In situ PEGylation of recombinant hirudin on an anion exchange chromatography column

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In situ PEGylation of recombinant hirudin on an anion exchange chromatography column Xudong Wang a,b , Xueqin Li a , Jun Zhao a , Li Lv c , Kairong Qin b , Hengli Yuan d , Zhilong Xiu a,∗ a

School of Life Science and Biotechnology, Dalian University of Technology, 2 Linggong Road, Dalian, 116024, PR China Department of Biomedical Engineering, Dalian University of Technology, 2 Linggong Road, Dalian, 116024, PR China c College of Pharmacy, Dalian Medical University, Western 9 Lvshunnan Road, Dalian, 116044, PR China d State Key Laboratory Cultivating Base for Long-acting Bio-medical Research of Jiangsu Province, Jiangsu Hansoh Pharmaceutical Group CO., LTD, Lianyunguang, 222000, PR China b

a r t i c l e

i n f o

Article history: Received 9 October 2016 Received in revised form 6 January 2017 Accepted 26 January 2017 Available online xxx Keywords: Recombinant hirudin PEGylation Ion exchange chromatography In situ PEGylation Solid-phase PEGylation On-column PEGylation

a b s t r a c t In this study, an integrated process was developed for successive solid-phase PEGylation of recombinant hirudin variant-2 (HV2) and separation of PEGylated HV2 species on an anion exchange chromatography column (so-called in situ PEGylation). The effects of different PEG sizes, ion exchange resins and reaction conditions on in situ PEGylation were investigated. The results showed that in situ PEGylation efficiently integrates the reaction, separation and purification into a single-unit operation using the same column. In situ PEGylation could improve the selectivity of PEGylation reactions by significantly reducing the formation of multi-PEG-HV2. The pore sizes and internal surface structures of different resins had a significant impact on the yield of mono-PEG-HV2. In contrast to liquid-phase PEGylation, the yield of mono-PEG-HV2 decreased as PEG size increased during the in situ PEGylation process, indicating that in situ PEGylation is a pore diffusion-controlled process. The in vitro and in vivo anticoagulant activities of mono-PEG-HV2 derived from in situ PEGylation were higher than those from liquid-phase PEGylation, indicating that in situ PEGylation could enhance the bioactivity retention of mono-PEG-HV2. The results of this study demonstrated that in situ PEGylation can be used as an effective approach for the development of PEGylated protein drugs. © 2017 Elsevier Ltd. All rights reserved.

1. Introduction Hirudin was initially found in leeches as a single-chain polypeptide consisting of 65 amino acids (molecular weight of 7 kDa). It exhibits potent anti-thrombin activity [1]. Recently, recombinant hirudin has been developed for therapeutic use because of the limited source of natural leeches. To date, two recombinant hirudins (lepirudin and desirudin) have been approved by the FDA. However, the clinical application of recombinant hirudin has been limited by its short plasma half-life and adverse side effects (such as bleeding and immunogenicity) [2,3]. PEGylation has been shown to be an effective strategy to improve the therapeutic efficacy of proteins such as recombinant hirudin [4,5]. Random PEGylation of the ␧-amino group of lysine residues is still the most commonly employed strategy for recombinant hirudin [6,7]. However, this

∗ Corresponding author. E-mail address: [email protected] (Z. Xiu).

strategy is limited due to the presence of several lysine residues in recombinant hirudin, which usually results in a complex mixture of various conjugates, a low yield of the desired mono-PEGylated form, and much difficulty in separating and purifying the desired mono-PEGylated form from the complex reaction mixtures. Additionally, the activity of recombinant hirudin in vitro was remarkably decreased by random PEGylation. Currently, to overcome these issues, site-specific PEGylation is frequently used. Unfortunately, common site-specific PEGylation methods (e.g., N-terminal, thiol and disulfide bridge PEGylation) might be not suitable for PEGylation of recombinant hirudin because of its special structure, which includes the following characteristics: (1) the N-terminus of recombinant hirudin is its activity site; (2) recombinant hirudin has no free cysteines; and (3) three disulfide bonds play an important role in stabilizing the secondary structure and tertiary structure of recombinant hirudin. To improve the selectivity of PEGylation, PEGylation with 5 kDa mPEG-SC at the only histidine residue (His 51) of recombinant hirudin variant-2 (HV2) was carried out in our previous study [8]. Compared to random PEGylation at the

http://dx.doi.org/10.1016/j.procbio.2017.01.024 1359-5113/© 2017 Elsevier Ltd. All rights reserved.

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Fig. 1. Purification of liquid-phase PEGylation reaction mixtures by anion exchange chromatography. (A) PEGylation of HV2 with 5 kDa mPEG-SC; (B) PEGylation of HV2 with 10 kDa mPEG-SC; (C) PEGylation of HV2 with 20 kDa mPEG-SC. Reaction conditions: HV2 = 1.5 mg/mL, pH = 8.0, temperature = 25 ◦ C, molar ratio of PEG to HV2 = 3, reaction time = 30 min. Purification procedures: Each sample (2 mL) of the reaction mixture was injected to a 5 mL HiTrap Q HP column pre-equilibrated in buffer A (20 mM sodium phosphate, pH 8.0) using an AKTA purifier 10 system at room temperature. After the column was washed by 3.6-fold column volumes of buffer A, PEGylated HV2 fractions were eluted with a linear salt gradient (20-fold column volumes) from 0% to 25% buffer B (20 mM sodium phosphate, 1 M NaCl, pH 8.0) over 100 min. The flow rate was 1 mL/min, and the detection wavelength was 280 nm. The product elution window is labeled as a blue square (elution volume from 20 to 120 mL). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

lysine residues of HV2, this strategy achieved increased selectivity of PEGylation, but decreased bioactivity of mono-PEG-HV2. As an alternative to the methods described above, solid-phase PEGylation is considered to be a site-selective PEGylation approach to some extent [4]. The advantages of solid-phase PEGylation can be summarized as follows: (1) some potential modification sites might be blocked and the frequency of protein contacting PEG molecules might be reduced due to steric hindrance between the protein and solid media, leading to more uniform degree of PEGylation [9]; (2) the active sites can be protected from modification by the oriented immobilization of the active region of the protein onto solid media in some cases, contributing to enhanced bioactivity retention of the desired mono-PEGylated form [10]; (3) the PEGylation reaction and primary separation of PEGylated products can be integrated into a single unit operation, resulting in a reduction operation steps

