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Expression, purification, and molecular analysis of the Necator americanus glutathione S-transferase 1 (Na-GST-1): A production process developed for a lead candidate recombinant hookworm vaccine antigen
Protein Expression and Purification 83 (2012) 145–151
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Expression, purification, and molecular analysis of the Necator americanus glutathione S-transferase 1 (Na-GST-1): A production process developed for a lead candidate recombinant hookworm vaccine antigen Gaddam Narsa Goud a, Vehid Deumic a, Richi Gupta a, Jill Brelsford a, Bin Zhan a,1, Portia Gillespie a,1, Jordan L. Plieskatt a, Eric I. Tsao c, Peter J. Hotez a,b,⇑,1, Maria Elena Bottazzi a,⇑,1 a
Department of Microbiology, Immunology & Tropical Medicine, George Washington University Medical Center, Washington, DC, USA Sabin Vaccine Institute and Texas Children’s Hospital Center for Vaccine Development, Houston, TX, USA c Aeras, Rockville, MD, USA b
a r t i c l e
i n f o
Article history: Received 20 March 2012 Available online 4 April 2012 Keywords: Hookworm Vaccine Vaccines Sabin Vaccine Institute Necator americanus Na-GST-1 Albendazole Mebendazole
a b s t r a c t The enzyme Necator americanus glutathione S-transferase 1 (Na-GST-1) belongs to a unique Nu class of GSTs and is a lead candidate antigen in a bivalent human hookworm vaccine. Here we describe the expression of Na-GST-1 in the yeast Pichia pastoris at the 20 L manufacturing scale and its purification process performed by three chromatographic steps, comprised of a Q Sepharose XL anion exchange column, followed by a Butyl Sepharose HP hydrophobic affinity column and a Superdex 75 size-exclusion column. Approximately 1.5 g of recombinant protein was recovered at an overall process yield of 51%, with a purity grade of 98% and the absence of detectable host cell protein. By mass spectrometry the recombinant protein exhibits a mass of 23,676 Da, which closely matches the predicted molecular mass of the protein. The expression and purification methods described here are suitable for further scale-up product development and for its use to design formulation processes suitable to generate a vaccine for clinical testing. Ó 2012 Elsevier Inc. All rights reserved.
Introduction Human hookworm infection is a major cause of iron deficiency anemia and protein malnutrition in the world’s low- and middleincome countries in Africa, Asia, and Latin America [1]. An estimated 600 million people are infected with hookworms worldwide, with most of the cases caused by Necator americanus [2]. Despite the widespread availability of benzimidazole anthelminthic drugs, hookworm infection remains a significant global health threat due to the high rates of mebendazole drug failure, and rapid posttreatment re-infection with albendazole (reviewed in Ref. [3]). Hence there is an urgent need for new control tools to combat hookworm infection including anthelminthic vaccine. A human hookworm vaccine is under development by the Sabin Vaccine Institute Product Development Partnership (Sabin PDP) [3]. The vaccine is comprised of two recombinant hookworm antigens, known as N. americanus aspartic protease 1 (Na-APR-1) and N. americanus ⇑ Corresponding authors. E-mail addresses: [email protected], [email protected] (P.J. Hotez), bottazzi @bcm.edu (M.E. Bottazzi). 1 Present address: Section Pediatric Tropical Medicine, Departments of Pediatrics and Molecular Virology & Microbiology at Baylor College of Medicine National School of Tropical Medicine, Houston, TX, USA. 1046-5928/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pep.2012.03.013
glutathione S-transferase 1 (Na-GST-1),2 each of which is a macromolecule involved in parasite blood feeding [3]. Na-APR-1 is an aspartic protease that degrades hemoglobin, but which has been modified through site directed mutagenesis to inactivate its hydrolytic function and yet retain its overall confirmation [3,4]. Na-GST-1 is a specially adapted Nu-class glutathione S-transferase that forms a heme- and hematin-binding pocket during homodimer formation [5–8]. Na-GST-1 is believed to function in heme detoxification during parasite blood feeding [5–8]. In preclinical testing recombinant Na-GST-1 expressed in yeast, as well as its orthologous enzyme from the dog hookworm Ancylostoma caninum (Ac-GST-1), elicit high levels of protective immunity against hookworm larval challenge infections, as evidenced by reductions in the numbers of adult hookworms relative to negative controls [5–8]. Here we describe the manufacturing of a 20 L scale fermentation process for the expression of Na-GST-1 in the yeast Pichia pastoris and its subsequent purification through three chromatographic steps. In addition, we provide in process characterization data to confirm the overall recovery (yields) and purity of the recombinant Na-GST-1 protein. The expression and purification 2 Abbreviations used: Na-APR-1, N. americanus aspartic protease 1; Na-GST-1, N. americanus glutathione S-transferase 1; cGMP, current good manufacturing practices; BMG, buffer media with glycerol; BSM, basal salt media; WCW, wet cell weight; CFS, concentrated fermentation supernatant.
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reported here were considered suitable for a pilot manufacture of a recombinant vaccine under current good manufacturing practices (cGMP). Methods Expression of Na-GST-1 The cloning of the gene encoding Na-GST-1 into P. pastoris has been reported previously [8]. A schematic overview of the processes used for the 20 L fermentor expression of Na-GST-1 is shown in Fig. 1A. Briefly, five vials of the working cell bank were grown to obtain a sufficient cell density for inoculation of the production fermentor. This step is performed in four 2-L buffered shake flasks containing 0.8 L of sterile buffer media with glycerol (BMG). Three of the shake flasks were harvested, while the fourth served as an optical density proxy and incubated at 30 ± 1 °C with agitation at 250 ± 10 rpm for approximately 27–30 h until the OD600 of the culture is 10.0 ± 4.0 was reached. Upon reaching the acceptable OD, a 20 L fermentor was inoculated containing 10 L of basal salt media (BSM) containing 3.5 ml/L of a 0.02% (D)-biotin solution. Fermentation was conducted in a 20 L Bioengineering fermentor model NLF-22 (Bioengineering, Switzerland). The pH of the BSM was adjusted to and maintained at 5.0 with 14% ammonium hydroxide feed. Cells were grown at 30 ± 1 °C in 30% dissolved oxygen and at an agitation speed of 450 rpm.
