into the yeast expression vector in the following manner. One plasmid, 276-2E, was selected, and a polymerase chain reaction product was obtained in a reaction using oligonucleotide primers.

The inhibitor DNA was subjected to polymerase chainreaction (PCR) -effected amplification (see United States Patent 4,800,159, column 2, lines 36-68, column 3, column 4, and column 5, lines 1-20, hereby incorporated by reference). The DNA strands were heat denatured in the presence of primers that bind to each strand. The primers instructed DNA polymerase, which performs its replication function, to copy a particular portion of the strand. The process was continued with a series of heating and cooling cycles, heating to separate strands, and cooling to allow annealing and primer extension forming copies of the desired sequences. The cycles were repeated to generate more and more copies of the specific sequences. Through amplification, the coding domain to which terminal restriction sites are appended was obtained.

The PCR product was used to generate pKH4 ^»TAP.

Construction of pKH4 2

Construction of pKH4 is described in Schultz, et al.. Gene 61 (1987) 123-133, which is incorporated by reference. The plasmid pJC197 (Schultz et al. Gene 54 (1987) 113-123) is an E. coli - S. cerevisiae shuttle vector which contains a unique BamHI cloning site between the yeast MFαl pre-pro leader and transcriptional terminator, originally derived in Kurjan and Herskowitz (1982) ibid. pJC197 was digested with EcoRI + Pstl. and the 0.7-kb Pstl-EcoRI fragment containing a portion of the MFαl pre-pro-leader, a three-frame translational terminator, and MFαl transcriptional terminator, was gel- purified. GAL 1 Op was isolated from YEP51 by digestion with Sau3A. flush-ending with Pollk. ligating with octameric BamHI linkers, and digestion with Sail.

The resulting 0.5-kb BamHI-Sall fragment bearing GALlOp was gel purified and ligated to a 35-bp Sall-Pstl synthetic oligodeoxynucleotide adapter encoding the first 1 1 bp of the MFαl nontranslated leader plus the ATG and first 8 aa of the MFαl pre-pro- leader. The resulting 0.5-kb fragment was digested with BamHI. gel- purified, and ligated together with the aforementioned 0.7 -kb Pstl- EcoRI fragment plus the 4.0kb EcoRI- BamHI vector fragment derived from pBR322. The resulting plasmid, pKH207-l, contains GAL 1 Op fused to the MFαl pre-pro- leader plus BamHI cloning site, translational termination codons, and MFαl transcriptional terminator. Upon digestion with EcoRI and partial digestion with BamHI. an expression cassette of GAL 1 Op fused to the yeast MFαl pre-pro-leader, a unique BamHI cloning site, translational termination codons in all three reading frames, and MFαl transcriptional terminator sequence was inserted into the yeast shuttle vector pCl/1 (Rosenberg et al. Nature 312 (1984) 77-80) which contains the yeast 2μ DNA sequence for stable propagation of the plasmid in yeast at high copy number, to form pKH4.

A 213-bp BamHI -Pstl fragment encoding aa 9-79 of the ppL was prepared from the plasmid pα2 (Bayne et al., Gene 66 (1988) 235-244). The plasmid pα2 contains a portion of the yeast MFαl pre- pro sequence (79aa) modified at codons 80 and 81 to create a BamHI site 6 aa upstream from the KEX2 processing site. The region corresponding to codon 9 (Pstl) of the ppL to the BamHI site of pKH4 was removed from pKH4 after digestion with BamHI followed by partial digestion with Pstl. The modified vector, pKH4α2 was prepared by replacement of this excised sequence with the BamHI -Pstl fragment from pα2. Plasmid pKH4α2 contains the yeast GAL10 promoter, a portion of the MFαl pre-pro leader (79 aa), a three-frame translational terminator and MFαl transcriptional terminator, the yeast LEU2 gene, yeast 2μ sequences, pBR322 derived sequences, including the Ap^ gene and the origin of DNA replication (on). Construction of pKH4»TAP

Polymerase chain reaction resulted in a blunt end fragment which was regenerated in the usual fashion by digestion with BamHI. The correct fragment was obtained after electrophoresis on a 2% agarose gel, excision of the band and electroelution. The purified fragment was ligated with the yeast expression vector pKH4α2 (Jacobson, M.A. et al. (1989) Gene 85: 513-518) that had been previously digested with BamHI and treated with calf alkaline phosphatase. The correct sequence of plasmid clones in the correct orientation was confirmed by DNA sequence analysis. Fusion products produced from pKH4»TAP are proteolytically processed by the Lys- Arg-cleaving endopeptidase (KEX2) encoded by the KEX2 gene and products are secreted into culture medium. KEX2 cleaves on the C- terminal side of Lys-Arg residues.