Fig. 2. A typical chromatographic process of in situ PEGylation of HV2 on an anion exchange chromatography column. (A) PEGylation of HV2 with 5 kDa mPEG-SC; (B) PEGylation of HV2 with 10 kDa mPEG-SC; (C) PEGylation of HV2 with 20 kDa mPEG-SC. In-situ PEGylation of HV2 were performed by using an AKTA purifier 10 system. The procedures are as follows: (1) A 5.0 mL HiTrap Q HP column was preequilibrated with buffer solution A (20 mM sodium phosphate buffer, pH 8.0). Then, 2.0 mL of HV2 (1.5 mg/mL) dissolved in the buffer solution A was injected into the column using a 2.0 mL sample loop. After this, the column was washed to remove unadsorbed HV2 with 2.6-fold column volumes of buffer solution A. (2) 5.0 mL of mPEG-SC (molar ratio of PEG to HV2 of 15:1) dissolved in the buffer solution A was subsequently injected into the column using a 5.0 mL sample loop. Then, the column was statically incubated for 60 min. (3) After the reaction, the column was washed with 3-fold column volumes of buffer solution A to remove the PEG and NHS. Then, the PEGylated HV2 fractions were eluted with a linear salt gradient (20-fold column volumes) from 0% to 25% buffer B (20 mM sodium phosphate, 1 M NaCl, pH 8.0) over 100 min. The flow rate was 1 mL/min, and the detection wavelength was 280 nm. The product elution window is labeled as a blue square (elution volume from 35 to 135 mL). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

and an easier process to control [11]. Based on the immobilization of PEGs or proteins onto solid media by covalent bond or adsorption techniques, solid-phase PEGylation can be divided into the following four types: (1) covalent bonding of PEGs onto solid media, e.g., PEGs grafted onto a modified Sephadex media [12]; (2) covalent bonding of proteins onto solid media, e.g., solid-phase synthesis for the PEGylation of small peptides [13]; (3) adsorption of PEGs onto solid media, e.g., PEG-ALD adsorbed onto hydrophobic interaction membranes [14]; (4) adsorption of proteins onto solid media, e.g., hemoglobin adsorbed onto CM Sepharose Fast Flow resin [15]. Among these four types of solid-phase PEGy-

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Fig. 3. SDS-PAGE (A) and RP-HPLC (B) analysis of elution fractions of in situ PEGylation of HV2. (A) SDS-PAGE analysis was performed according to our previously reported method [28]. The polyacrylamide concentration was 5% in the stacking gel and 15% in the separation gel. Afterwards, fractions were identified by coomassie blue staining with protein (a) and barium-iodine staining with PEG (b), in which the protein and PEG were stained, respectively. Accordingly, protein marker and PEG marker were used as molecular weight standards, respectively. (B) RP-HPLC analysis was performed according to our previously reported method [7,28]. The analysis was performed on a LiChrospher 100 RP-18 column (250 mm × 4.0 mm, 5 ␮m) by using an Agilent 1100 HPLC system. The procedures: 50 ␮L of each sample was injected into the column pre-equilibrated with 85% solvent A (0.1% (v/v) TFA in water) and 15% solvent B (0.1% (v/v) TFA in acetonitrile). Then, the PEGylated HV2 fractions were eluted with a linear gradient from 15% to 60% solvent B over 45 min. The column temperature was set at 40 ◦ C. The flow rate was 1 mL/min, and the detection wavelength was 215 nm. Curves 1, 2, 3 and 4 corresponded to the elution fractions of peak 1, 2, 3 and 4 in Fig. 2.

lation, the adsorption of proteins onto solid media is the most commonly employed strategy. Because use of a chromatography column is dominant among the media used for solid-phase PEGylation, chromatography column-aided solid-phase PEGylation is also called on-column PEGylation. In recent years, on-column PEGylation has been developed using size-exclusion chromatography [16], hydrophobic interaction chromatography [17], affinity chromatography [10,18–22] and ion exchange chromatography (IEC) [9,11,15,23–27]. Among these four media, IEC is the most widely used. In some cases of on-column PEGylation, the PEGylation reaction, separation and purification of the desired PEGylated protein form can be achieved in a single-step operation in the same column (so-called in situ PEGylation). Although on-column PEGylation has been investigated by several researchers, the mechanism of

in situ PEGylation is still not clearly understood. Thus, more detailed investigations into in situ PEGylation should be carried out to determine the crucial parameters. In this study, in situ PEGylation of recombinant hirudin variant-2 (HV2) on an anion exchange chromatography column was developed to increase the homogeneity and bioactivity retention of the desired mono-PEGylated form. First, the effects of different PEG sizes, ion exchange resins and reaction conditions (pH, molar ratio of PEG to HV2 and reaction time) on in situ PEGylation were investigated. Second, elution fractions collected from in situ PEGylation were characterized by SDS-PAGE and RP-HPLC analysis. Finally, the in vitro anticoagulant activity and in vivo pharmacological efficacy of mono-PEG-HV2 was further characterized. Additionally,