Approximately 18 h into the glycerol phase and after a sharp increase in the percentage of dissolved oxygen (indicating depletion of glycerol), 50% (v/v) glycerol was introduced into the cell culture medium at a set flow rate of 15 g/L/h, for 6 h. The pH of the culture was increased linearly from 5.0 to 6.0 by adding 14% ammonium hydroxide and the temperature linearly decreased from 30 to 26 °C over a 2-hour period before the completion of the fed-batch glycerol phase. Excessive foaming was controlled with 10% (v/v) antifoam KFO673 (KABO Chemicals Inc.) in deionized water. The agitation speed was increased from 450 to 700 rpm. The methanol induction phase was initiated at a wet cell weight (WCW) of approximately 200 g/L and increased from 1.5 to 11.0 ml/L/h over an 8-hour period. Methanol induction was continued for another 57 h by pumping 100% methanol at a flow rate of 11 ml/L of BSM/h until harvest at a WCW of approximately 463 g/L. Centrifugation [@7000 rpm and 4°C for 30 min using Avanti J-26 XPI and JLA 8.1000 rotor (Beckman)] was used to remove the cells and cellular debris and to recover the supernatant (17 L) containing the recombinant Na-GST-1. This supernatant was filtered using a 0.8 and 0.2 lm sterile depth filter and then concentrated to 4 L by using an ultrafiltration unit consisting of a Masterflex Pump and a 3 kDa hollow fiber cartridge (UFP-3C-55-GE Healthcare). Concentrated fermentation supernatant (CFS) was washed using ultrafiltration with additional volume of 20 mM Tris HCl buffer, pH 8.5 to decrease the conductivity 5.0 mS and increase the pH 8.5. Finally, this CFS
Fig. 1. Flow diagram of the steps used to express and purify recombinant Na-GST-1. (A) Shows the expression of Na-GST-1 in P. pastoris beginning in the shaker flask using the research cell bank of pPicZalpha-A containing the DNA sequence encoding Na-GST-1. Following inoculation of a 10 L fermenter with 5 L basal salt medium (BSM) the cells were grown in a glycerol batch phase followed by induction of protein expression with methanol. After harvest, microfiltration and ultrafiltration prepare the target antigen for subsequent purification. (B) Shows the downstream purification utilized including a capture chromatography step via ion exchange chromatography on Q XL media. A butyl HP step served as an additional polishing step followed by removal of high molecular weight host cell proteins (HCPs) via Superdex 75 size exclusion chromatography. The final Na-GST-1 recombinant protein was sterile filtered and subsequently stored.
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was filtered using a 0.22 lm sterile filter and stored at 4 °C until further purification. Purification of recombinant Na-GST-1 consisted of three column chromatography steps Fig. 1B shows the purification steps used to purify recombinant Na-GST-1. First, a Q Sepharose XL anion exchange column was used to capture recombinant Na-GST-1. This column (7 21.6 cm) was prepared according to the manufacturer’s instructions, sanitized with NaOH, and equilibrated with 20 mM Tris–HCl, 45 mM NaCl, at pH 8.5. All steps were performed at room temperature at a flow rate of 180 cm/h (116 ml/min). After adjustment of pH and the conductivity to 8.5 and 5.4 mS, respectively (with 6 N HCl or 5 N NaOH, and 4 M NaCl), the CFS was loaded onto the Q Sepharose XL column and washed with 20 mM Tris–HCl containing 100 mM NaCl, pH 8.5, to remove unbound material. Recombinant Na-GST1 was then eluted with 20 mM Tris–HCl/300 mM NaCl, pH 8.5. The eluate was collected while monitoring the absorbance at 280 nm and evaluated by SDS–PAGE and size exclusion high performance liquid chromatography (SE-HPLC). Eluted protein was stored at 2–8 °C for the subsequent purification step. As a second chromatographic step, pooled material from the Q XL column was passed through a Butyl Sepharose HP hydrophobic affinity column at room temperature using a flow rate of 150 cm/h (38 ml/min) (Fig. 1B). The Butyl Sepharose HP column (4.4 15 cm) was prepared according to the manufacturer’s instructions using a Vantage L column (Millipore). After sanitization, the column was equilibrated with 20 mM Tris–HCl/2 M ammonium sulfate, pH 8.5. A solution of 20 mM Tris–HCl containing 3 M ammonium sulfate was added to the pooled material from the Q XL column to achieve a concentration of 2 M ammonium sulfate, and the pH was adjusted to 8.5. The Na-GST-1 Q XL elution pool was loaded onto the Butyl Sepharose HP column, and washed with buffer (20 mM Tris–HCl/1.4 M ammonium sulfate, pH 8.5). After the absorbance of the eluate had returned to baseline, recombinant NaGST-1 was eluted from the column by decreasing the concentration of ammonium sulfate in the buffer to 0.7 M. The eluate was collected and analyzed by SDS–PAGE and SE-HPLC. The eluted protein was stored at 2–8 °C for the subsequent purification step. As a third and final chromatographic and polishing step, a Superdex 75 size-exclusion column was used to remove high molecular weight host cell impurities and to perform buffer exchange into the final buffer. An A2 Vantage Column (VA 130 500 with column tube extension kit VTK 130 500, Millipore) was packed according to the manufacturer’s instructions, sanitized, and equilibrated with 10 mM imidazole, 10% D-glucose, pH 7.4, buffer. The Na-GST-1 pool from the Butyl Sepharose HP column was divided into two pools and loaded onto the prepared Superdex 75 column over two cycles. For each run, the column eluate was collected and analyzed by SDS– PAGE and SE-HPLC. Sample eluates from two cycles of purification containing Na-GST-1 were pooled and filtered through a 0.22 lm filter. Characterization of recombinant Na-GST-1 SDS–PAGE of Na-GST-1 was performed on 4–20% gradient Tris– glycine polyacrylamide gels using an X-cell mini cell apparatus (Invitrogen) with Mark 12 unstained marker (Invitrogen) under denaturing and both non-reducing and reducing conditions. The gels were stained for 30 min (after bringing to a boil) in Coomassie Brilliant Blue R-250 (GE Healthcare) and destained twice for 15 min (after bringing to a boil). Gels were scanned using a calibrated densitometer (Bio-Rad) and analyzed using Quantity One Software (Bio-Rad).
