INTRODUCTION

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The gram-positive organism Bacillus anthracis, the etiologic agent of anthrax, is the only member of the genus Bacillus capable of causing epidemic disease in humans and other mammals. B. anthracis grows in long chains and is nonmotile; virulent strains harbor two endogenous plasmids, pXO1 (29, 43) and pXO2 (10, 46), which code for the major known virulence factors of this organism. Plasmid pXO2 harbors the genes responsible for the synthesis of the glutamyl polypeptide capsule, which gives the strains their characteristic mucoid appearance in the presence of bicarbonate (10, 24, 25). Plasmid pXO1 harbors the structural genes for toxin production and regulation (30, 39-42, 48). Toxigenic B. anthracis strains secrete two bipartite exotoxins, lethal toxin and edema toxin. The secreted M_r 83,000 protein, known as protective antigen (PA), serves as the B component of both toxins (20). It binds to an unidentified receptor on the cell surface, where it is cleaved by cellular protease(s) to an M_r 63,000 form that ^{exposes the binding site for the A components lethal factor (LF) and edema factor (EF), which bind competitively (4, 9, 17).}

It was discovered during the late 1800s and early 1900s that cultures of virulent B. anthracis could be attenuated by growth at 42 to 43°C. The attenuation observed with such Pasteur-type vaccine strains resulted from the loss of plasmid pXO1. Fully virulent pXO2⁺ pXO1⁺ strains were thus attenuated by conversion to the pXO2⁺ pXO1 genotype. Other attenuated strains, such as the Sterne strain, spontaneously lost pXO2 while retaining pXO1. Culturing the Sterne strain at 42°C resulted in the loss of pXO1 and produced the avirulent pXO1 pXO2 strain referred to as Sterne-1 (11).

The currently licensed human vaccine is produced by growing the pXO1⁺ pXO2 V770-NP1-R strain of B. anthracis in minimal medium in the presence of bicarbonate under microaerophilic conditions and adsorbing the sterile filtered culture supernatant to aluminum oxyhydroxide adjuvant (36, 37). The protective component of the vaccine appears to be PA. Although the vaccine has proven efficacious (7, 13, 14, 37), the current vaccine strain has several limitations, including a sporogenic and fully toxigenic genotype. Production of vaccine from this strain results in lot-to-lot variability due to inconsistent PA production levels, inclusion of undefined PA proteolytic degradation products, and inclusion of other bacterial products including LF and EF (31).

To eliminate these limitations, an avirulent, nontoxigenic strain, Sterne-1, was selected as a host for PA expression. The recombinant plasmid pPA102 was created by subcloning a 6-kb BamHI fragment harboring the PA structural gene and flanking sequence originally cloned from the endogenous B. anthracis plasmid pXO1 (15). The 6-kb fragment was inserted into the gram-positive vector pUB110 and transformed into B. subtilis 1S53, and PA-positive transformants were selected (15). Subsequent characterization of the B. subtilis transformants revealed that spontaneous deletions had occurred, resulting in the loss of substantial portions of the original 6-kb insert, including the bicarbonate regulation (42) of PA production. A stable kanamycin-resistant, PA-positive version of the plasmid was isolated and termed pPA102 (15). This plasmid was electrotransformed into B. anthracis Sterne-1 to specifically restore constitutive PA production (12). Subsequently, an asporogenic variant was selected and characterized (49). We describe here the fermentation, purification, and characterization of recombinant PA produced from Sterne-1(pPA102)CR4.

	MATERIALS AND METHODS

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Bacterial strains and culture conditions. The B. anthracis pXO1 pXO2 Sterne-1 strain used in this study (11) was electrotransformed with pPA102 (15), and transformants displaying a stable PA-positive, kanamycin-resistant, LF-negative, EF-negative, and capsule-negative phenotype were selected (12). An asporogenic variant, Sterne-1(pPA102)CR4, was then selected and provided by Worsham and Sowers (49). The working stock was stored at 70°C in 50% (vol/vol) glycerol.