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Fig. 4. Effect of different anion exchange resins on in situ PEGylation of HV2. The molecular weight of mPEG-SC was 10 kDa. Except for the columns and PEG sizes, the other procedures for in situ PEGylation were the same as described in Fig. 2. The conversion, yield and selectivity were calculated according to Equation (1), (2) and (3) in Section 2.9, respectively.

liquid-phase PEGylation of HV2 was also carried out to compare its characteristics with in situ PEGylation. 2. Materials and methods 2.1. Materials Recombinant hirudin variant-2 (HV2) (>95% pure) was purchased from Chongqing Kerun Biomedical R&D Co., Ltd. (Chongqing, China). Monomethoxy-PEG-succinimidyl carbonate (mPEG-SC, MW = 5, 10 and 20 kDa) was purchased from Beijing Kaizheng Biotech Development Co., Ltd. (Beijing, China). Thrombin (Sigma-T4648) and fibrinogen (Sigma-F8630) were purchased from Sigma-Aldrich (St. Louis, USA). Acetonitrile (HPLC grade), trifluoroacetic acid (TFA) and other chemicals of analytical grade were also purchased from Sigma-Aldrich. A LiChrospher 100 RP-18 column (250 mm × 4.6 mm, 5 ␮m) was obtained from Merck (Darmstadt, Germany). HiTrap Q HP (5 mL), HiTrap Q FF (5 mL), HiTrap DEAE FF (5 mL), HiTrap Q XL (5 mL) and HiTrap ANX FF (5 mL) were obtained from GE Healthcare (Piscataway, New Jersey, USA). Thrombin time (TT) assay kits were purchased from Nanjing Jiancheng Biotechnology Co. Ltd. (Nanjing, China). 2.2. Animals Male New Zealand White rabbits (2.0 ± 0.2 kg) were obtained from the Experimental Animal Center of Dalian Medical University (Dalian, China, quality certificate number: SCXK (Liao) 2008-0002). They were housed under standard conditions of a 12 h dark-light cycle at 25 ± 2 ◦ C and maintained on a standard diet with water ad libitum. All of the animal experiments were performed according to the Animal Guidelines of Dalian Medical University and were approved by the Ethics Committee of Dalian Medical University. 2.3. Liquid-phase PEGylation of HV2 and separation of the PEGylated HV2 species HV2 (1.5 mg/mL) dissolved in a 20 mM sodium phosphate buffer (pH 8.0) was, respectively, reacted with 5, 10 or 20 kDa mPEG-SC at a molar ratio of PEG to HV2 of 3:1. The reaction mixtures were incubated at room temperature with a stirring speed of 150 rpm for 30 min. The reactions were stopped by adding an excess of glycine solution. Samples obtained from the quenched reaction mixtures were immediately analyzed by RP-HPLC to calculate the yields of PEGylated HV2 species according to our previous reports [7,28]. The remaining samples of the quenched reaction mixtures were purified by anion exchange chromatography according to our previously reported method [28]. All of the elution fractions collected from anion exchange chromatography were concentrated

Fig. 5. Effect of pH (A), molar ratio of PEG to HV2 (B) and reaction time (C) on in situ PEGylation of HV2. The molecular weight of mPEG-SC was 10 kDa. Except for the investigated factor, the other procedures of in situ PEGylation were the same as described in Fig. 2. The conversion, yield and selectivity were calculated according to Equation (1), (2) and (3) in Section 2.9, respectively.

and stored at −20 ◦ C for SDS-PAGE and RP-HPLC analysis. The in vitro anticoagulant activity and in vivo pharmacological efficacy of identified mono-PEG-HV2 was further characterized. 2.4. In situ PEGylation of HV2 on anion exchange chromatography columns In situ PEGylation of HV2 on anion exchange chromatography columns were performed using an AKTA purifier 10 system (GE Healthcare, USA) at room temperature. The effects of different PEG sizes, ion exchange resins and reaction conditions on the in situ PEGylation were systematically investigated. The procedures for in situ PEGylation of HV2 are as follows: (1) a 5.0 mL column (HiTrap Q HP, HiTrap Q FF, HiTrap DEAE FF, HiTrap Q XL or HiTrap ANX FF) was pre-equilibrated with buffer solution A (20 mM sodium phosphate buffer, pH 8.0, 7.0 or 6.0). Then, 2.0 mL of HV2 (1.5 mg/mL) dissolved in the buffer solution A was injected into the column

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Fig. 6. A possible pore diffusion-controlled process of in situ PEGylation on an anion exchange chromatography column. The in situ PEGylation of HV2 with 5, 10 or 20 kDa mPEG-SC was performed as described in Section 2.4. The chromatographic processes are shown in Fig. 2. (A) During the in situ PEGylation process, mass transfers of HV2 and PEG molecules inside a resin (microsphere) are mainly driven by pore diffusion. (B) HV2 molecules were injected into the column within 2 min, and the column was washed for 13 min at 1 mL/min. (C) PEG molecules were injected into the column within 5 min, and then the column was statically incubated for 60 min. (D) PEG molecules diffused into a pore and contacted adsorbed HV2 molecules. R represents the particle radius of a resin (microsphere). R-r1 represents the pore diffusion distance of HV2 molecules. R-r2 represents the pore diffusion distance of PEG molecules. The pore-diffusion distance of PEG reduced as PEG size increased.

using a 2.0 mL sample loop. After this, the column was washed to remove unadsorbed HV2 with 2.6-fold column volumes of buffer solution A. (2) Then, 5.0 mL of mPEG-SC (molecular weight 5, 10 or 20 kDa) dissolved in the buffer solution A was subsequently injected into the column using a 5.0 mL sample loop. To reduce

waste of mPEG-SC during the hydrolysis reaction itself, mPEG-SC should be immediately injected into the column after being dissolved in buffer solution A, and the molar ratio of PEG to HV2 was 5:1, 10:1 or 15:1. After the mPEG-SC solution completely entered the column, the column was statically incubated for 30, 60, 90