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Size exclusion-high performance liquid chromatography (SE-HPLC) An Alliance 2695 system with an in-line photo-diode array detector (Waters) was used with a G2000SWXL TSK-GEL-HPLC column (TOSOH Bioscience) fitted with a guard column. A mobile phase of 50 mM sodium acetate, pH 4.8, was used at a flow rate of 0.25 ml/min. The spectrum from 210 to 400 nm was recorded and ultraviolet absorbance of the resultant peaks was monitored at 215 and 280 nm. To determine system suitability, 50 lg gel filtration standards (Bio-Rad) were injected before each sample set. To measure the amount of protein, a standard curve was generated by loading Na-GST-1 standards from 0.1 to 75 lg in 50 lL. The NaGST-1 peak was analyzed after extracting the 280 nM channel and a linear curve generated using Empower 2 (Waters) software. Inprocess samples were analyzed at two dilutions (neat or 1:5) depending on their expected concentration as to fall within the linear range. Final purified Na-GST-1 was evaluated at an injection of 25 lg in 50 lL and percent purity determined using Empower 2 software. Mass spectrometry A MicroMass Q-ToF spectrometer (Waters) was used to determine the molecular mass of the recombinant Na-GST-1 molecule. The drug substance was desalted and diluted into formic acid for analysis. Buffer solution was removed by ZipTip desalting prior to analysis. The mobile phase for this analysis was 50% acetonitrile/0.1% formic acid or various ratios of methanol/water with 0.1% formic acid. For mass spectrometry analysis, 2–3 lL of the eluted sample was introduced into the mass spectrometer. The mass spectrometer was controlled and the data processed using the MassLynx 4.0 operating system. Approximately 100 scans were combined (to average out fluctuations in the electrospray), baseline subtracted, smoothed and the centroid of each observed peak determined. The software was used to calculate the mass of the protein from two consecutive multiply charged ions in the spectrum that were manually defined. Results The overall recovery of recombinant Na-GST-1 after the purification process was 51% with a purity of 98% At the conclusion of 20 L scale fermentation, approximately 3 g of secreted soluble Na-GST-1 protein was produced (150 mg/L). Table 1 summarizes the mass balance results of the three major chromatographic steps as determined using an SE-HPLC method for the quantification of Na-GST-1 (in-process samples). The three chromatographic steps resulted in a product with 98% purity and approximately 50% recovery. Fig. 2 shows representative data of the use of the in-process SE-HPLC assay for quantitation of Na-GST-1 during expression and purification and monitoring the efficiency of each chromatography step. Fig. 2A shows the SE-HPLC results of concentrated fermentation supernatant from two 10 Lscale process development runs. Similar results were obtained from the 20 L run (data not shown). The major peak at a retention time of approximately 37 min represents the recombinant antigen Na-GST1. Analysis of the eluate following passage through the Q Sepharose XL column during purification of Na-GST-1, obtained from two 10 L process development runs is also shown in Fig. 2B, which also demonstrates the removal of both high and low molecular weight protein impurities during this capture step. After the Butyl chromatography step, the purity of Na-GST-1 was >90% (Fig. 2C). These remaining host cell proteins were subsequently removed during the size-exclusion chromatography step using Superdex 75 resin
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Table 1 Quantification of recombinant Na-GST-1 using the SE-HPLC method: in-process samples were analyzed from each purification step to monitor mass balance and determine column efficiency and recovery. Purity of the Na-GST-1 target protein is reported as a percentage of total protein. The overall recovery of the process was 51.4%, yielding protein of 98% purity. Column
Sample description
HPLC concentration (mg/ml)
Amount of Na-GST-1 (g)
Purity (%)
Step recovery (%)
Overall recovery (%)
QXL
Concentration of fermentation supernatant Diluted fermentation of supernatant Flowthrough Wash #1 Elution Flowthrough Column strip Starting material Flowthrough Wash #1 Elution Flowthrough Column strip Flowthrough Fraction #1 (high kDa cutoff) Elution Filtered bulk protein
1.95 1.7 0.0 0.0 7.16 0.052 0.125 2.39 0.0 0.004 5.47 0.382 1.68 0.001 0.05 1.74 1.74
2.979 2.885 0.000 0.000 2.549 0.069 0.103 2.572 0.000 0.009 1.633 0.170 0.299 0.002 0.005 1.534 1.530
79
86
51.4
94
64
98
94
BHP
S75
QXL: Q Sepharose XL anion exchange column, BHP: Butyl Sepharose HP hydrophobic affinity column, S75: Superdex 75 size-exclusion column.
Fig. 2. SE-HPLC of in-process samples during recovery and downstream processing of Na-GST-1 during two process development runs (overlayed). Analysis of concentrated fermentation supernatant (A), Q XL eluate (B), Butyl HP eluate (C) and Superdex 75 eluate (D) show the separation used for quantitation of Na-GST-1 at a retention time of approximately 37 min and relative purity calculations using Empower 2 software.
(Fig. 2D). Further analysis of in-process samples during Superdex 75 chromatography revealed that this step removed contaminating proteins in the observed peak shoulder during purification (Fig. 3A) with only nominal loss (6%) of the target Na-GST-1 in this fraction. These results were confirmed using a host cell protein assay that relies on rat antibody to P. pastoris proteins (data not shown).
The predicted mass of Na-GST-1, without an intra-molecular disulfide bond, is 23,679 Da By mass spectrometry the recombinant protein exhibits a mass of 23,699 Da (Fig. 4), with a smaller second peak with a molecular weight of 23,679 Da. This peak is +20 Da relative to the smaller peak, and therefore likely corresponds to a single oxidation event,
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Fig. 3. Chromatogram of purification of Na-GST-1 during Superdex 75 chromatography (A). The shoulder was collected as a separate fraction from the eluant peak and evaluated by SE-HPLC (B), showing the removal of high molecular weight proteins in the eluant peak, yielding with a final protein purity of 98%.
or sodium or potassium addition. Other minor peaks were observed that differ from the predominant peak, suggesting gylcosylation or sugar addition combined with other oxidation or sodium events. Na-GST-1 contains four methionine residues, each of which is a possible site for oxidation. Under reducing conditions, SDS–PAGE analysis of recombinant Na-GST-1 demonstrated a major band that was present at the expected molecular weight (approximately 24 kDa). A Coomassie-stained SDS–PAGE gel under both reducing and non-reducing conditions and loaded with Na-GST-1 recombinant protein (2 lg per load) obtained from one 10 L fermentation and purification run and as compared to the 20 L manufacturing run is shown in Fig. 5. Under non-reducing conditions, the major band is seen as a doublet. The primary amino acid sequence of Na-GST-1 contains two cysteines, and the doublet may result from the presence and absence of an intra-molecular disulfide bond. The absence
of the doublet in the presence of a reducing agent is consistent with this hypothesis. The gels were analyzed on a Bio-Rad GS-800 densitometer to determine the relative amounts of the main doublet (non-reduced) or singlet (reduced) band migrating at 24 kDa (Fig. 5). The percentage migrating as the main bands (doublet) under non-reducing conditions was approximately 96% and the percent protein migrating as the main band under reducing conditions was approximately 97%.
Discussion Both heme and hematin are potentially toxic products released as a consequence of hemoglobin breakdown and digestion. Heme and hematin can result in the production of oxygen radicals that are toxic to blood feeding parasites, including malaria parasites
Fig. 4. Mass spectrometry of Na-GST-1. Mass spectrometry results showing a predominant peak at 23,699 Da.