Fermentation conditions. The fermentations were performed with a Micros I top-drive fermentor (New Brunswick Scientific, New Brunswick, N.J.) with a 20-liter-working-volume 316-L stainless steel vessel equipped with two Rushton impellers whose diameter was equal to one-third the vessel diameter. The lower impeller was positioned on the drive shaft at a distance equal to the impeller diameter from the bottom of vessel, while the remaining impeller was positioned 1.5 times the impeller diameter above the lower impeller. The fermentor was also equipped with an ML4100 process controller, a two-gas mixer, and Advanced Fermentation software (New Brunswick Scientific). The vessel and medium were sterilized by exposure to 121°C for 15 min. The short sterilization cycle was required to minimize Millard-type and other medium degradation reactions. The total elapsed time at temperatures in excess of 37°C was less than 45 min. Subsequent testing revealed that medium sterility was maintained for more than 48 h under growth conditions.

Protease activity determinations. Protease activity in culture supernatants was determined with resorufin-labeled casein (Boehringer Mannheim Biochemicals, Indianapolis, Ind.). Cultures were grown in baffled shake flasks with shaking at 150 rpm at 37°C until the late log phase or early stationary phase. The cells were removed by sterile filtration of the culture with low-protein-binding 0.22-µm-pore-size cellulose acetate filters (Millipore Corp.). The sterile supernatant was used immediately in the assays, which were performed as described by Twining (45), except that no calcium was added other than the amount present in the medium. Protease activity was measured spectroscopically at 574 nm.

Assay of the M_r 83,000 PA in crude fermentation liquor by SDS-PAGE. Samples for PA determination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were filtered through 0.22-µm-pore-size filters and stored at 70°C after the addition of 2 mM EDTA and 20 mM HEPES (pH 7.3). The samples were later concentrated approximately 10-fold with Centricon 30 concentrators (Amicon, Beverly, Mass.) by centrifugation at 4,900 × g and were desalted twice by diluting to the original volume with 20 mM HEPES (pH 7.3)-5 mM NaCl-2 mM EDTA and repeating the concentration step. The samples were frozen, lyophilized, dissolved in 50 µl of the HEPES buffer described above, and diluted 1:1 with a twofold-concentrated SDS solubilization buffer consisting of 450 mM Tris-HCl, 4% (wt/vol) SDS, 12% (vol/vol) glycerol, 0.0025% (wt/vol) bromphenol blue, and 0.0025% (wt/vol) phenol red before being heated at 95°C for 5 min. Appropriate dilutions of the solubilized fermentation samples were applied in a total volume of 10 to 15 µl to 10% Tris-Tricine gels (Novex, San Diego, Calif.). The gels were fixed in 10% (vol/vol) acetic acid-50% (vol/vol) methanol (MeOH), stained with 0.05% (wt/vol) Coomassie brilliant blue in 10% (vol/vol) acetic acid for a minimum of 16 h, and destained in 10% (vol/vol) acetic acid. The gels were scanned with a model 420oe optically enhanced, 42-µm-resolution scanner (PDI, Huntington, N.Y.) with QS30 software (PDI). Portions of the gel without protein were randomly chosen and scanned to accurately subtract any background absorption. Standard curves were generated with 100 to 1,000 ng of purified PA on each gel used for the assay. Plots of PA band absorbance against the amount loaded proved linear, with r values of 0.992 to 0.996 from linear regression analysis.

Fermentation harvest conditions. At an OD₆₀₀ of 10 to 12, when we noted a pronounced drop in oxygen consumption, the fermentor was cooled to 10°C and EDTA was added to a final concentration of 24 mM. The culture was collected with a peristaltic pump at 25 liters/h through the fermentor harvest valve to a CEPA LE continuous-flow centrifuge (New Brunswick Scientific) equipped with a clarification bowl and centrifuged at 40,000 × g. The clarified supernatant was subsequently sterile filtered with a Pellicon cassette system equipped with two 5-ft², 0.45-µm-pore-size cellulose acetate cartridges (Millipore Corp).