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or 120 min. (3) After the reaction, the column was washed with 3-fold column volumes of buffer solution A to remove the PEG and NHS. Then, PEGylated HV2 fractions were eluted with a linear salt gradient (20-fold column volumes) from 0% to 25% buffer B (20 mM sodium phosphate, 1 M NaCl, pH 8.0, 7.0 or 6.0) over 100 min. The flow rate was 1 mL/min, and the detection wavelength was 280 nm. All the elution fractions were collected, then concentrated and stored at −20 ◦ C for SDS-PAGE and RP-HPLC analysis. The in vitro anticoagulant activity and in vivo pharmacological efficacy of the identified mono-PEG-HV2 was further characterized. The concentrations of separated PEGylated HV2 species with different degrees of modification were calculated based on the peak area. The standard reaction condition for investigating the effect factors is as follows: HiTrap Q HP column, pH 8.0, molar ratio of PEG to HV2 of 15:1, reaction time of 60 min and PEG size of 10 kDa.

Fig. 7. Comparison of the in vitro anticoagulant activity of mono-PEG-HV2 obtained from liquid-phase and in situ PEGylation of HV2. The in vitro anticoagulant activity was assessed by the thrombin titration method according to our previous report [28]. The retention of bioactivity was calculated according to Eq. (4) in Section 2.9.

2.5. SDS-PAGE analysis Elution fractions collected from anion exchange chromatography were analyzed by SDS-PAGE according to our previous report [28].

tivity of PEGylation and bioactivity retention of mono-PEG-HV2. These parameters were calculated as follows: conversion of HV2 =

the total PEGylated HV2 (mono- and multi-PEG-HV2) (mol) the initial HV2 for PEGylation(mol)

(1)

2.6. RP-HPLC analysis Reaction mixtures collected from liquid-phase PEGylation and the elution fractions collected from anion exchange chromatography were analyzed by RP-HPLC according to our previous reports [7,28].

yield of mono-PEG-HV2 =

mono-PEG-HV2(mol) the initial HV2 for PEGylation(mol)

(2) selectivity of PEGylation

2.7. In vitro anticoagulant activity analysis The in vitro anticoagulant activities of the unmodified HV2 and mono-PEG-HV2 (derived from liquid-phase and in situ PEGylation) were assessed by the thrombin titration method according to our previous report [28].

=

mono-PEG-HV2(mol) the total PEGylated HV2 (mono- and multi-PEG-HV2) (mol)

bioactivity retention of mono-PEG-HV2 =

(3)

bioactivity of mono-PEG-HV2 bioactivity of HV2

(4)

2.8. In vivo pharmacological efficacy analysis 2.10. Data analysis Seven groups of male New Zealand White rabbit (five rabbits per group) were randomly assigned to be injected with HV2 formulations as follows: (1) HV2, (2) mono-PEG5k-HV2 derived from liquid-phase PEGylation, (3) mono-PEG5k-HV2 derived from in situ PEGylation, (4) mono-PEG10k-HV2 derived from liquid-phase PEGylation, (5) mono-PEG10k-HV2 derived from in situ PEGylation, (6) mono-PEG20k-HV2 derived from liquid-phase PEGylation, (7) mono-PEG20k-HV2 derived from in situ PEGylation. The rabbits were administered intravenous ear vein injections of HV2 formulations (containing 0.1 mg/mL HV2), each at a dose of 0.1 mg/kg body weight. Blood samples (1 mL) were collected from the rabbit hearts before and at selected time points after administration. The blood samples were immediately placed into centrifuge tubes containing 3.8% sodium citrate (blood sample/sodium citrate = 9:1 (v/v)) and centrifuged at 5000 rpm for 15 min. The supernatants were collected and stored at −20 ◦ C until assay. The thrombin time (TT) for each sample was determined using commercially available assay kits following the manufacturer’s instructions. The assay was performed using a C2000-A Automatic Blood Coagulation Instrument (Beijing Precil Instrument Co., Ltd., China). The in vivo pharmacological efficacy of HV2 for different groups was expressed as prolongation in TT based on the normal level before injection for each rabbit. 2.9. PEGylation efficiency evaluation The PEGylation efficiency of HV2 was evaluated using the parameters of conversion of HV2, yield of mono-PEG-HV2, selec-

All of the experimental data were obtained from three independently repeated experiments and are represented as the mean ± SD unless particularly outlined. Statistical analysis was performed by one-way analysis of variance (ANOVA) and Student’s t-test using the software SPSS11.5. The significance level was set to be P < 0.05. 3. Results and discussion 3.1. Liquid-phase PEGylation of HV2 To evaluate the efficiency of in situ PEGylation of HV2, liquidphase PEGylation of HV2 was performed as a control. The reaction mixtures were obtained under relatively optimal reaction conditions according to our previous report [7]. The PEGylation degrees of different PEGylated HV2 forms (unmodified HV2, mono-PEGHV2, di-PEG-HV2 or tri-PEG-HV2) were analyzed by RP-HPLC. Moreover, the reaction mixtures were purified by anion exchange chromatography (IEC) according to our previously reported method [28] (Fig. 1). All of the elution fractions collected from IEC were analyzed by SDS-PAGE and RP-HPLC [28]. SDS-PAGE and RP-HPLC analysis indicated that peak 1 and peak 2 in Fig. 1 corresponded to unmodified HV2 and mono-PEG-HV2. The target product of monoPEG-HV2 was labeled in Fig. 1. Fractions of unmodified HV2 and mono-PEG-HV2 were efficiently separated from multi-PEG-HV2 (di-PEG-HV2 and tri-PEG-HV2). The PEGylation degree of multiPEG-HV2 decreased as the PEG size increased, and the results obtained from RP-HPLC and IEC were consistent. The collected