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Fig. 5. SDS–PAGE with Coomassie staining of recombinant Na-GST-1. Sample Lanes A and B run under non-reducing conditions show 2 lg protein loads obtained from one 10 L and one 20 L fermentation and purification runs, while Lanes C and D show the respective samples evaluated under reducing (with 10 mM DTT) conditions. NaGST-1 appears as a doublet at approximately 24 kDa under non-reducing conditions and as a single band at similar molecular weight in the presence of a reducing agent.
and hookworms [9]. Malaria parasites respond to this threat by polymerizing heme and hematin into an inert pigment known as hemozoin [9], whereas blood-feeding nematode parasites (including hookworms) have been shown to produce a unique Nu class of GSTs used to putatively transport, remove, and detoxify heme and hematin [5–8,10–14]. In hamsters and dogs challenged with hookworm larvae we have shown that antibodies to hookworm GSTs are associated with reductions in the number of larvae that become adult blood-feeding parasites [5–8]. It is presumed that the protective effect is the result of anti-Na-GST-1 antibodies interfering with the ability of hookworms to detoxify heme and establish in the gut during the earliest stages of blood feeding [3]. Here we described the expression of Na-GST-1 in the yeast P. pastoris expression system at the 20 L scale and its purification by three chromatographic steps, comprised of a Q Sepharose XL anion exchange column, followed by a Butyl Sepharose HP hydrophobic affinity column and a Superdex 75 size-exclusion column. Approximately 1.5 g of bulk protein was recovered at a yield of 51%, and with a purity of 98%. Based on in process assessments and the results from the mass balance it became evident that approximately 14% protein loss is observed during the Q Sepharose XL anion exchange column and 22% loss during the Butyl Sepharose HP hydrophobic affinity column step. These recovery rates are considered suitable for transfer to production scales needed for the generation of material suitable for Phase 1 trial. By mass spectrometry the recombinant protein exhibits a mass of 23,676 Da, which closely matches the predicted molecular mass of the protein. In selecting both a proven expression system such as the yeast P. pastoris and easily scalable chromatography steps, Na-GST-1 was produced as a proof of principle for scalable process development and for use in preclinical studies, and ultimately for Phase 1 clinical testing. The most significant challenge to overcome in designing an acceptable purification process was the tendency for high molecular weight host cell proteins to co-elute with Na-GST-1. These proteins were successfully eliminated by using size-exclusion chromatography with Superdex 75 resin, yielding protein of high purity (98%) with no detectable host cell protein. By incorporating SE-HPLC as an in-process assay, we were also able to monitor the target protein
throughout the purification process, thus generating further understanding of the robustness of each purification step. Studies are in progress to evaluate the stability of the Na-GST-1 generated through these processes, as well as its stability after its binding to alum. Ultimately, the human hookworm vaccine is envisioned as a bivalent recombinant protein vaccine on alum, possibly together with a second immunostimulant such as a synthetic lipid A as a toll-like 4 receptor agonist [3]. As Na-GST-1 advances through clinical development it will become necessary to revise the processes described here, including possible improvements in yield and reductions in cost suitable for large scale manufacturing. With respect to improving yield the greatest protein losses were noted following elution of the protein from Butyl-Sepharose – therefore process improvements may require modifying or substituting this step. With regards to cost, altering or substituting the Superdex-75 column may be advantageous. Because hookworm infection is almost exclusively an infection affecting the world’s poorest people [2,15], it is anticipated that the human hookworm vaccine will need to be manufactured for the lowest possible costs [16]. In summary, we have developed a process for the pilot expression of Na-GST-1 in yeast and its purification using a series of three chromatography steps. The process is suitable for producing purified protein that could be formulated with alum and other adjuvants. Finally this process is also suitable for technology transfer for manufacture under current good manufacturing practices (cGMP) for Phase 1 testing. Acknowledgments This work is supported by the Sabin Vaccine Institute through grants obtained from the Bill & Melinda Gates Foundation (Grant #32472 and #38988) and the Dutch Ministry of Foreign Affairs. References [1] D.W. Crompton, The public health importance of hookworm disease, Parasitology 121 (Suppl) (2000) S39–S50. [2] P.J. Hotez, P.J. Brindley, J.M. Bethony, C.H. King, E.J. Pearce, J. Jacobson, Helminth infections: the great neglected tropical diseases, J. Clin. Invest. 118 (2008) 1311–1321. [3] P.J. Hotez, J.M. Bethony, D.J. Diemert, M. Pearson, A. Loukas, Developing vaccines to combat hookworm infection and intestinal schistosomiasis, Nat. Rev. Microbiol. 8 (2010) 814–826. [4] M.S. Pearson, J.M. Bethony, D.A. Pickering, L.M. de Oliveira, A. Jariwala, H. Santiago, A.P. Miles, B. Zhan, D. Jiang, N. Ranjit, J. Mulvenna, L. Tribolet, J. Plieskatt, T. Smith, M.E. Bottazzi, K. Jones, B. Keegan, P. Hotez, A. Loukas, An enzymatically inactivated hemoglobinase from Necator americanus induces neutralizing antibodies against hookworm species and protects dogs against heterologous hookworm infection, FASEB J. 23 (2009) 3007–3019. [5] B. Zhan, S. Liu, S. Perally, J. Xue, R. Fujiwara, P. Brophy, S. Xiao, Y. Liu, J. Feng, A. Williamson, Y. Wang, L.L. Bueno, S. Mendez, G. Goud, J.M. Bethony, J.M. Hawdon, A. Loukas, K. Jones, P.J. Hotez, Biochemical characterization and vaccine potential of a heme-binding glutathione transferase from the adult hookworm Ancylostoma caninum, Infect. Immun. 73 (2005) 6903–6911. [6] O.A. Asojo, K. Homma, M. Sedlacek, M. Ngamelue, G.N. Goud, V. Deumic, O. Asojo, P.J. Hotez, X-ray structures of Na-GST-1 and Na-GST-2 two glutathione S-transferase from the human hookworm Necator americanus, BMC Struct. Biol. 7 (2007) 42. [7] S. Xiao, B. Zhan, G.N. Goud, A. Loukas, Y. Liu, A. Williamson, S. Liu, V. Deumic, P. Hotez, The evaluation of recombinant hookworm antigens as vaccines in hamsters (Mesocricetus auratus) challenged with human hookworm, Necator americanus, Exp. Parasitol. 118 (2008) 32–40. [8] B. Zhan, S. Perally, P.M. Brophy, J. Xue, G. Goud, S. Liu, V. Deumic, L.M. de Oliveira, J. Bethony, M.E. Bottazzi, D. Jiang, P. Gillespie, S.H. Xiao, R. Gupta, A. Loukas, N. Ranjit, S. Lustigman, Y. Oksov, P. Hotez, Molecular cloning, biochemical characterization, and partial protective immunity of the hemebinding glutathione S-transferases from the human hookworm Necator americanus, Infect. Immun. 78 (2010) 1552–1563. [9] N. Klonis, R. Dilanian, E. Hanssen, C. Darmanin, V. Streitsov, S. Deed, H. Quiney, L. Tilley, Hematin-hematin self-association states involved in the formation and reactivity of the malaria parasite pigment, hemozoin, Biochemistry 49 (2010) 6804–6811. [10] P.M. Brophy, J. Barrett, Gultathione transferase in helminths, Parasitology 100 (Pt. 2) (1990) 345–349.