Transformation of DMY6

Diploid yeast strain DMY6 (Schultz, L.D. (1987) Gene 61 : 123-133) was transformed with pKH4»TAP using standard protocols (Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75: 1929-1933). One isolate was chosen and designated S. cerevisiae MY 2030 9718P281-3. The isolate was deposited with the American Type Culture Collection and identified ATCC No. 20984.

Deposit

S. cerevisiae MY 2030 9718P281 -3, deposited with the American Type Culture Collection, Rockville, MD, USA, is designated ATCC 20984. The deposit was made February 21, 1990 under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and the Regulations thereunder (Budapest Treaty). Maintenance of a viable culture is assured for 30 years from date of deposit. The organisms will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Applicants and ATCC which assures unrestricted availability upon issuance of the pertinent U.S. patent. Availability of the deposited strains is not to be construed as a license to practice the invention in contravention rights granted under the authority of any government in accordance with its patent laws.

EXAMPLE 2

Fermentation

rTAP culture 281 -3 was inoculated onto leu^" agar medium (Schulman et al., J. Biotechnology 21 : 109-126) and incubated for 3 days at 28 °C. Cells were removed from the plate and inoculated into a 2-L flask containing 500 mL of 5X leu^" liquid medium (Jacobson et al. Gene 85:511-516) containing 4% glucose. The flask was incubated for 15 h at 28°C at 300 rpm in a New Brunswick G26 rotary shaker. The flask served as second-stage inoculum which was transferred (5% v/v) to a New Brunswick 16-L Microferm fermenter containing 12 L 5X leu^" medium, 8% galactose. Operating conditions were 28°C, 5 L air/min, 500 rpm, 123 h.

Initial processing of fermentation broth was accomplished by crossflow filtration with 0.19 wr Millipore 200,000 molecular mass cut-off (MrCO) PZHK Prostak cassette membrane. The broth was concentrated 4-fold, and the concentrated cells were diafiltered with 12 L cold, sterile distilled water. The permeate (diafiltered fermentation broth) was stored at -70°C.

The inoculum development for the 250-L fermentation was identical to that used for the 16-L fermentation except that a third seed stage was incorporated after the 2-L flask by using it to inoculate a 15-L New Brunswick Micros fermenter at 5% v/v. The fermenter contained 10 L of 5X leu^" medium, 4% glucose. Operating conditions were 28°C, 500 rpm, 5 L air/min, 9 h. Ten L of fermentation broth were inoculated into a 250-L New Brunswick Magnaferm fermenter. Conditions were 200 L of 5X leu^" medium containing 8% galactose, 28°C, 260 rpm, 100 L air/min, for 92 h. Dissolved oxygen was maintained at >30% saturation by automatic increases in agitation.

Recovery of rTAP from the fermentation broth was accomplished by crossflow filtration with 4.64 m^ Millipore 200,000 MrCO PZHK Prostak cassette membrane. The broth was concentrated 4-fold, and the concentrated cells were diafiltered with 185 L of cold, sterile distilled water. The permeate (diafiltered fermentation broth) was collected in a jacketed, sterile process vessel and maintained at 8°C until it was processed by SCX chromatography.

EXAMPLE 3

Purification following cross-flow filtration

Purification involves the use of POROS II HS/P SCX sulfopropyl strong cation-exchange (SCX) resin and POROS R/M reverse phase resin. These media are composed of a poly(styrenedivinylbenzene) matrix transected by 6,000-8,000 A throughpores which permit convective flow within the particle. These convective pores are lined with 500-1500 A diffusive pores containing functional groups that make a certain portion of the internal surface available for solute binding as a result of convective flow of solvent through the particle. The end result is that the media displays augmented dynamic capacity and high resolution at substantially greater linear flow rates while exhibiting significantly less back- pressure.

Small-scale purification - 10 liters

Small-scale purification process development was done with recombinant yeast broth fermented at the 10-1 scale and harvested by cross-flow microfiltration.