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mono-PEG-HV2 had a high purity based on both electrophoresis and HPLC analysis (both >95%) and could be used for subsequent characterization. 3.2. In situ PEGylation of HV2 To explore the feasibility of in situ PEGylation, HV2 was conjugated with mPEG-SC on an anion exchange chromatography column using an AKTA purifier 10 system. Moreover, the effects of different PEG sizes, ion exchange resins and reaction conditions on in situ PEGylation were systematically investigated to deeply understand this process and determine the crucial parameters. 3.2.1. Effects of different PEG sizes In situ PEGylation of HV2 with 5, 10 or 20 kDa mPEG-SC was investigated using a HiTrap Q HP column. The chromatographic process is shown in Fig. 2. All the elution fractions of PEGylated HV2 species were collected and analyzed by SDS-PAGE and RP-HPLC (Fig. 3). As shown in Fig. 3, SDS-PAGE and RP-HPLC analysis indicated that peak 1 and peak 2 in Fig. 2 corresponded to unmodified HV2 and mono-PEG-HV2. The target product of monoPEG-HV2 was labeled in Fig. 2. Peaks of different fractions at the linear gradient elution stage (elution volume from 35 to 135 mL) of in situ PEGylation were consistent with those of liquid-phase PEGylation (elution volume from 20 to 120 mL). In situ PEGylation achieved higher resolution separation of different PEGylated HV2 species than those from liquid-phase PEGylation due to the reduced formation of multi-PEG-HV2. Moreover, the collected mono-PEGHV2 had a high purity based on both electrophoresis and HPLC analysis (both > 95%) and could be used for subsequent characterization (Fig. 3). In recent reports [20,21], solid-phase PEGylation (on-column PEGylation) could only achieve the integration of PEGylation reaction and primary separation of PEGylated products using one column. Further purification using another column is required to obtain the desired mono-PEGylated product at high purity. In this study, in situ PEGylation integrated the PEGylation reaction, separation and purification of the desired mono-PEG-HV2 into a single-unit operation using the same HiTrap Q HP column. The effects of different PEG sizes on in situ PEGylation of HV2 are discussed in the subsequent section (Section 3.3). 3.2.2. Effects of different ion exchange resins Anion exchange resins were used for in situ PEGylation of HV2 because the isoelectric point of HV2 is nearly 4.0, and PEGylation with mPEG-SC is usually performed at pH > 4.0. Five columns (HiTrap Q HP, HiTrap Q FF, HiTrap Q XL, HiTrap DEAE FF and HiTrap ANX FF) were selected according to the properties of different anion exchange resins (Table 1). The effects of different ion exchange resins on in situ PEGylation of HV2 are shown in Fig. 4. The yields of mono-PEG-HV2 obtained from HiTrap Q HP, HiTrap Q FF, HiTrap Q XL, HiTrap DEAE FF and HiTrap ANX FF columns were 22.4%, 24.7%, 5.7%, 23.0% and 32.7%, respectively. Compared to the HiTrap DEAE FF column, the HiTrap ANX FF column displayed higher PEGylation efficiency probably due to its larger pore size, which probably leads to increasing internal mass transfer of mPEG-SC, reducing steric hindrance between PEG molecules and HV2 molecules [29], and thus increasing the contacts between HV2 and mPEG-SC. Compared to the HiTrap Q FF column, the HiTrap Q XL column displayed lower PEGylation efficiency, probably due to its different internal surface structures (matrix and dynamic binding capacity). HiTrap Q XL column has long dextran chains coupled to a 6% cross-linked agarose matrix and the charged ligands (quaternary amine, Q) are linked to the dextran. The dextran chains increase the exposure of charged ligands (Q), which leads to increasing dynamic binding capacity and protein density on the internal surface of the resin, and thus reducing protein accessibility to PEG [11]. Due to the flexibility

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of the long dextran chains, the ligands (Q) are able to bind to more amino groups in HV2, so more potential modification sites in HV2 are likely to be blocked. Moreover, the steric hindrance between the dextran and PEG can also prevent the PEG from contacting HV2, because both of them are flexible long- chain molecules. The HiTrap Q HP, HiTrap Q FF and HiTrap DEAE FF columns displayed similar PEGylation efficiency, although they have different particle sizes, ligands or ionic capacities. The above results indicated that the effects of pore sizes and internal surface structures of different resins on the in situ PEGylation of HV2 were more significant than the particle sizes, ligands and ionic capacities of the resins. It is worth noting that the HiTrap Q HP column, which has a smaller particle size, could achieve a higher resolution separation of PEGylated HV2 species than the other columns listed in Table 1 (data not shown). Thus, the HiTrap Q HP column might be a better choice to achieve the integration of PEGylation reaction, separation and purification of the desired mono-PEG-HV2 into a single-unit operation. 3.2.3. Effects of different reaction conditions In our previous study [7], we demonstrated that reaction conditions (e.g., pH, molar ratio of PEG to HV2 and reaction time) had significant effects on liquid-phase PEGylation of HV2. Likewise, the effects of pH, molar ratio of PEG to HV2 and reaction time on in situ PEGylation of HV2 were investigated, and the results are shown in Fig. 5. As the pH increased, the conversion and yield increased, whereas the selectivity decreased. This pattern is logical because the PEGylation rates increase as the pH increases [7]. The effect of the molar ratio of PEG to HV2 on the in situ PEGylation of HV2 displayed a trend similar to pH. Theoretically, increased conversion and yield can be achieved by further increasing the molar ratio of PEG to HV2 (>15:1). However, it would also result in a waste of mPEG-SC and increased formation of multi-PEG-HV2. Therefore, the molar ratio of PEG to HV2 should be carefully taken into consideration. As the reaction time increased, the conversion increased, while the selectivity decreased. The yield initially increased and then plateaued with increasing reaction time. After reaction for 60 min, the yield remained almost constant, but the conversion increased slightly, indicating an increased formation of multi-PEGHV2. Therefore, the optimal reaction time was 60 min. Compared to liquid-phase PEGylation of HV2 in our previous study [7], the different PEGylated HV2 forms (unmodified HV2, mono-PEG-HV2 and multi-PEG-HV2) of in situ PEGylation changed more slowly as the reaction conditions (especially, molar ratio of PEG to HV2 and reaction time), indicating that in situ PEGylation is mainly limited by the diffusion of the reactants. 3.3. Comparison between liquid-phase PEGylation and in situ PEGylation 3.3.1. Conversion, yield, and selectivity of PEGylation of HV2 Compared with liquid-phase PEGylation, in situ PEGylation achieved lower conversion and yield, but higher selectivity (Table 2). Similar results have been reported by many authors in the literatures [10,11,25], although the reasons are still not completely clear. A possible explanation is reduced reactivity due to steric hindrance between HV2 and resin. The effects of steric hindrance on in situ PEGylation are as follows: (1) when HV2 adsorbed onto the resin, some potential modification sites in the resin-binding region are prevented from reacting with mPEG-SC; (2) the mobility of HV2 adsorbed onto the resin was limited to reduce the reaction between HV2 and mPEG-SC; (3) once the HV2 molecule bound one PEG molecule, the attached PEG molecule would hinder another PEG molecule to react with the same HV2 molecule as well as the neighboring HV2 molecules adsorbed onto the resin.