G.N. Goud et al. / Protein Expression and Purification 83 (2012) 145–151 [11] S. Perally, E.J. Lacourse, A.M. Campbell, P.M. Brophy, Heme transport and detoxification in nematodes: subproteomics evidence of differential role of glutathione transferases, J. Proteome Res. 7 (2008) 4557–4565. [12] D.J. Schuller, Q. Liu, I.A. Kriksunov, A.M. Campbell, J. Barrett, P.M. Brophy, Q. Hao, Crystal structure of a new class of glutathione transferase from the model human hookworm nematode Heligmosomoides polygyrus, Proteins 61 (2005) 1024–1031. [13] A.M. Campbell, P.H. Teesdale-Spittle, J. Barrett, E. Liebau, J.R. Jeffries, P.M. Brophy, A common class of nematode glutathione S-transferase (GST) revealed by the theoretical proteome of the model organism Caenorhabditis elegans, Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 128 (2001) 701–708.
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Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
Expression, purification, and molecular analysis of the Necator americanus glutathione S-transferase 1 (Na-GST-1): A production process developed for a lead candidate recombinant hookworm vaccine antigen Gaddam Narsa Goud a, Vehid Deumic a, Richi Gupta a, Jill Brelsford a, Bin Zhan a,1, Portia Gillespie a,1, Jordan L. Plieskatt a, Eric I. Tsao c, Peter J. Hotez a,b,⇑,1, Maria Elena Bottazzi a,⇑,1 a
Department of Microbiology, Immunology & Tropical Medicine, George Washington University Medical Center, Washington, DC, USA Sabin Vaccine Institute and Texas Children’s Hospital Center for Vaccine Development, Houston, TX, USA c Aeras, Rockville, MD, USA b
a r t i c l e
i n f o
Article history: Received 20 March 2012 Available online 4 April 2012 Keywords: Hookworm Vaccine Vaccines Sabin Vaccine Institute Necator americanus Na-GST-1 Albendazole Mebendazole
a b s t r a c t The enzyme Necator americanus glutathione S-transferase 1 (Na-GST-1) belongs to a unique Nu class of GSTs and is a lead candidate antigen in a bivalent human hookworm vaccine. Here we describe the expression of Na-GST-1 in the yeast Pichia pastoris at the 20 L manufacturing scale and its purification process performed by three chromatographic steps, comprised of a Q Sepharose XL anion exchange column, followed by a Butyl Sepharose HP hydrophobic affinity column and a Superdex 75 size-exclusion column. Approximately 1.5 g of recombinant protein was recovered at an overall process yield of 51%, with a purity grade of 98% and the absence of detectable host cell protein. By mass spectrometry the recombinant protein exhibits a mass of 23,676 Da, which closely matches the predicted molecular mass of the protein. The expression and purification methods described here are suitable for further scale-up product development and for its use to design formulation processes suitable to generate a vaccine for clinical testing. Ó 2012 Elsevier Inc. All rights reserved.
Introduction Human hookworm infection is a major cause of iron deficiency anemia and protein malnutrition in the world’s low- and middleincome countries in Africa, Asia, and Latin America [1]. An estimated 600 million people are infected with hookworms worldwide, with most of the cases caused by Necator americanus [2]. Despite the widespread availability of benzimidazole anthelminthic drugs, hookworm infection remains a significant global health threat due to the high rates of mebendazole drug failure, and rapid posttreatment re-infection with albendazole (reviewed in Ref. [3]). Hence there is an urgent need for new control tools to combat hookworm infection including anthelminthic vaccine. A human hookworm vaccine is under development by the Sabin Vaccine Institute Product Development Partnership (Sabin PDP) [3]. The vaccine is comprised of two recombinant hookworm antigens, known as N. americanus aspartic protease 1 (Na-APR-1) and N. americanus ⇑ Corresponding authors. E-mail addresses: [email protected], [email protected] (P.J. Hotez), bottazzi @bcm.edu (M.E. Bottazzi). 1 Present address: Section Pediatric Tropical Medicine, Departments of Pediatrics and Molecular Virology & Microbiology at Baylor College of Medicine National School of Tropical Medicine, Houston, TX, USA. 1046-5928/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pep.2012.03.013
glutathione S-transferase 1 (Na-GST-1),2 each of which is a macromolecule involved in parasite blood feeding [3]. Na-APR-1 is an aspartic protease that degrades hemoglobin, but which has been modified through site directed mutagenesis to inactivate its hydrolytic function and yet retain its overall confirmation [3,4]. Na-GST-1 is a specially adapted Nu-class glutathione S-transferase that forms a heme- and hematin-binding pocket during homodimer formation [5–8]. Na-GST-1 is believed to function in heme detoxification during parasite blood feeding [5–8]. In preclinical testing recombinant Na-GST-1 expressed in yeast, as well as its orthologous enzyme from the dog hookworm Ancylostoma caninum (Ac-GST-1), elicit high levels of protective immunity against hookworm larval challenge infections, as evidenced by reductions in the numbers of adult hookworms relative to negative controls [5–8]. Here we describe the manufacturing of a 20 L scale fermentation process for the expression of Na-GST-1 in the yeast Pichia pastoris and its subsequent purification through three chromatographic steps. In addition, we provide in process characterization data to confirm the overall recovery (yields) and purity of the recombinant Na-GST-1 protein. The expression and purification 2 Abbreviations used: Na-APR-1, N. americanus aspartic protease 1; Na-GST-1, N. americanus glutathione S-transferase 1; cGMP, current good manufacturing practices; BMG, buffer media with glycerol; BSM, basal salt media; WCW, wet cell weight; CFS, concentrated fermentation supernatant.
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reported here were considered suitable for a pilot manufacture of a recombinant vaccine under current good manufacturing practices (cGMP). Methods Expression of Na-GST-1 The cloning of the gene encoding Na-GST-1 into P. pastoris has been reported previously [8]. A schematic overview of the processes used for the 20 L fermentor expression of Na-GST-1 is shown in Fig. 1A. Briefly, five vials of the working cell bank were grown to obtain a sufficient cell density for inoculation of the production fermentor. This step is performed in four 2-L buffered shake flasks containing 0.8 L of sterile buffer media with glycerol (BMG). Three of the shake flasks were harvested, while the fourth served as an optical density proxy and incubated at 30 ± 1 °C with agitation at 250 ± 10 rpm for approximately 27–30 h until the OD600 of the culture is 10.0 ± 4.0 was reached. Upon reaching the acceptable OD, a 20 L fermentor was inoculated containing 10 L of basal salt media (BSM) containing 3.5 ml/L of a 0.02% (D)-biotin solution. Fermentation was conducted in a 20 L Bioengineering fermentor model NLF-22 (Bioengineering, Switzerland). The pH of the BSM was adjusted to and maintained at 5.0 with 14% ammonium hydroxide feed. Cells were grown at 30 ± 1 °C in 30% dissolved oxygen and at an agitation speed of 450 rpm.
