Stability Improvement of a Therapeutic Protein by Reducing Agent Pretreatment

CHINESE JOURNAL OF BIOTECHNOLOGY Volume 24, Issue 12, December 2008 Online English edition of the Chinese language journal RESEARCH PAPER

Cite this article as: Chin J Biotech, 2008, 24(12), 2142í2143.

Stability Improvement of a Therapeutic Protein by Reducing Agent Pretreatment Lin Zhang, Murray Moo-Young, and C. Perry Chou Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1

Abstract: A major concern in developing protein-based biopharmaceuticals is protein instability. A strategy with the use of reducing agent pretreatment to improve protein stability was developed for recombinant hCD83ext (i.e. the extracellular domain of human CD83) with a potential therapeutic activity. Under physiological conditions, the therapeutic product tended to denature, form aggregates and precipitates, and eventually degrade. The reducing agent pretreatment was demonstrated to be effective in improving the protein stability. Keywords: degradation; protein stability; hCD83ext; reducing agent pretreatment

Introduction Therapeutic proteins made by recombinant DNA technology are gaining importance in pharmaceutical research and industry. Approximately 150 therapeutic proteins with a market volume of $50–60 billion have already been approved for practical applications and at least 500 candidates are under clinical trials all over the world[1,2]. Stabilization of protein molecules has been recognized as a key issue in biopharmaceutical research and development since denatured and instable proteins can potentially jeopardize the safety and efficacy of therapeutic products during the stages of manufacturing, storage, and administration[3]. In general, protein instability was mediated by two mechanisms: chemical degradation pathways (such as oxidation, deamidation, and disulfide scrambling) and physical degradation pathways (such as denaturation, aggregation and precipitation)[4,5]. At present, the most popular stabilizing approaches for therapeutic proteins are adding excipients into liquid formulations and lyophilizing them into solid

formulations[6]. The impact of disulfide bond on protein stability has been intensively studied. It has been demonstrated that native disulfide bonds improve protein stability, whereas nonnative disulfide bonds often impair it[7]. Protein stabilization can be an empirical science because of the unique nature of this biomolecule and the wide range of agents/conditions that can result in protein denaturation and instability. The therapeutic potential of extracellular domain of human CD83 (hCD83ext) has been proposed for treating autoimmune disorder[8]. Structurally, hCD83ext contains five cysteine residues which can mediate various intramolecular and intermolecular disulfide bonds. Disulfide bond formation has been shown to play a crucial role in molecular behavior of hCD83ext[9]. We observed that hCD83ext was unstable during room-temperature storage. Therefore, an effective strategy needs to be established to improve the stability of hCD83ext as a novel therapeutic protein. In this study, improving hCD83ext stability with disulfide reducing agent pretreatment was explored.

Received: October 17, 2008; Accepted: November 25, 2008 Supported by: the Natural Sciences and Engineering Research Council (NSERC) and the Canada Research Chair (CRC) program of the Canadian government. Corresponding author: C. Perry Chou. Tel: +519-888-4567 x33310; Fax: 519-746-4979; E-mail: [email protected] Copyright © 2008, Institute of Microbiology, Chinese Academy of Sciences and Chinese Society for Microbiology. Published by Elsevier BV. All rights reserved.