Purification of PA from fermentor cultures. The sterile supernatant was concentrated with an Amicon DC10L concentrator equipped with two cellulose acetate spiral-wound 30-kDa cutoff cartridges (Amicon) at 5°C from 21 to less than 2 liters at a back pressure of less than 30 lb/in². The concentrate was diafiltered at constant retentate volume against 10 volumes of 25 mM diethanolamine 50 mM NaCl-2 mM EDTA-30 mM KCl (pH 8.9). The diafiltered PA was chromatographed at 30 cm/h and 5°C through a 9- by 10-cm packed bed of quaternized amine Macro Prep 50Q resin (Bio-Rad, Hercules, Calif.) in an Vantage-S column (Amicon) previously equilibrated in the same buffer used for the diafiltration. The eluate was concentrated and diafiltered with an S1Y30 30-kDa cutoff cellulose acetate spiral-wound cartridge (Amicon) at an operating pressure of 20 lb/in². The final concentrate of ca. 300 ml was diafiltered against 10 volumes of 145 mM ammonium acetate-2 mM EDTA (pH 10.0) and passed through a 0.2-µm-pore-size cellulose acetate filter.

Analysis of purified M_r 83,000 PA. The purity of the PA was assessed by several different methods. SDS-PAGE was performed as described above, except that the samples were not lyophilized. Native PAGE was performed as recommended by the manufacturer, with 4 to 15% acrylamide Phast gels (Pharmacia Biotech, Piscataway, N.J.).

	RESULTS

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Medium effects and growth. The first step in the development of the PA production method was to assess the effects of growth medium on bacterial cell growth, PA production, and protease activity. Good growth rates and high biomass were achieved previously with the Sterne-1 strain in a high-tryptone, high-yeast-extract medium (5). When the same medium was investigated with the Sterne-1(pPA102)CR4 strain under conditions of low or high aeration in shake flask experiments, the highest cell densities and PA production levels were achieved under aerobic conditions. PA production levels were compared in late-log- to early-stationary-phase aerobic cultures in complete medium (Fig. 1, lane 1), in medium with 25% of the total yeast extract found in complete medium (lane 2), and in medium with 25% of the total tryptone found in complete medium (Fig. 1, lane 3). The largest amount of intact M_r 83,000 PA was observed in complete medium (lane 1), while smaller amounts were generated with the reduced-yeast-extract medium shown in lane 2 and little, if any, intact M_r 83,000 PA was found in the reduced-tryptone medium (lane 3). However, the cell growth in all three media was comparable to the growth in the complete culture (lane 1), reaching 7 OD₆₀₀ units, while the reduced-yeast-extract (lane 2) and reduced-tryptone (lane 3) cultures reached 10 and 8 OD₆₀₀ units, respectively.

View larger version (60K): [in this window] [in a new window]   FIG. 1.   SDS-PAGE analysis of Sterne-1(pPA102)CR4 culture supernatants from complete, reduced-yeast-extract, and reduced-tryptone media. Lanes: 1, complete medium with 33 g of tryptone/liter and 20 g of yeast extract/liter; 2, complete reduced yeast extract medium with 33 g of tryptone/liter and 5 g of yeast extract/liter; 3, complete reduced tryptone medium with 8.25 g of tryptone/liter and 20 g of yeast extract/liter. A 1-ml sample was removed from each culture at late log to early stationary phase. The samples were sterile filtered, buffer exchanged, and concentrated to equivalent volumes. Equivalent volumes were loaded for SDS-PAGE analysis.