The capacity of POROS II HS/P SCX resin to bind rTAP from diafiltered fermentation broth was determined by doing frontal loading studies with 10.6 ml of resin in a low-pressure chromatography column (Pharmacia XK26), 2.0 x 2.6 cm internal diameter (I.D.), and a peristaltic pump fitted with silicone tubing. The resin was equilibrated with 100 mM NaCl, 50 mM sodium formate, pH 3.5 (Starting Buffer), and then diafiltered fermentation broth was adjusted to pH 3.5 and pumped onto the column at various flow rates of 60-760 cm/h. Breakthrough of rTAP in the column effluent was detected by analytical SCX-HPLC. It was determined that POROS B HS/P could capture >35 mg rTAP/ml column volume (CV) from the diafiltered broth and that the capacity of the resin was independent of the flow rate within this range. In addition to efficiently capturing rTAP, the capture step also accomplished a significant purification, as shown by analytical SCX- HPLC, such that the product of the capture step was 85% homogeneous for rTAP as compared to 3% for the diafiltered broth.

The major contaminant in the product of the capture step was a peptide which eluted after rTAP during SCX-HPLC chromatography and it was shown by N-terminal sequencing to be the incompletely processed fusion peptide of rTAP with a segment of the MFαl prepro leader (S-L-A-L-R-TAP). Also present was a peak of UV-absorbing contaminating materials that eluted before rTAP, and N- terminal sequencing indicated that it contained N-terminal- truncated forms of rTAP. This data showed that SCX chromatography was capable of separating rTAP from these impurities. Additionally, previous experiments had shown that anion-exchange and RP-HPLC were not capable of doing so. Therefore, the same POROS II HS/P chromatographic media that was used for capture was also used in a high resolution mode to remove the contaminating fusion and N- terminal truncated peptides. An 8 -ml column of resin was prepared and equilibrated with Starting Buffer. Baseline resolution of all peptides was obtained in experiments in which the peptides (<375μg/ml CV) were bound to POROS II HS/P in Starting Buffer and then eluted with a linear 0.4-0.7 M NaCl gradient over 10 CV. However, resolution was unsatisfactory when >1 mg/mL CV was loaded. Improved resolution with the higher loadings was obtained when isocratic elution conditions, instead of a linear gradient, were employed to elute the peptides. Small- scale, high resolution preparative separations were conducted in which the peptides were bound onto the column and then eluted with varying concentrations of NaCl in 50 mM sodium formate, pH 3.5. rTAP was not eluted from the column with <20 CV of 0.45 M NaCl, but was eluted in <20 CV with NaCl concentrations of > 0.5 M. The separation of rTAP from the fusion peptide was greater with 0.5 M NaCl than with 0.6 or 0.65 M NaCl .

Loading studies then were done with the same column to determine the maximal amount of peptide that could be purified for a given volume of resin, while maintaining satisfactory resolution. Amounts of rTAP equivalent to 1 , 2, or 3 mg/ml CV were bound to the column and eluted with 0.5 M NaCl, 50 mM sodium formate, pH 3.5. Fractions of 1 CV each were collected and analyzed for purity by SCX- HPLC. Satisfactory resolution of the peptides was obtained with loadings of <1 mg/ml of CV, and purity of the product was >99% by peptide composition when analyzed by SCX/HPLC. However, loadings of 2 or 3 mg/ml CV caused the peptides to fuse together and SCX-HPLC showed that there were significant amounts of the fusion peptide in the latter half of the rTAP peak at these loadings.

Large-scale purification - 200 liters.

After determining the capacity of the SCX resin for capturing rTAP from the fermentation broth, as well as the optimal NaCl concentration and protein loading for the high-resolution SCX purification, the fermentation was scaled-up to the 200 1 scale and chromatography columns were scaled-up ca. 100-fold. Recoveries and yields of rTAP from each step of the purification process are given in Table 1.

1. With respect to peptide content.

2. Determined by SCX HPLC, as decribed in Experimental Protocol.

3. N.D.= not determined.

4. 11.1 L, 16.7 g rTAP, obtained during the capture step, was purified by high-resolution SCX.