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8 Table 1 Properties of different anion exchange resins.a Column

Type

Matrix

Ligand (Functional group)

Average pore size (nm)

Particle size (␮m)

Ionic capacity (mmol Cl− /mL medium)

Dynamic binding capacity (mg HSA/mL medium)

HiTrap Q HP

Strong

6% cross-linked agarose

30

34 (24–44)

0.14–0.20

70

HiTrap Q FF HiTrap Q XL

Strong

30

90 (45–165) 90 (45–165)

0.18–0.25

120

0.18–0.26

>130

HiTrap DEAE FF

Weak

6% cross-linked agarose 6% cross-linked agarose with dextran surface extenders 6% cross-linked agarose

Quaternary −Oamine (CH3 )3 N+ CH2 Quaternary amine (CH3 )3 N+ CH2 −OQuaternary amine (CH3 )3 N+ CH2 O-

30

90 (45–165)

0.11–0.16

47

HiTrap ANX FF

Weak

Diethylaminoethyl (CH3 CH2 )2 H N+ CH2 CH2 ODiethylaminopropyl (CH3 CH2 )2 H N+ CH2 CHOHCH2 O-

45

90 (45–165)

0.13–0.17

43

a

Strong

4% cross-linked agarose

30

The data were collected from the manufacturer’s (GE Healthcare) instructions.

Table 2 Comparison of the yield, conversion and selectivity between liquid-phase and in situ PEGylation of HV2.a Types of PEGylation

PEG size (kDa)

molar ratio of PEG to HV2

Reactiontime (min)

Yield (%)

Conversion (%)

Selectivity(%)

Liquid phase In situ Liquid phase In situ Liquid phase In situ

5 5 10 10 20 20

3 15 3 15 3 15

30 60 30 60 30 60

48.1 ± 1.4 36.2 ± 1.1 53.1 ± 1.2 22.4 ± 1.0 58.8 ± 1.7 19.3 ± 0.8

73.7 ± 1.8 48.1 ± 1.3 71.6 ± 1.6 26.4 ± 1.5 70.5 ± 1.4 20.3 ± 0.7

65.2 ± 2.5 75.4 ± 2.7 74.1 ± 1.8 84.8 ± 2.1 83.8 ± 3.2 95.2 ± 1.7

a Reaction conditions of liquid-phase and in situ PEGylation of HV2 were described in Fig. 1 and Fig. 2, respectively. The conversion, yield and selectivity were calculated according to Eqs. (1)–(3) in Section 2.9, respectively.

In addition to steric hindrance, another possible reason is the slow pore-diffusion velocity of mPEG-SC into the internal pores of the resin. For liquid-phase PEGylation, the HV2 and PEG were fully mixed, and the diffusion of the reactants could be ignored. Thus, liquid-phase PEGylation is defined as a reaction-controlled process. For in situ PEGylation, HV2 molecules were pre-adsorbed onto the internal pore surfaces of the resin (Fig. 6B), and then PEG molecules diffused into these regions of the resin (microsphere) and contacted adsorbed HV2 molecules (Fig. 6C). Because the hydrodynamic radius of the HV2 molecule is much smaller than the pore size of the resin, HV2 quickly diffuses into the resin and is adsorbed onto the internal pore surfaces of the resin throughout (Fig. 6B). However, the PEG molecule is 5-to-10 fold as large as a protein with equivalent molecular weight, due to both the binding capability to water molecules (2–3 water molecules bound per ethylene oxide unit) and the high flexibility of the long chain [30]. Therefore, mPEG-SC molecules diffuse into the resin much more slowly, and not completely enter to the center region of the resin (microsphere), resulting in unmodified HV2 molecules in this region (Fig. 6C). In addition, PEGylated HV2 species formed near the pore entrance might hinder the diffusion of mPEG-SC into the pore (Fig. 6D). For the above reasons, the diffusion of mPEG-SC into the resin becomes a limiting step, thus in situ PEGylation is defined as a pore diffusion-controlled process. As shown in Table 2, the conversions of both liquid-phase and in situ PEGylation decreased as PEG size increased, whereas their selectivities increased. However, the effect of PEG size on the yield of in situ PEGylation displayed an opposite trend to that of liquidphase PEGylation. The yield of liquid-phase PEGylation increased as PEG size increased, whereas the yield of in situ PEGylation decreased. The result of liquid-phase PEGylation was consistent with that of the PEGylation kinetics obtained in our previous study [7]. Likewise, the in situ PEGylation was similar to the previously reported results concerning solid-phase or on-column PEGylation