Fermentation. Figure 2 shows the pH, DO₂, percent O₂, and agitation values for a representative fermentation at the 20-liter level. The pH was monitored but not controlled during the course of the fermentation. A small drop in pH can be seen between elapsed fermentation times (EFT) of 50 and 175 min, which we attribute to the production of organic acids from the metabolism of carbohydrates supplied with the yeast extract. After an approximate EFT of 190 to 200 min, the pH began to increase, consistent with the release of ammonium from the aerobic metabolism of amino acids and peptides. After the pH had increased by 0.01 unit, a second addition of tryptone was made as a 10-fold concentrate of the amount added initially to ensure that tryptone never became limiting during the fermentation. Once the tryptone had been added, the pH decreased transiently at 200 to 205 min due to minor temperature and pH differences between the added tryptone and the vessel. The pH increased to a maximum of 8.5 during the course of the fermentation but remained within the acceptable range of 6 to 9 for B. anthracis Sterne-1(pPA102)CR4 (data not shown).

View larger version (23K): [in this window] [in a new window]   FIG. 2.   Physical-chemical parameters from aerobic Sterne-1(pPA102)CR4 fermentation. Symbols: open circles, DO2; solid line, pH; open squares, agitation; solid squares, percent oxygen. The tryptone addition during the fermentation is indicated by the arrow above the percent oxygen data at 200 min. The temperature range was 36.7 to 37.4°C. Pressure was constant at 2.0 lb/in2, and the sparge rate was 1 vol/vol/min. The sharp positive spike in the DO2 values at 350 min was due to the addition of antifoam KFO673.

1

View larger version (16K): [in this window] [in a new window]   FIG. 3.   PA production versus cell density. Symbols: solid circles, growth data 7 July fermentation; open circles, growth data 4 August fermentation; solid triangles, PA yield data 7 July fermentation; open triangles, PA yield data 4 August fermentation. Equivalent volumes were sampled from the 20-liter fermentor for each EFT. The OD600 was determined for each EFT, and the samples were sterile filtered. The filtrates were desalted and concentrated to equivalent final volumes before a 1:1 dilution with twofold-concentrated SDS solubilization buffer and analysis by SDS-PAGE. Coomassie blue-stained gels were digitally scanned as described in Materials and Methods, and amounts of Mr 83,000 PA were determined for each EFT.

View larger version (52K): [in this window] [in a new window]   FIG. 4.   SDS-PAGE results reflecting the time course of PA production from aerobic fermentation of Sterne-1(pPA102)CR4. Lanes: 1, molecular weight standards; 2, EFT 1 h; 3, EFT 2 h; 4, EFT 3 h; 5, EFT 4 h; 6, EFT 5 h; 7, EFT 6 h; 8, immunoblot of EFT 6 h developed with a pool of four monoclonal antibodies. Equivalent sample volumes were taken for each EFT. Samples were sterile filtered, and the filtrate was desalted, lyophilized, and resuspended in buffer to the same final volume before a 1:1 dilution with twofold-concentrated SDS solubilization buffer and analysis by SDS-PAGE. Equal volumes of each sample were applied to the gel. The immunoblot was developed with 4-chloro-1-naphthol after incubation with goat-anti mouse antibody linked to horseradish peroxidase. Bio-Rad low-molecular-weight standards with the following Mrs were used: phosphorylase b, 97,400; serum albumin, 66,200; ovalbumin, 45,000; carbonic anhydrase, 31,000; trypsin inhibitor, 21,500; and lysozyme, 14,400.

Purification. The crude fermentation supernatant was sterilized by filtration and then concentrated and diafiltered into ammonium acetate buffer with ultrafiltration spiral-wound cartridges with a molecular mass cutoff of 30 kDa. The diafiltration step was introduced primarily for buffer exchange but was also a critical purification step, as shown by the decrease in total A₂₆₀ and A₂₈₀ in Fig. 5. The decreased UV absorbance reflected the loss of protein, DNA, and RNA. A total diafiltration volume corresponding to 10 times the volume of the protein concentrate was required for the maximum 70- to 80-fold reduction in the UV absorbance seen in Fig. 5. SDS-PAGE analysis of the permeate, shown in the inset in Fig. 5, revealed an enrichment in proteins with M_rs less than 30,000 and little M_r 83,000 PA. Consistent with this, the calculated yield of the product was 90 to 95%. Analysis of the A₂₆₀/A₂₈₀ ratio for the diafiltered sample containing the product revealed a value of 1.93, which indicates protein that was still contaminated with 69% (by weight) of nucleic acids (8, 26).