Recombinant S. cerevisiae secreting rTAP was harvested by cross-flow microfiltration and diafiltration. To capture rTAP from the diafiltered broth at this scale, a 1.25-1 column (2.5 X 25.2 cm I.D.) of POROS II HS/P was packed and equilibrated in Starting Buffer. Diafiltered fermentation broth (386 1 containing 37.5 g rTAP) was adjusted to pH 3.5 and pumped onto the column at 1.5 1/min (180 cm/h) with a peristaltic pump fitted with silicone tubing. Monitoring of the effluent by SCX-HPLC during the loading showed that no detectable amounts of rTAP were coming through the column unadsorbed. The backpressure of the system throughout the loading operation was 140- 155 KPa, indicating that no appreciable amount of fouling of the resin was occurring. After all the diafiltered fermentation broth had been pumped onto the column, it was washed with 36 1 of Starting Buffer to remove any unadsorbed proteins. rTAP (24.3 g) was then eluted with 1 M NaCl , 50 mM sodium formate, pH 3.5, in a volume of 16.1 1.

To do high resolution purification of the rTAP that was eluted from the capture column, the same resin that was used for the capture step was removed from the capture column, cleaned as described in the experimental protocol, and re-packed into an 800-ml high-pressure column. Methods development with the 8-ml column had shown that the capacity of the resin to do high-resolution purification of rTAP was 1 mg of peptides/ml of CV. Therefore the 800-ml column could purify 800 mg of peptide per cycle, and purification of rTAP that had been eluted from the SCX capture column would require multiple cycles of the 800-ml column. The sample to be processed, 16.7 g rTAP, was divided into aliquots of <800 mg. Each aliquot was diluted 10-fold with 50 mM sodium formate, pH 3.5, immediately before being processed, and pumped onto the SCX high-resolution column at flow rate of 1 CV/min. After washing the column with 2 CV Starting Buffer, rTAP was eluted with 0.5 M NaCl, 50 mM sodium formate, pH 3.5. Separation obtained at this scale was equivalent to that obtained at the 8-ml scale at the same loading. Each cycle required 28 minutes and purification of 16.7 g of rTAP yielded 15.6 g (93.4% step yield).

The volume obtained from high resolution purification was 55.1 1, and the preparation contained salts and uncharacterized yellow pigments from the fermentation broth which had been incompletely removed during previous purification steps. Therefore, RP-HPLC on a 10 x 5 cm I.D. column (200 ml CV) of POROS R/M media was used to concen- trate, decolorize, and purify the protein. The yellow pigment eluted on the leading edge of the rTAP peak and could be separated from it when a linear gradient of 0-40% acetonitrile, 0.1 % TFA, over 10 min at 200 ml/min, was used. Twenty-five cycles of 23 min duration each were done to process 15.6 g of rTAP and the step-yield was 82%. Finally, all the pools from RP-HPLC were combined (9.2 1 of volume), concentrated by vacuum distillation on a Savant Speed- Vac and lyophilized. The total amount of rTAP purified was 12.8 g (46.9% yield).

Quantitative amino acid analysis of purified rTAP gave results that indicated a homogeneous preparation with composition consistent with that predicted from the cloned gene. It was >99% homogeneous by N-terminal sequencing and analytical SCX-HPLC, and

>96% by WAX -HPLC and capillary zonal electrophoresis (CZE). The in vitro inhibitory activity of the purified protein was assessed by titration of HfXa, 0.500 nM, with rTAP and 50% of the non-inhibited activity (IC50) was achieved with 0.359 nM rTAP, which is reasonably close to the value of 0.250 n expected for stoichiometric binding, and showed that it was fully active

Costs can be reduced by operating a smaller column at high flow rates through multiple cycles, as opposed to operating a larger, more costly one through a lengthy cycle. Additionally, using a smaller column permits the design of a more compact process, and the space requirements are reduced proportionately. Table 2 compares the productivities of the present and a previous large-scale purification of rTAP (Lehman et al. J. Chromatography 574:225-235). For the method using the Perfusion media, the capture of rTAP from fermentation broth and subsequent high-resolution purification was done in two chromatographic steps and the total time required to process 37.5 g was 15 h. Pooling of column fractions was done based upon the A280 elution profile. The previous process used a single 10-1