Lin Zhang et al. / Chinese Journal of Biotechnology, 2008, 24(12): 2142–2143

1

Materials and methods

1.1 Bacterial strain and plasmids Escherichia coli BL21(DE3)pLysS (Novagen, USA) was used as the host strain. The plasmid of pGEX2ThCD83ext containing the cDNA encoding hCD83ext fused with GST[10] was used as the expression vector for the production of recombinant GST-hCD83ext. 1.2 Expression, recovery, and purification of hCD83ext Cells were revived by streaking the stock culture stored at 80°C on a Luria-Bertani (LB) agar plate (5 g/L of NaCl, 5 g/L of Bacto yeast extract, 10 g/L of Bacto tryptone, and 15 g/L of Bacto agar). An isolated colony was picked to inoculate into 150 mL LB medium supplemented with 50 μg/mL ampicillin. The seed culture was inoculated into a table-top bioreactor (Omni-Culture, VirTis, USA) containing 2 L LB medium (20 g/L Bacto yeast extract, 20 g/L Bacto tryptone, and 5 g/L NaCl) supplemented with 5 g/L glucose. When cells grew to 1.8 OD600, 0.5 mM isopropyl-E-D- thiogalactopyranoside (IPTG) was added to induce the expression of GST-hCD83ext. To avoid excessive foaming, 10 PL/L Antifoam 289 (Sigma-Aldrich, USA) was added. The bioreactor was sparged with filter-sterilized air at 2 L/min. The bioreactor was operated at 28°C, 800 rpm and pH 7.0 for 6 h after induction. At the end of cultivation, the cells were pelleted at 6000 × g and 4°C, followed by resuspension in PBS (Phosphate buffered saline) buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4, pH 7.3). Probe sonication was used to obtain cell lysate, and the lysate was then spun at 30 000 × g and 4°C for 15 min. The supernatant containing soluble proteins was collected and filtrated through 0.45-Pm pore size syringe filter (Whatman, USA) before downstream processing. The downstream processing was divided into three stages. Initially, the expressed GST-hCD83ext were captured in a GSTPrep FF 16/10 column (GE Healthcare, Canada) using PBS buffer as the binding buffer at a flow rate of 4 mL/min. Subsequently, the bound fusion proteins were in situ cleaved with thrombin (Sigma-Aldrich, USA) while GST was bound to GSTPrep column and hCD83ext was eluted to bulk liquid phase containing thrombin. Finally, thrombin and other contaminants were removed by anion exchange chromatography conducted on a BioLogic Work Station (Bio-Rad, USA) equipped with a Q-Sepharose column (GE Healthcare, Canada) at a flow rate of 5 mL/min. Tris·HCl buffer with 50 mM NaCl (pH 7.0) was used as the binding buffer, while Tris·HCl buffer with 1 M NaCl (pH 7.0) was the elution buffer. The purity of product was testified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to be higher than 99%. 1.3 Reduction treatment Incubation of the protein product samples (1 mg/mL)

with 5 mM dithiothreitol (DTT) was carried out for 3 hours at room temperature. Removal of residual DTT was conducted by diafiltration using a high-pressurized stirred cell (Amicon, Model 8010 with YM10 disk, Millipore Canada) against Tris·HCl buffer containing 50 mM NaCl, pH 7.5 at 4°C. 1.3 SDS-PAGE SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed under reducing and non-reducing conditions in a Mini-PROTEAN®II electrophoresis cell (Bio-Rad, USA) using a 12.5% polyacrylamide separating gel and a 4% polyacrylamide stacking gel in 0.75 mm thickness. 0.25 μg of protein was loaded in each lane. Electrophoresis was conducted under a constant voltage of 200 V for approximately 45 min. The bands were observed by silver stain. 1.4 Protein concentration measurement Protein concentration was determined by measuring its absorbance at 280 nm (OD280) on a ND-1000 spectrophotometer (NanoDrop Technologies, USA). The extinction coefficient was determined to be approximately 1.16 OD280-mL/mg/cm by bicinchoninic acid assay (Sigma, Canada). 1.5 Circular dichroism (CD) spectrometry Far-UV CD spectra of protein samples were measured on a Jasco-815 spectropolarimeter (Jasco, Japan) from 250 nm down to 190 nm using a quartz cuvette with 0.1 mm pathlength. Baseline correction using 20 mM Tris·HCl buffer, 50 mM NaCl, pH 7.5, and averaged over 2 scans was applied for far-UV CD measurements at room temperature, 23°C, with a 3-nm band width. 1.6 Fluorescence spectrometry Fluorescence measurement of tryptophan residues was conducted on a Jasco FP-6500 spectrofluorometer (Jasco) using a microquartz cuvette. Excitation wavelength was set at 295 nm in order to avoid disturbance of fluorescence generated by tyrosine residuals. Emission spectra from 300 nm to 420 nm were recorded with a 5-nm band width at room temperature.

2

Results

2.1 Time course stability of hCD83ext Destabilization of hCD83ext was demonstrated by incubation of protein samples exposed to air at 25°C. Fig. 1A shows hCD83ext degradation during a 12-h incubation period as indicated by the appearance of a lower-molecularweight band under the regular monomer (~15 kD). Fig. 1B shows that an aggregate with a molecular weight smaller than dimer (~30 kD) appeared after 12 h and the amount of this aggregate increased with prolonged incubation up to 60 h. Then, the aggregate was subject to degradation. The absorbance of the protein sample at 280 nm (OD280)