View larger version (28K): [in this window] [in a new window]   FIG. 5.   UV-spectrophotometric and SDS-PAGE analysis of the diafiltration permeate. Symbols: solid circles, A260; open circles, A280. Permeate from the Amicon 30-kDa-cutoff spiral-wound cartridge during the first diafiltration step was collected, diluted with the diafiltration buffer, and analyzed by UV spectroscopy at 280 and 260 nm. The spectrophotometer was blanked against the diafiltration buffer. Values at 350 and 320 nm were measured to confirm that sample turbidity was negligible. The inset shows a Coomassie blue-stained SDS-PAGE gel of a 10-fold concentrate of the diafiltration permeate collected at the outset of the diafiltration step.

View larger version (73K): [in this window] [in a new window]   FIG. 6.   SDS-PAGE analysis of PA purity after chromatographic purification steps. Lanes: 1, molecular weight standards; 2, PA after Macro-Prep 50 Q chromatography; 3, PA after HQ chromatography; 4, PA after QE chromatography; 5, PA after ether HIC chromatography. PA samples from each purification step were desalted, concentrated, and solubilized in SDS buffer. The total protein loaded in lanes 2 to 5 was 2 µg. Molecular weight standards were as in Fig. 4.

Analysis of product purity. The final product purity described above was determined by SDS-CE and reversed-phase chromatography. Figure 7A shows the SDS-CE analysis of QE-purified recombinant PA monitored at 215 nm. The main M_r 83,000 PA peak at 10.8 min is labeled 3, while the other major peak, labeled 1, at 3.65 min is the mellitic acid internal standard. The impurities can be seen as multiple single and double peaks collectively labeled 2 between 7.5 and 10.5 min. The peak labeled 4 at 11.99 min was investigated further and found to be identical to a contaminant with an M_r of 80,000 by SDS-PAGE. The reason for the increased estimated mass relative to the M_r 83,000 PA with SDS-CE was not clear, but the observed baseline resolution of SDS-CE made an assay of this 1 to 3% contaminant possible. Overall integration of the peak area of the M_r 83,000 PA and the sum of all the contaminants yielded a final PA purity of 90% for this lot. Analysis of the same PA after HIC purification (Fig. 7B) revealed a final purity of 98% with trace contaminants between 8 and 10.5 min at the threshold of detection. The only significant resolved contaminant peak was seen as a shoulder of the PA peak at 10.6 min, which was believed to be a minor related impurity that was also visualized as a slightly faster-migrating minor band by SDS-PAGE and immunoblotting (data not shown). The HIC step was extremely effective in removing the contaminant labeled 4 in Fig. 7A. The results shown in Fig. 7A and B also indicated that neither sample contained detectable peptide contamination since such impurities eluted after the internal standard in the 4- to 7-min range (data not shown).

View larger version (11K): [in this window] [in a new window]   FIG. 7.   Determination of PA purity by SDS capillary electrophoresis. PA samples were analyzed after QE (A) or ether HIC (B) chromatography steps. The samples were desalted and concentrated to 3 to 4 mg/ml before being solubilized with SDS as described in Materials and Methods. The samples were applied by electrokinetic injection at 5 kV for 5 to 10 s. Separations were performed at 10 kV for 15 min, and the A215 was monitored.

View larger version (20K): [in this window] [in a new window]   FIG. 8.   Reversed-phase HPLC analysis of PA. A 100-µg sample of POROS QE-purified PA was subjected to reversed-phase HPLC analysis. The three main absorbance peaks labeled 1 were collected and pooled, and peak 2 was collected separately. Fractions collected for SDS-PAGE were immediately neutralized by adding 20 mM HEPES (pH 7.3) and diluting 1:1 with MQ water before concentrating. The results of Coomassie blue-stained SDS-PAGE are shown in the inset. The numbering of the inset lanes corresponds to the peak labeling. The Mr 80,000 contaminant in lane 1 is identified by an arrow to the left of the inset.