SCX column for combined capture of rTAP and its separation from the contaminating peptides. This required 29.3 h. Additionally, it was necessary to do analytical HPLC to determine which fractions were pure enough (>95%) to be included in the rTAP pool, which added another 24 h and the process was done in a room maintained at 4-8°C in consideration of proteolytic degradation that might occur in yeast broth before contaminating proteases were removed. Therefore, the time required for capture and initial purification were reduced in the present process. The use of a 4-8°C room was completely eliminated due to the use of a smaller column (1.25 1) and shorter time was required to separate rTAP from proteolytic enzymes (4.5 h) so that the capture step could be done in the fermenter facility next to a chilled holding tank. The subsequent high-resolution purification step was then performed at 20-23 °C. Time required for preparative RP-HPLC was reduced by 30% in the present process, and the fact that residual contaminating pigments were removed by RP-HPLC in the present process, but were removed by charcoal adsorption in the previous, meant that the lyophilization time was reduced from two-cycles to one. Additionally, the present process has increased the yield of rTAP from 32 to 47%.

a: Capture and high-resolution purification was combined.

b: Includes 24 h required for analytical HPLC to aid in pooling fractions.

c: Not applicable.

Capture of rTAP from 200 liters diafiltered fermentation broth was done with a Pharmacia Bioprocess Column (2.5 X 25.2 cm I.D.) whose flow adapter and end piece were modified by placing a nylon mesh with 10 micron mesh openings (Spectra/ Mesh, no. 124084) between the net and support screen. Chromatography resin was suspended in 2.5% NaCl to make a 50% (w/v) slurry and poured into the column. The flow adapter was put into place, and the column was washed with 9 L of 2.5% (w/v) saline at 1.5 L/min with a Masterflex peristaltic pump (Cole-Parmer cat. no. 07549-30) fitted with a 7019 pump head and silicone tubing. The column then was equilibrated with

Starting Buffer. The diafiltered fermentation broth (368 L) was maintained at 2-8°C in a 400-L jacketed stainless steel holding tank. Its pH was adjusted to 3.5 by adding 1.8 L of 98% formic acid, after which the conductivity was determined to be 7.0 mS. The broth was pumped onto the capture column at 1.5 L/min, and UV absorbance was monitored at 280 nm with a Pharmacia UV1 monitor equipped with an industrial flow cell. After all the broth had been pumped onto the column, it was washed with 36 L of starting buffer to elute any unadsorbed proteins. rTAP was desorbed with 1.0 M NaCl in 50 mM sodium formate, pH 3.5.

For high-resolution purification of rTAP, the resin was removed from the capture column and cleaned sequentially with 0.5 M

NaOH and acetone. It then was resuspended in 30% glycerol, 2.5%

NaCl (packing buffer) and packed into a 10 X 10 cm I.D. stainless steel column fitted with a 25-cm packing reservoir and straight adapter

(ModCol, St. Louis, Mo.). A flow of 1 L/min was used, and the packing buffer was pumped onto the column until the back- pressure had stabilized. Then the excess resin was removed from the top of the column, and the top end piece and frit were put into place. The column then was equilibrated with Starting Buffer until the backpressure had stabilized and the pH and conductivity of the column effluent were the same as the starting buffer. Pumping was done with an IBF HPLC

Pump Biopressing Unit 260 (Sepracor, Inc., Marlborough, MA.) Sample detection was by A200 using a Linear UVIS 200 detector with a

3 mm light path and Pharmacia LKB REC 1 recorder.

Preparative RP-HPLC was done with a POROS R/M stainless steel column, 10 X 5 cm I.D., using the IBF unit.

Quantitation of rTAP by SCX-HPLC was done with a Supelco Progel-TSK SP-NPR column (35 X 4.6 mm I.D.), flow rate of 1 mL/min on Spectra-Physics chromatography system consisting of model 8800 gradient pump, model 8880 autosampler and a Spectrafocus detector and software. The column initially was equilibrated with 50 mM sodium formate, pH 3.5. The sample was injected and the column was operated isocratically with the same buffer for 2 min, after which a linear gradient of 0-1.0 M NaCl in 50 mM sodium formate, pH 3.5, was developed. The column then was re-equilibrated with starting buffer for 2.9 min. In some cases, the peptides were separated isocratically with 0.38 M NaCl in 50 mM sodium formate, pH 3.5 instead of with linear gradient. The concentrations of rTAP in sample fluids were determined with reference to a standard curve made with purified rTAP. Purity by peptide composition was determined from the area percent of the peptide relative to the total area of all UV (280 nm) absorbing components.