Lin Zhang et al. / Chinese Journal of Biotechnology, 2008, 24(12): 2142–2143

increased to 0.14 after 36 h and continued rising with prolonged incubation. Notably, the protein solution became turbid and precipitates were present after two weeks. CD spectra (Fig. 1C) displayed a typical characteristic of ȕ-sheet mixed with type II ȕ-turn (defined by backbone torsional angle values), which has a negative signal at 220 nm and a positive signal of comparable magnitude near 203 nm[11]. Significant changes in the magnitude of CD signal were also observed. The signal at 203 nm increased continuously (ǻCD=4.5–5.5 mdeg) throughout the incubation, while the signal at 220 nm displayed a red shift (ǻȜ=6 nm) after 36 h till the end. Fluorescence spectra (Fig. 1D) showed that tertiary structure was stable only within the first 12 h. The data implied that hCD83ext was relatively stable within 12 h, and then degradation of both monomer and dimer was triggered as represented by the downward migrating bands in the SDS-PAGE, red-shift of band at 220 nm in CD spectra, loss of tryptophan fluorescence features in fluorescence spectra. Within the whole incubation period, the breakdown of the degradants became increasingly prevalent during incubation. Steep increases of UV absorbance at 280 nm (OD280) were observed over this degradation process.

Fig. 1

2.2 Time course stability of DTT-pretreated hCD83ext Profiles corresponding to incubation of hCD83ext pretreated with DTT are included in Fig. 2. The stable period was significantly extended up to 125 h. OD280 fluctuated around 0.10 throughout the incubation period. In Fig. 2C, the secondary structure is shown to be stable with prolonged incubation, except that the band at 203 nm dramatically increased over the first 6 h. There was almost no red shift of the band at 220 nm from 6 h to the end. A drop in fluorescence signal at approximately 450 AU during the first 6 h was observed in Fig. 2D. After that, the fluorescence signal was maintained around (500±50)AU up to 230 h.

3

Discussion

The decrease in molecular weight of dimer and monomer shown on SDS-PAGE can imply a major cleavage of peptide backbone. It was reported that Asp-X bond is more susceptible to hydrolysis than any other peptide bond[12,13]. Particularly, the Asp-Pro bond was found to be extremely labile and tended to be hydrolyzed under the conditions in which other aspartic acid bonds are stable. In the primary

Stability analysis during incubation at 25°C

(A) SDS-PAGE under reducing conditions; (B) SDS-PAGE under nonreducing conditions; (C) Time profile of CD spectra; (D) Time profile of fluorescence spectra

Lin Zhang et al. / Chinese Journal of Biotechnology, 2008, 24(12): 2142–2143

Fig. 2 Stability analysis of DTT-pretreated sample during incubation at 25°C (A) SDS-PAGE under reducing conditions; (B) SDS-PAGE under non-reducing conditions; (C) Time profile of CD spectra; (D) Time profile of fluorescence spectra

structure of hCD83ext, there are seven Asp-X bonds and two of them are Asp-Pro. All these sites could be possible cleavage targets. The impact of degradation on tertiary structure of hCD83ext was clearly characterized by fluorescence emission spectra of tryptophan residues in which one emission maximum appeared at 340 nm and the other one was around 400 nm upon degradation. The oxidation product of tryptophan, formylkynurenine and kynurenine has been reported to emit maximally above 400 nm[14], suggesting that oxidation might be a possible source causing degradation in hCD83ext. On the other hand, sulfhydryl groups in cysteine residues can be oxidized to form disulfide bond when exposed to atmospheric oxygen. It is hypothesized that the oxidation-induced degradation could be retarded by prior oxidation of sulfhydryl groups. Thus, disulfide reducing agent was used to pretreat hCD83ext. The significantly enhanced stability in DTT-pretreated hCD83ext was observed with an extended stable period. The underlying mechanism was speculated to be that 1) oxidation of sulfhydryl groups occurred prior to oxidation of the other residues, such as methionine, histidine, tryptophan, tyrosine and phenylalanine, resulting in a prolonged stable period; and 2) native disulfide bond was probably formed during this reducing-oxidizing process

which improved the stability of hCD83ext. Notably, a 28% increase in OD280 was suddenly observed after 36 h incubation in hCD83ext accompanied by the debut of degraded species detected in SDS-PAGE. Furthermore, OD280 increased exponentially to 6-fold of original readings at the end of incubation when substantial protein degradation was observed. Similar phenomenon was reported during degradation of protein basic fibroblast growth factor[15]. Hence, OD280 can be used as an indicator for monitoring the degradation of hCD83ext.

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