View larger version (52K): [in this window] [in a new window]   FIG. 9.   Immunoblot analysis of the total impurities in Mr 83,000 PA purified by QE anion-exchange chromatography. The pooled protein contaminants from three separate lots of PA were purified by reversed-phase HPLC, separated by SDS-PAGE, and transferred electrophoretically to nitrocellulose membranes. The immunoreactive bands were detected with the ECL reagent and a pool of monoclonal antibodies as described in Materials and Methods.

	DISCUSSION

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The major rationale for the continued use of a B. anthracis expression system was the direct secretion and accumulation of the desired protein into culture medium in a relatively pure state. In addition, the secretion apparatus was the native secretion system for PA, ensuring that the secreted protein would have the signal sequence correctly removed and that the protein would be correctly folded. An additional rationale for selecting the B. anthracis strain was the observed low protease activity in culture supernatants. The greater than 95% inhibition of supernatant protease activity by EDTA and the low (less than 30%) inhibition by PMSF alone made it unlikely that B. anthracis excreted significant subtilisin or related alkaline protease activity under the conditions used here. In addition, the lack of additional inhibition when PMSF, benzamidine, or 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF) was added in addition to EDTA suggested the presence of fewer proteases under the growth conditions and medium used here than observed for other bacilli such as B. subtilis (1, 34). The inhibition of EDTA was consistent with a neutral or metalloprotease(s) (16, 27), although the presence of a calcium-dependent, serine-type protease (2, 34), as described for B. subtilis, could not be ruled out. The plausibility of omitting PMSF from the entire process was confirmed when no loss of product, increase in related impurities, or decrease in stability upon storage at 4°C was observed when only EDTA was added. The lack of a PMSF requirement was a key factor in the selection of B. anthracis as a host over B. subtilis, since the addition of this toxic inhibitor was essential for stable PA when it was isolated from B. subtilis strains. This included the recombinant strain B. subtilis WB600, which had six unique protease genes deleted but still required PMSF addition to inhibit its remaining activity (50).

Fermentation of the asporogenic variant Sterne-1(pPA102)CR4 under the conditions described here resulted in no detectable spore production and had the secondary effect of greatly reducing the amounts of surface array protein normally released into the supernatant under the conditions used here (5). The reduction in the amount of the surface array protein was not investigated further but was attributed to pleiotropic effects of the spontaneous mutation selected on Congo Red or to a second, uncharacterized mutation.

Comparison of growth in complete medium, reduced yeast extract, or reduced tryptone revealed no change in cell density or growth rate, suggesting that the observed decrease in oxygen use observed at approximately 10 to 12 OD₆₀₀ units in complete medium was not attributable to substrate limitation. Attempts at medium supplementation at this point did not increase overall cell densities or eliminate the drop in oxygen consumption. These observations were most consistent with the accumulation of a toxic metabolic by-product rather than the shortage of a critical nutrient as the cause of the observed cessation of growth. Because the aerobic fermentation of amino acids and peptides increases culture pH due to released ammonium, the ammonium levels (47) generated during fermentation were determined (data not shown). The final concentration of ammonium found during the fermentations and a twofold-higher concentration were tested as potential growth inhibitors by adding ammonium sulfate to aerobic shake flask cultures. Neither concentration had any effect on growth, suggesting that the accumulation of ammonium was not the reason for the cessation of growth under the fermentation conditions described here.

Although maintaining the high DO₂ set point of 75% optimized the growth rate, it may also have led to the observed limitation in cell density by facilitating the rapid accumulation of metabolic by-products. However, the susceptibility of PA to proteolytic degradation even in the presence of tryptone necessitated the development of a rapid fermentation process. The rationale for optimizing growth rates can be seen from the M_r 83,000 PA yield data. Clearly, the yield of M_r 83,000 PA reached a peak during the deceleration (early stationary) phase and then declined rapidly. This decline in the M_r 83,000 PA level directly reduced yield and complicated the purification of M_r 83,000 PA from the increasingly complex mixture of proteolytic degradation products.