[10,25]. The decreased yield of in situ PEGylation could be explained by increased steric hindrance and reduced pore-diffusion velocity of mPEG-SC inside the resin with the increasing PEG size (Fig. 6C and D). 3.3.2. In vitro and in vivo bioactivity of mono-PEG-HV2 The in vitro anticoagulant activities of the mono-PEG-HV2 derived from liquid-phase and in situ PEGylation were evaluated by the thrombin titration method (Fig. 7). Compared to unmodified HV2, the in vitro anticoagulant activities of the mono-PEG-HV2 decreased due to the active sites of HV2 shielded by the binding PEG molecules. The greater the PEG size is, the stronger the steric hindrance is. The in vivo pharmacological efficacy of the mono-PEGHV2 was evaluated by thrombin time (TT) prolongation in rabbits (Fig. 8). After the injection of unmodified HV2, mono-PEG5k-HV2, mono-PEG10k-HV2 and mono-PEG20k-HV2, their TT prolongation reached a maximal level after 5, 5, 10, and 10 min, respectively. Additionally, the TT returned to the normal level after 40, 40, 120, and 180 min, respectively. For mono-PEG5k-HV2, the maximal level of TT prolongation decreased compared with unmodified HV2, although TT prolongation dropped slowly from the maximal level to normal level. This indicated that mono-PEG5k-HV2 did not have significantly enhanced in vivo pharmacological efficacy. Compared to unmodified HV2, the maximal level and action time of TT prolongation increased for both mono-PEG10k-HV2 and monoPEG20k-HV2, and thus, the in vivo pharmacological efficacy was significantly enhanced. These results are similar to the previous reports concerning other mono-PEGylated proteins [31]. Interestingly, mono-PEG-HV2 derived from in situ PEGylation displayed significantly higher in vitro and in vivo bioactivities than those derived from liquid-phase PEGylation (Figs. 7 and 8). Similar results have been reported in some cases of solid-phase PEGylation [10,24]. A potential explanation for solid-phase PEGylation is the site-selective, in another words, some active sites of the protein

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9

near disulfide bridges (C16-C28), and the optimum site for PEGylation is Lys 35 located in a prominent loop [33]. To experimentally confirm our speculation, the identification of modified sites and mono-PEG-HV2 isoforms should be taken into consideration in future studies.

3.4. Strategies for improving the yield of mono-PEGylated form of in situ PEGylation In this study, the yield of mono-PEG-HV2 obtained from in situ PEGylation is lower than that of liquid-phase PEGylation. Future work will be focused on improving the yield of the desired monoPEG-HV2. Possible improvement strategies are summarized as follows:

Fig. 8. Comparison of the in vivo pharmacological efficacy of mono-PEG-HV2 obtained from liquid-phase and in situ PEGylation of HV2. (A) PEGylation of HV2 with 5 kDa mPEG-SC; (B) PEGylation of HV2 with 10 kDa mPEG-SC; (C) PEGylation of HV2 with 20 kDa mPEG-SC. The in vivo pharmacological efficacy of mono-PEG-HV2 was evaluated by the thrombin time (TT) prolongation versus time after intravenous injection of different HV2 formulations (HV2, mono-PEG-HV2 obtained from liquidphase PEGylation and mono-PEG-HV2 obtained from in situ PEGylation) in five rabbits at doses of 0.1 mg/kg. Each point was represented as the mean ± SD (n = 5). The enlarged parts on the right top of each figure are used for clearer illustration.

towards the solid media are protected from reacting with PEG. The effect of solid-phase PEGylation on the distribution of the monoPEGylated isoforms has been confirmed by previous reports [10,11]. In this study, HV2 contains two functional domains, i.e. a compact NH2 -terminal domain (N-domain) stabilized by three disulfide bonds and a disordered COOH-terminal tail (C-domain) [32]. It has four active lysine residues (Lys 24, Lys 27, Lys 35 and Lys 47) available for potential PEGylation. During the in situ PEGylation of HV2 at pH 8.0, the C-domain, containing abundant acidic amino acids (negatively charged), might bind to the functional groups of the anion exchange resin by static interactions like in thrombin/HV2 complex [33]. Lys 24 and Lys 47 are likely buried by the electrostatic interactions between the HV2 and the resin. Lys 27 and Lys 35 are relatively free and likely to be modified by mPEG-SC. Therefore, mono-PEG-HV2 obtained from in situ PEGylation of HV2 could achieve higher bioactivity than liquid-phase PEGylation. This speculation was supported by calculating the solvent accessible surface area (SASA) of lysine residues of HV2 using molecular dynamics simulation in our previous study [33]. PEGylation at Lys 27 would reduce the anticoagulant activity of hirudin because it is located