Approximately 18 h into the glycerol phase and after a sharp increase in the percentage of dissolved oxygen (indicating depletion of glycerol), 50% (v/v) glycerol was introduced into the cell culture medium at a set flow rate of 15 g/L/h, for 6 h. The pH of the culture was increased linearly from 5.0 to 6.0 by adding 14% ammonium hydroxide and the temperature linearly decreased from 30 to 26 °C over a 2-hour period before the completion of the fed-batch glycerol phase. Excessive foaming was controlled with 10% (v/v) antifoam KFO673 (KABO Chemicals Inc.) in deionized water. The agitation speed was increased from 450 to 700 rpm. The methanol induction phase was initiated at a wet cell weight (WCW) of approximately 200 g/L and increased from 1.5 to 11.0 ml/L/h over an 8-hour period. Methanol induction was continued for another 57 h by pumping 100% methanol at a flow rate of 11 ml/L of BSM/h until harvest at a WCW of approximately 463 g/L. Centrifugation [@7000 rpm and 4°C for 30 min using Avanti J-26 XPI and JLA 8.1000 rotor (Beckman)] was used to remove the cells and cellular debris and to recover the supernatant (17 L) containing the recombinant Na-GST-1. This supernatant was filtered using a 0.8 and 0.2 lm sterile depth filter and then concentrated to 4 L by using an ultrafiltration unit consisting of a Masterflex Pump and a 3 kDa hollow fiber cartridge (UFP-3C-55-GE Healthcare). Concentrated fermentation supernatant (CFS) was washed using ultrafiltration with additional volume of 20 mM Tris HCl buffer, pH 8.5 to decrease the conductivity 5.0 mS and increase the pH 8.5. Finally, this CFS
Fig. 1. Flow diagram of the steps used to express and purify recombinant Na-GST-1. (A) Shows the expression of Na-GST-1 in P. pastoris beginning in the shaker flask using the research cell bank of pPicZalpha-A containing the DNA sequence encoding Na-GST-1. Following inoculation of a 10 L fermenter with 5 L basal salt medium (BSM) the cells were grown in a glycerol batch phase followed by induction of protein expression with methanol. After harvest, microfiltration and ultrafiltration prepare the target antigen for subsequent purification. (B) Shows the downstream purification utilized including a capture chromatography step via ion exchange chromatography on Q XL media. A butyl HP step served as an additional polishing step followed by removal of high molecular weight host cell proteins (HCPs) via Superdex 75 size exclusion chromatography. The final Na-GST-1 recombinant protein was sterile filtered and subsequently stored.
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was filtered using a 0.22 lm sterile filter and stored at 4 °C until further purification. Purification of recombinant Na-GST-1 consisted of three column chromatography steps Fig. 1B shows the purification steps used to purify recombinant Na-GST-1. First, a Q Sepharose XL anion exchange column was used to capture recombinant Na-GST-1. This column (7 21.6 cm) was prepared according to the manufacturer’s instructions, sanitized with NaOH, and equilibrated with 20 mM Tris–HCl, 45 mM NaCl, at pH 8.5. All steps were performed at room temperature at a flow rate of 180 cm/h (116 ml/min). After adjustment of pH and the conductivity to 8.5 and 5.4 mS, respectively (with 6 N HCl or 5 N NaOH, and 4 M NaCl), the CFS was loaded onto the Q Sepharose XL column and washed with 20 mM Tris–HCl containing 100 mM NaCl, pH 8.5, to remove unbound material. Recombinant Na-GST1 was then eluted with 20 mM Tris–HCl/300 mM NaCl, pH 8.5. The eluate was collected while monitoring the absorbance at 280 nm and evaluated by SDS–PAGE and size exclusion high performance liquid chromatography (SE-HPLC). Eluted protein was stored at 2–8 °C for the subsequent purification step. As a second chromatographic step, pooled material from the Q XL column was passed through a Butyl Sepharose HP hydrophobic affinity column at room temperature using a flow rate of 150 cm/h (38 ml/min) (Fig. 1B). The Butyl Sepharose HP column (4.4 15 cm) was prepared according to the manufacturer’s instructions using a Vantage L column (Millipore). After sanitization, the column was equilibrated with 20 mM Tris–HCl/2 M ammonium sulfate, pH 8.5. A solution of 20 mM Tris–HCl containing 3 M ammonium sulfate was added to the pooled material from the Q XL column to achieve a concentration of 2 M ammonium sulfate, and the pH was adjusted to 8.5. The Na-GST-1 Q XL elution pool was loaded onto the Butyl Sepharose HP column, and washed with buffer (20 mM Tris–HCl/1.4 M ammonium sulfate, pH 8.5). After the absorbance of the eluate had returned to baseline, recombinant NaGST-1 was eluted from the column by decreasing the concentration of ammonium sulfate in the buffer to 0.7 M. The eluate was collected and analyzed by SDS–PAGE and SE-HPLC. The eluted protein was stored at 2–8 °C for the subsequent purification step. As a third and final chromatographic and polishing step, a Superdex 75 size-exclusion column was used to remove high molecular weight host cell impurities and to perform buffer exchange into the final buffer. An A2 Vantage Column (VA 130 500 with column tube extension kit VTK 130 500, Millipore) was packed according to the manufacturer’s instructions, sanitized, and equilibrated with 10 mM imidazole, 10% D-glucose, pH 7.4, buffer. The Na-GST-1 pool from the Butyl Sepharose HP column was divided into two pools and loaded onto the prepared Superdex 75 column over two cycles. For each run, the column eluate was collected and analyzed by SDS– PAGE and SE-HPLC. Sample eluates from two cycles of purification containing Na-GST-1 were pooled and filtered through a 0.22 lm filter. Characterization of recombinant Na-GST-1 SDS–PAGE of Na-GST-1 was performed on 4–20% gradient Tris– glycine polyacrylamide gels using an X-cell mini cell apparatus (Invitrogen) with Mark 12 unstained marker (Invitrogen) under denaturing and both non-reducing and reducing conditions. The gels were stained for 30 min (after bringing to a boil) in Coomassie Brilliant Blue R-250 (GE Healthcare) and destained twice for 15 min (after bringing to a boil). Gels were scanned using a calibrated densitometer (Bio-Rad) and analyzed using Quantity One Software (Bio-Rad).