Our results demonstrated that tryptone was essential for reducing the measured protease activity and maximizing product recovery at the end of the fermentation. SDS-CE analysis of filtered, nondialyzed tryptone revealed the presence of multiple species, with the majority of the material having a mass less than 15 kDa with minor contributions of higher-mass species. The addition of completely hydrolyzed protein in the form of Casamino Acids was ineffective in protecting PA from proteolytic degradation. These results suggest that it was the polypeptides in tryptone that were crucial to the observed effects on blocking of protease activity. It remains unclear whether the proteolysis is blocked by polypeptides interacting with the protease active site(s) or whether tryptone contributes to a reduction in the actual amounts of protease released into the medium by this organism.

The combination of diafiltration and the initial ion-exchange chromatography resulted in the removal of more than 99% of the nucleic acids and 70% of the carbohydrates. These steps also contributed significantly to the overall purification by eliminating up to 50% of the contaminating protein in the crude fermentation liquor while M_r 83,000 PA recoveries were consistently around 90%. The subsequent ion-exchange purification yielded a product with 88 to 93% purity. The major losses in the whole process occurred during the second of these column purifications, with recoveries of 30 to 40% of the total initial product. These losses were incurred in part because of microheterogeneity in PA, which is readily observed in the form of multiple discrete bands or isoforms by native PAGE (19). The isoforms with higher mobility on native gels coeluted with lower-M_r proteins, reducing the yield, while additional losses were incurred due to coelution of the M_r 80,000 nonrelated impurity with the isoforms of lower mobility. The net result was a loss of 40 to 50% of the PA recovered from the HQ ion-exchange step and a product enriched in three of the initial isoforms.

The alkaline pH used during the purification was chosen for three reasons: (i) to avoid the pH optimum for the remaining contaminating protease(s), (ii) to maintain a sufficient difference from the pI of PA to minimize charge differences between isoforms, and (iii) to minimize protein-protein interactions between intact PA and proteolytic degradation products. The alkaline buffers were instituted with caution since biological activity was a criterion for final product and since exposure of PA to alkaline pH values greater than 8.9 to 9.0 was not previously reported. Comparing the biological activity of samples of PA purified under the conditions described here with PA purified under more physiological pH conditions revealed equivalent activities in the macrophage lysis assay. The data suggest that the exposure to alkaline conditions did not affect the native folding state of PA, or did so in a manner that was not apparent in the cell lysis assay. We also compared the isoform content to that of PA purified under physiological pH conditions, since prolonged exposure of proteins to alkaline pH can drive the nonenzymatic deamination of asparagine residues to aspartate and isoaspartate residues (33). No shift in the isoform content or in the relative proportions of the isoforms was observed after exposure to buffers used here.

We also investigated the HIC method as a substitute for QE chromatography. The M_r 83,000 PA was purified from the multiple small contaminants with excellent yield, but the product was simultaneously enriched with M_r 37,000 and M_r 47,000 proteolytic fragments of PA that did not dissociate under nondenaturing conditions (32). The copurification of the proteolytically cut species of PA indicated that the HIC method was most useful as a final step.

Clearly, the investigation reported here was undertaken to improve on the undesirable aspects of the current vaccine production system. The production process described here successfully met a number of those goals, including production from an avirulent, asporogenic, nontoxigenic strain; fermentation in the absence of added antibiotic; and minimization of product proteolysis without the addition of the toxic protease inhibitor PMSF. In addition, we designed a 4-day purification process which is robust and flexible enough to overcome potential changes in the amount and ratio of impurities in the starting material yet produces the desired product in adequate yield and desired purity at the desired scale. The purities given were defined on the basis of new and orthogonally related SDS-CE and reversed-phase methods that agreed within 1 to 2% of each other. Finally, immunization studies carried out with PA produced by Sterne-1(pPA102)CR4 and purified by the methods described here have proved comparable in efficacy to studies with the current licensed vaccine as a control (12, 14, 35).