(1) Selection of a more suitable anion exchange chromatography column. The pore size of the resin is a crucial parameter because in situ PEGylation is a diffusion-controlled process (Figs. 4 and 6). To promote the faster diffusion of large PEG molecules into the resin, a larger pore size resin is required. Recently, a novel gigaporous ion exchange media with a 100 nm level pore size have been developed for the rapid mass transfer of large biomolecules [34,35]. For example, the mass transfer of mono-PEG30k-G-CSF into the SP-GP resin (100–500 nm pore size) was much faster than that into SP-6FF resin (30 nm pore size) [35]. Theoretically, the yield of mono-PEG-HV2 would significantly increase if in situ PEGylation was performed using such a gigaporous ion exchange media. To avoid diffusion limitations in particles, monolithic media was recommended for solid-phase PEGylation by several authors [11,25]. However, the expected high yield was not achieved [11]. In addition to pore size, other properties of the media (e.g., ligand density, particle size and protein binding capacity) should be taken into consideration simultaneously. (2) Optimization of reaction conditions. From the view of process optimization, the maximum yield of mono-PEG-HV2 was not achieved in the selected range of different factors, although the effects of pH, molar ratio of PEG to HV2 and reaction time on in situ PEGylation of HV2 were investigated. Therefore, these factors (especially, molar ratio of PEG to HV2) should be further optimized. It is worth noting that pH can also affect the adsorption orientation of HV2 onto the resin, and thus affect the isoform distribution and bioactivity of mono-PEG-HV2, which should be considered more carefully. Other reaction conditions (e.g., ionic strength of buffer solution, protein loading and flow rate of PEG) should also be further optimized. Considering the interactions of different factors, bioprocess optimization methods such as response surface analysis might be a better choice than a single factor experiment for future optimization. (3) Recycling of the unmodified protein. In this study, the fractions of unmodified HV2 and mono-PEG-HV2 were efficiently separated from multi-PEG-HV2 during a single batch in situ PEGylation (Fig. 2). This result indicated that the total yield of mono-PEG-HV2 could be improved after repeated recycling of unmodified HV2. Interestingly, a continuous multi-column chromatographic process was developed to recycle the unmodified protein [27]. In this process, the yield and productivity of mono-PEGylated protein (85% and 0.9 mg/h) were significantly increased compared to the batch on-column process (44% and 0.3 mg/h) at the equivalent purity of mono-PEGylated protein (95%).

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4. Conclusions To enhance the homogeneity and bioactivity retention of mono-PEG-HV2, an in situ PEGylation of HV2 was developed on anion exchange chromatography columns. In situ PEGylation could achieve the integration of PEGylation reaction, separation and purification of the desired mono-PEG-HV2 into a single-unit operation using the same column. In the in situ PEGylation, both the pore size and internal surface structure of different resins had significant impact on the yield of mono-PEG-HV2. Unlike reactioncontrolled liquid-phase PEGylation, in situ PEGylation was a pore diffusion-controlled process, in which the yield of mono-PEG-HV2 decreased as PEG size increased. Compared to liquid-phase PEGylation, in situ PEGylation could improve the selectivity of PEGylation, reduce the formation of multi-PEGylated conjugates, and enhance the in vitro and in vivo bioactivity of mono-PEG-HV2. Future work will focus on improving the yield of the desired mono-PEGylated form derived from in situ PEGylation and promoting this technology for the development of PEGylated protein drugs. Acknowledgments This research was financially supported by the National Natural Science Foundation of China (Grant No. 81072590 and 21606038) and the Jiangsu Province State Key Laboratory Cultivating Base for Long-acting Bio-medical Research of Jiangsu Hansoh Pharmaceutical Group CO., LTD. The authors would like to thank Prof. Zhiguo Su of Institute for Processing Engineering, Chinese Academy of Sciences for his thoughtful suggestions and discussions. References [1] A. Greinacher, T.E. Warkentin, The direct thrombin inhibitor hirudin, Thromb. Haemost. 99 (2008) 819–829. [2] W.E. Dager, R.H. White, Treatment of heparin-induced thrombocytopenia, Ann. Pharmacother. 36 (2002) 489–503. [3] T.E. Warkentin, Bivalent direct thrombin inhibitors: hirudin and bivalirudin, Best Pract. Res. Clin. Haematol. 17 (2004) 105–125. [4] D. Pfister, M. Morbidelli, Process for protein PEGylation, J. Controll. Release 180 (2014) 134–149. [5] M. Lopez, A. Koehler, G. Nowak, Biochemical and pharmacokinetic characterisation of two PEGylated variants of dipetarudin, Thromb. Haemost. 102 (2009) 454–459. [6] G.C. Avgerinos, B.G. Turner, K.J. Gorelick, A. Papendieck, U. Weydemann, G. Gellissen, Production and clinical development of a Hansenula polymorpha-derived PEGylated hirudin, Semin. Thromb. Hemost. 27 (2001) 357–371. [7] X.D. Wang, J.J. Hu, D.T. Pan, H. Teng, Z.L. Xiu, PEGylation kinetics of recombinant hirudin and its application for the production of PEGylated HV2 species, Biochem. Eng. J. 85 (2014) 38–48. [8] B.B. Hou, S.R. Li, X.H. Li, Z.L. Xiu, Design, preparation and in vitro bioactivity of mono-PEGylated recombinant hirudin, Chin. J. Chem. Eng. 15 (2007) 775–780. [9] B.K. Lee, J.S. Kwon, H.J. Kim, S. Yamamoto, E.K. Lee, Solid-phase PEGylation of recombinant interferon alpha-2a for site-specific modification: process performance, characterization, and in vitro bioactivity, Bioconjug. Chem. 18 (2007) 1728–1734. [10] J. Wang, Y.J. Wang, T. Hu, X.N. Li, Y.D. Huang, Y.D. Liu, G.H. Ma, Z.G. Su, An oriented adsorption strategy for efficient solid phase PEGylation of recombinant staphylokinase by immobilized metal-ion affinity chromatography, Process Biochem. 47 (2012) 106–112. [11] B. Maiser, K. Baumgartner, F. Dismer, J. Hubbuch, Effect of lysozyme solid-phase PEGylation on reaction kinetics and isoform distribution, J. Chromatogr. B 1002 (2015) 313–318.

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