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Size exclusion-high performance liquid chromatography (SE-HPLC) An Alliance 2695 system with an in-line photo-diode array detector (Waters) was used with a G2000SWXL TSK-GEL-HPLC column (TOSOH Bioscience) fitted with a guard column. A mobile phase of 50 mM sodium acetate, pH 4.8, was used at a flow rate of 0.25 ml/min. The spectrum from 210 to 400 nm was recorded and ultraviolet absorbance of the resultant peaks was monitored at 215 and 280 nm. To determine system suitability, 50 lg gel filtration standards (Bio-Rad) were injected before each sample set. To measure the amount of protein, a standard curve was generated by loading Na-GST-1 standards from 0.1 to 75 lg in 50 lL. The NaGST-1 peak was analyzed after extracting the 280 nM channel and a linear curve generated using Empower 2 (Waters) software. Inprocess samples were analyzed at two dilutions (neat or 1:5) depending on their expected concentration as to fall within the linear range. Final purified Na-GST-1 was evaluated at an injection of 25 lg in 50 lL and percent purity determined using Empower 2 software. Mass spectrometry A MicroMass Q-ToF spectrometer (Waters) was used to determine the molecular mass of the recombinant Na-GST-1 molecule. The drug substance was desalted and diluted into formic acid for analysis. Buffer solution was removed by ZipTip desalting prior to analysis. The mobile phase for this analysis was 50% acetonitrile/0.1% formic acid or various ratios of methanol/water with 0.1% formic acid. For mass spectrometry analysis, 2–3 lL of the eluted sample was introduced into the mass spectrometer. The mass spectrometer was controlled and the data processed using the MassLynx 4.0 operating system. Approximately 100 scans were combined (to average out fluctuations in the electrospray), baseline subtracted, smoothed and the centroid of each observed peak determined. The software was used to calculate the mass of the protein from two consecutive multiply charged ions in the spectrum that were manually defined. Results The overall recovery of recombinant Na-GST-1 after the purification process was 51% with a purity of 98% At the conclusion of 20 L scale fermentation, approximately 3 g of secreted soluble Na-GST-1 protein was produced (150 mg/L). Table 1 summarizes the mass balance results of the three major chromatographic steps as determined using an SE-HPLC method for the quantification of Na-GST-1 (in-process samples). The three chromatographic steps resulted in a product with 98% purity and approximately 50% recovery. Fig. 2 shows representative data of the use of the in-process SE-HPLC assay for quantitation of Na-GST-1 during expression and purification and monitoring the efficiency of each chromatography step. Fig. 2A shows the SE-HPLC results of concentrated fermentation supernatant from two 10 Lscale process development runs. Similar results were obtained from the 20 L run (data not shown). The major peak at a retention time of approximately 37 min represents the recombinant antigen Na-GST1. Analysis of the eluate following passage through the Q Sepharose XL column during purification of Na-GST-1, obtained from two 10 L process development runs is also shown in Fig. 2B, which also demonstrates the removal of both high and low molecular weight protein impurities during this capture step. After the Butyl chromatography step, the purity of Na-GST-1 was >90% (Fig. 2C). These remaining host cell proteins were subsequently removed during the size-exclusion chromatography step using Superdex 75 resin
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Table 1 Quantification of recombinant Na-GST-1 using the SE-HPLC method: in-process samples were analyzed from each purification step to monitor mass balance and determine column efficiency and recovery. Purity of the Na-GST-1 target protein is reported as a percentage of total protein. The overall recovery of the process was 51.4%, yielding protein of 98% purity. Column
Sample description
HPLC concentration (mg/ml)
Amount of Na-GST-1 (g)
Purity (%)
Step recovery (%)
Overall recovery (%)
QXL
Concentration of fermentation supernatant Diluted fermentation of supernatant Flowthrough Wash #1 Elution Flowthrough Column strip Starting material Flowthrough Wash #1 Elution Flowthrough Column strip Flowthrough Fraction #1 (high kDa cutoff) Elution Filtered bulk protein
1.95 1.7 0.0 0.0 7.16 0.052 0.125 2.39 0.0 0.004 5.47 0.382 1.68 0.001 0.05 1.74 1.74
2.979 2.885 0.000 0.000 2.549 0.069 0.103 2.572 0.000 0.009 1.633 0.170 0.299 0.002 0.005 1.534 1.530
79
86
51.4
94
64
98
94
BHP
S75
QXL: Q Sepharose XL anion exchange column, BHP: Butyl Sepharose HP hydrophobic affinity column, S75: Superdex 75 size-exclusion column.
Fig. 2. SE-HPLC of in-process samples during recovery and downstream processing of Na-GST-1 during two process development runs (overlayed). Analysis of concentrated fermentation supernatant (A), Q XL eluate (B), Butyl HP eluate (C) and Superdex 75 eluate (D) show the separation used for quantitation of Na-GST-1 at a retention time of approximately 37 min and relative purity calculations using Empower 2 software.
(Fig. 2D). Further analysis of in-process samples during Superdex 75 chromatography revealed that this step removed contaminating proteins in the observed peak shoulder during purification (Fig. 3A) with only nominal loss (6%) of the target Na-GST-1 in this fraction. These results were confirmed using a host cell protein assay that relies on rat antibody to P. pastoris proteins (data not shown).
The predicted mass of Na-GST-1, without an intra-molecular disulfide bond, is 23,679 Da By mass spectrometry the recombinant protein exhibits a mass of 23,699 Da (Fig. 4), with a smaller second peak with a molecular weight of 23,679 Da. This peak is +20 Da relative to the smaller peak, and therefore likely corresponds to a single oxidation event,
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Fig. 3. Chromatogram of purification of Na-GST-1 during Superdex 75 chromatography (A). The shoulder was collected as a separate fraction from the eluant peak and evaluated by SE-HPLC (B), showing the removal of high molecular weight proteins in the eluant peak, yielding with a final protein purity of 98%.
or sodium or potassium addition. Other minor peaks were observed that differ from the predominant peak, suggesting gylcosylation or sugar addition combined with other oxidation or sodium events. Na-GST-1 contains four methionine residues, each of which is a possible site for oxidation. Under reducing conditions, SDS–PAGE analysis of recombinant Na-GST-1 demonstrated a major band that was present at the expected molecular weight (approximately 24 kDa). A Coomassie-stained SDS–PAGE gel under both reducing and non-reducing conditions and loaded with Na-GST-1 recombinant protein (2 lg per load) obtained from one 10 L fermentation and purification run and as compared to the 20 L manufacturing run is shown in Fig. 5. Under non-reducing conditions, the major band is seen as a doublet. The primary amino acid sequence of Na-GST-1 contains two cysteines, and the doublet may result from the presence and absence of an intra-molecular disulfide bond. The absence
of the doublet in the presence of a reducing agent is consistent with this hypothesis. The gels were analyzed on a Bio-Rad GS-800 densitometer to determine the relative amounts of the main doublet (non-reduced) or singlet (reduced) band migrating at 24 kDa (Fig. 5). The percentage migrating as the main bands (doublet) under non-reducing conditions was approximately 96% and the percent protein migrating as the main band under reducing conditions was approximately 97%.
Discussion Both heme and hematin are potentially toxic products released as a consequence of hemoglobin breakdown and digestion. Heme and hematin can result in the production of oxygen radicals that are toxic to blood feeding parasites, including malaria parasites
Fig. 4. Mass spectrometry of Na-GST-1. Mass spectrometry results showing a predominant peak at 23,699 Da.