Analytical weak anion-exchange chromatography (WAX- HPLC) was done on the same system with a Supelco Progel-TSK DEAE-NPR column (35 X 4.6 mm I.D.), flow rate of 1.0 mL/min. The column initially was equilibrated with 20 mM bis-Tris, pH 6.0. Sample was injected at 0 min, the column was eluted isocratically with the same solvent for 1 min, and then proteins were eluted with a linear gradient to 0.5 M NaCl, 20 mM bis-Tris, pH 6.0, over 4 min. Sample detection was by A2 o-

Capillary zonal electrophoresis (CZE) analysis of purified rTAP was performed in an Applied Biosystems Model 270A CZE system using an uncoated open capillary 72 cm long (50 cm working length) with a 50-μm bore. Samples were analyzed at 30°C using 25 mM sodium phosphate, pH 7.25 (Running Buffer). A 27 kV electric field was applied across the capillary, with the detector end of the capillary being at negative potential with respect to the inlet of the capillary. Typical running current was ca. 30μA. Sample injection time was 1.5 s using vacuum, which results in a ca. 15 nL sample. A 2- min wash with 0.1 M NaOH followed by a 3 -min wash with Running Buffer preceeded each sample injection. Sample detection was by measuring A200-

Quantitative amino acid analysis, except cysteine/cystine, was done after 20 h hydrolysis in vacuo in 6M HC1, 0.1% phenol at 110°C, with a Beckman Model 6300 amino acid analyzer with ninny drin post- column detection as specified by the manufacturer. Automated N-terminal sequence analysis was done with an Applied Biosystems Model 470A sequenator.

Inhibition of HfXa activity was determined with the chromogenic substrate Spectrozyme fXa (O'Neill Palladino et al., Protein Expression and Purification 2:37-42), using purified r-TAP as standard.

SEQUENCE LISTING

(1 ) GENERAL INFORMATION:

(i) APPLICANT: Lehman, E. Dale

Freymeyer, Daniel K.

(ii) TITLE OF INVENTION: A method for purifying Recombinant TAP

° (iii) NUMBER OF SEQUENCES : 1

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Merck & Co., Inc.

(B) STREET: P.O. Box 2000 ⁵ (C) CITY: Rahway

(D) STATE: NJ

(E) COUNTRY: USA

(F) ZIP: 07065

° (v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, ⁵ Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US

(B) FILING DATE: ⁰ (C) CLASSIFICATION:

(vϋi) ATTORNEY/AGENT INFORMATION:

(A) NAME: Parr, Richard S.

(B) REGISTRATION NUMBER: 32,586

(C) REFERENCE/DOCKET NUMBER: 18712 (ix) TELECOMMUNICAΗON INFORMAΗON:

(A) TELEPHONE: (908) 594-4958

(B) TELEFAX: (908) 594-4720

(C) TELEX: 138825

(2) INFORMAΗON FOR SEQ ID NO: 1 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Tyr Asn Arg Leu Cys He Lys Pro Arg Asp Trp He Asp Glu Cys Asp 1 5 10 15

Ser Asn Glu Gly Gly Glu Arg Ala Tyr Phe Arg Asn Gly Lys Gly Gly 20 25 30

Cys Asp Ser Phe Trp He Cys Pro Glu Asp His Thr Gly Ala Asp Tyr

35 40 45

Tyr Ser Ser Tyr Arg Asp Cys Phe Asn Ala Cys He 50 55

Claims

WHAT IS CLAIMED IS:

1. A method for producing TAP comprising the steps of:

(a) culturing S. cerevisiae ATCC 20984 yeast cells in yeast cell culture media suitable for expressing TAP;

(b) separating the cells from expressed protein by crossflow microfiltration and diafiltration to clarify the broth;

(c) concentrating and partially purifying rTAP from the clarified broth by binding the protein onto perfusion chromatography chromatographic media;

(d) eluting the protein with a concentrated salt buffer;

(e) purifying the eluate to greater than 96% homogeneity with a step gradient generated with a preparative HPLC pump;

(f) desalting and concentrating the protein on a reverse phase chromatography column; and

(g) lyophilizing the protein.

2. A method of claim 1 wherein the clarified broth resulting from step (b) is adjusted to a pH of about 3.5.

3. A method of claim 1 wherein the perfusion chromatography chromatographic media is a POROS II HS/P sulfopropyl cation-exchange resin.

4. A method of claim 1 wherein the broth is pumped onto the cation-exchange resin column using a peristaltic pump.

5. A method of claim 1 wherein the concentrated salt is about 1 M NaCl.

6. A method of claim 1 wherein the reverse phase chromatograp phhyy ccoolluummnn i is a POROS R/M reverse phase chromatography column.