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Fig. 5. SDS–PAGE with Coomassie staining of recombinant Na-GST-1. Sample Lanes A and B run under non-reducing conditions show 2 lg protein loads obtained from one 10 L and one 20 L fermentation and purification runs, while Lanes C and D show the respective samples evaluated under reducing (with 10 mM DTT) conditions. NaGST-1 appears as a doublet at approximately 24 kDa under non-reducing conditions and as a single band at similar molecular weight in the presence of a reducing agent.
and hookworms [9]. Malaria parasites respond to this threat by polymerizing heme and hematin into an inert pigment known as hemozoin [9], whereas blood-feeding nematode parasites (including hookworms) have been shown to produce a unique Nu class of GSTs used to putatively transport, remove, and detoxify heme and hematin [5–8,10–14]. In hamsters and dogs challenged with hookworm larvae we have shown that antibodies to hookworm GSTs are associated with reductions in the number of larvae that become adult blood-feeding parasites [5–8]. It is presumed that the protective effect is the result of anti-Na-GST-1 antibodies interfering with the ability of hookworms to detoxify heme and establish in the gut during the earliest stages of blood feeding [3]. Here we described the expression of Na-GST-1 in the yeast P. pastoris expression system at the 20 L scale and its purification by three chromatographic steps, comprised of a Q Sepharose XL anion exchange column, followed by a Butyl Sepharose HP hydrophobic affinity column and a Superdex 75 size-exclusion column. Approximately 1.5 g of bulk protein was recovered at a yield of 51%, and with a purity of 98%. Based on in process assessments and the results from the mass balance it became evident that approximately 14% protein loss is observed during the Q Sepharose XL anion exchange column and 22% loss during the Butyl Sepharose HP hydrophobic affinity column step. These recovery rates are considered suitable for transfer to production scales needed for the generation of material suitable for Phase 1 trial. By mass spectrometry the recombinant protein exhibits a mass of 23,676 Da, which closely matches the predicted molecular mass of the protein. In selecting both a proven expression system such as the yeast P. pastoris and easily scalable chromatography steps, Na-GST-1 was produced as a proof of principle for scalable process development and for use in preclinical studies, and ultimately for Phase 1 clinical testing. The most significant challenge to overcome in designing an acceptable purification process was the tendency for high molecular weight host cell proteins to co-elute with Na-GST-1. These proteins were successfully eliminated by using size-exclusion chromatography with Superdex 75 resin, yielding protein of high purity (98%) with no detectable host cell protein. By incorporating SE-HPLC as an in-process assay, we were also able to monitor the target protein
throughout the purification process, thus generating further understanding of the robustness of each purification step. Studies are in progress to evaluate the stability of the Na-GST-1 generated through these processes, as well as its stability after its binding to alum. Ultimately, the human hookworm vaccine is envisioned as a bivalent recombinant protein vaccine on alum, possibly together with a second immunostimulant such as a synthetic lipid A as a toll-like 4 receptor agonist [3]. As Na-GST-1 advances through clinical development it will become necessary to revise the processes described here, including possible improvements in yield and reductions in cost suitable for large scale manufacturing. With respect to improving yield the greatest protein losses were noted following elution of the protein from Butyl-Sepharose – therefore process improvements may require modifying or substituting this step. With regards to cost, altering or substituting the Superdex-75 column may be advantageous. Because hookworm infection is almost exclusively an infection affecting the world’s poorest people [2,15], it is anticipated that the human hookworm vaccine will need to be manufactured for the lowest possible costs [16]. In summary, we have developed a process for the pilot expression of Na-GST-1 in yeast and its purification using a series of three chromatography steps. The process is suitable for producing purified protein that could be formulated with alum and other adjuvants. Finally this process is also suitable for technology transfer for manufacture under current good manufacturing practices (cGMP) for Phase 1 testing. Acknowledgments This work is supported by the Sabin Vaccine Institute through grants obtained from the Bill & Melinda Gates Foundation (Grant #32472 and #38988) and the Dutch Ministry of Foreign Affairs. References [1] D.W. Crompton, The public health importance of hookworm disease, Parasitology 121 (Suppl) (2000) S39–S50. [2] P.J. Hotez, P.J. Brindley, J.M. Bethony, C.H. King, E.J. Pearce, J. Jacobson, Helminth infections: the great neglected tropical diseases, J. Clin. Invest. 118 (2008) 1311–1321. [3] P.J. Hotez, J.M. Bethony, D.J. Diemert, M. Pearson, A. Loukas, Developing vaccines to combat hookworm infection and intestinal schistosomiasis, Nat. Rev. Microbiol. 8 (2010) 814–826. [4] M.S. Pearson, J.M. Bethony, D.A. Pickering, L.M. de Oliveira, A. Jariwala, H. Santiago, A.P. Miles, B. Zhan, D. Jiang, N. Ranjit, J. Mulvenna, L. Tribolet, J. Plieskatt, T. Smith, M.E. Bottazzi, K. Jones, B. Keegan, P. Hotez, A. Loukas, An enzymatically inactivated hemoglobinase from Necator americanus induces neutralizing antibodies against hookworm species and protects dogs against heterologous hookworm infection, FASEB J. 23 (2009) 3007–3019. [5] B. Zhan, S. Liu, S. Perally, J. Xue, R. Fujiwara, P. Brophy, S. Xiao, Y. Liu, J. Feng, A. Williamson, Y. Wang, L.L. Bueno, S. Mendez, G. Goud, J.M. Bethony, J.M. Hawdon, A. Loukas, K. Jones, P.J. Hotez, Biochemical characterization and vaccine potential of a heme-binding glutathione transferase from the adult hookworm Ancylostoma caninum, Infect. Immun. 73 (2005) 6903–6911. [6] O.A. Asojo, K. Homma, M. Sedlacek, M. Ngamelue, G.N. Goud, V. Deumic, O. Asojo, P.J. Hotez, X-ray structures of Na-GST-1 and Na-GST-2 two glutathione S-transferase from the human hookworm Necator americanus, BMC Struct. Biol. 7 (2007) 42. [7] S. Xiao, B. Zhan, G.N. Goud, A. Loukas, Y. Liu, A. Williamson, S. Liu, V. Deumic, P. Hotez, The evaluation of recombinant hookworm antigens as vaccines in hamsters (Mesocricetus auratus) challenged with human hookworm, Necator americanus, Exp. Parasitol. 118 (2008) 32–40. [8] B. Zhan, S. Perally, P.M. Brophy, J. Xue, G. Goud, S. Liu, V. Deumic, L.M. de Oliveira, J. Bethony, M.E. Bottazzi, D. Jiang, P. Gillespie, S.H. Xiao, R. Gupta, A. Loukas, N. Ranjit, S. Lustigman, Y. Oksov, P. Hotez, Molecular cloning, biochemical characterization, and partial protective immunity of the hemebinding glutathione S-transferases from the human hookworm Necator americanus, Infect. Immun. 78 (2010) 1552–1563. [9] N. Klonis, R. Dilanian, E. Hanssen, C. Darmanin, V. Streitsov, S. Deed, H. Quiney, L. Tilley, Hematin-hematin self-association states involved in the formation and reactivity of the malaria parasite pigment, hemozoin, Biochemistry 49 (2010) 6804–6811. [10] P.M. Brophy, J. Barrett, Gultathione transferase in helminths, Parasitology 100 (Pt. 2) (1990) 345–349.
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