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REGULAR ARTICLE Large-scale preparation of thrombin from human plasma☆ Peter Aizawa, Stefan Winge, Göran Karlsson Octapharma AB, SE-11275 Stockholm, Sweden Received 27 August 2007; received in revised form 18 December 2007; accepted 27 December 2007 Abstract Thrombin was prepared from human blood plasma (batch size 1200 L). First, prothrombin was isolated by the following separation techniques: cryoprecipitation, ion-exchange chromatography (diethyl aminoethyl, DEAE-IEX), heparin affinity chromatography, a second DEAE-IEX step, and immobilized metal-affinity chromatography (IMAC). Prothrombin was then activated to thrombin, which was purified by hydrophobic interaction chromatography (HIC) and concentrated by ultrafiltration. This process is cost-effective because a waste fraction can be used from one of the steps (heparin affinity chromatography) in the commercial production of plasma-derived Factor IX (FIX). The final thrombin preparation had a purity of approximately 75% (specific activity approximately 2400 IU/mg protein), which is sufficient for its intended purpose in a fibrin glue. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Factor X (FX) activity analysis, and analytical HIC were also used to characterize the thrombin. Three substantially different techniques were used to reduce any viral activity, namely: solvent/detergent (S/D) treatment, pasteurization, and virus filtration (nanofiltration). The manufacturing process presented here would be suitable for large-scale production of thrombin with a high degree of virus safety. © 2008 Elsevier Ltd. All rights reserved. KEYWORDS Thrombin;Prothrombin;Fibrin glue;Fibrin sealant Abbreviations: APC, activated protein C; DEAE, diethyl aminoethyl; ELISA, enzyme-linked immunoabsorbent assay; FXIII, Factor XIII (other coagulation factors are abbreviated in the same way); GFC, gel filtration chromatography; HIC, hydrophobic interaction chromatography; HPLC, high-performance liquid chromatography; HSA, human serumalbumin; IDA, iminodiacetic acid; IEX, ion-exchange chromatography; IMAC, immobilized metal-affinity chromatography; MW, molecular weight; PCR, polymerase chain reaction; S/D, solvent/detergent; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; Tris, tris(hydroxymethyl) aminomethane. ☆ This work was initiated by the Plasma Products R&D Section, at Pharmacia AB (Stockholm), which was later spun off and integrated with Biovitrum AB (Stockholm). The purification process presented here was part of two patent applications by Biovitrum AB as the applicant, before PlasmaProducts R&Dwas sold to OctapharmaAB. The authors of this paper are also the inventors of the published patent applications. ⁎ Corresponding author. Tel.: +46 8 56643000. E-mail address: goran.karlsson@octapharma.se (G. Karlsson). intl.elsevierhealth.com/journals/thre 0049-3848/$ - see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2007.12.027 Thrombosis Research (2008) xx, xxx–xxx TR-03349; No of Pages 8 Please cite this article as: Aizawa P, et al, Large-scale preparation of thrombin from human plasma, Thromb Res (2008), doi:10.1016/ j.thromres.2007.12.027 Introduction Thrombin activates fibrinogen, FV, FVIII, FXI, and FXIII, initiates platelet secretion and aggregation, and is inhibited by antithrombin [1,2]. In addition to its ability to activate coagulation, thrombin can produce feedback inhibition of coagulation by binding to thrombomodulin, which then activates protein C, in a complex with protein S, to form activated protein C (APC), which finally cleaves and inactivates FVa and FVIIIa [3]. Thus, thrombin plays a crucial role in the hemostasis process. Human thrombin exists in three forms: α-, β-, and γ-thrombin [1]. Of these, α-thrombin is the enzymatically active form in coagulation. α-Thrombin consists of two polypeptides, the A and B chains, which are linked by a disulfide bridge to give a total molecular weight of 37 kDa. By autolytic cleavage, α-thrombin is converted to β-thrombin, and subsequently to γ-thrombin. The β- and γ-thrombin forms have no known physiological function and exhibit very reduced clotting activities compared to α-thrombin (b1%), though they retain most of their activities towards chromogenic peptide substrates [4]. Unless otherwise indicated, in this work, α-thrombin is referred to as thrombin. In human plasma, thrombin exists as the inactive form prothrombin, which may be converted to the active form thrombin by enzymatic cleavage. Cryoprecipitation of plasma is widely used as the initial step during manufacturing of plasma-derivatives, and is followed by several chromatography steps. In addition to conventional ion-exchange chromatography (IEX) and gel filtration chromatography (GFC) [1,5,6], thrombin has been successfully purified by heparin- [5], benzamidine- [7], hydrophobic- affinity chromatography [8], and also by hydrophobic interaction chromatography (HIC), using a column with bound phenyl groups [9]. Furthermore, prothrombin has been separated from other plasma proteins by HIC [10] and immobilized metal-affinity chromatography (IMAC) [11]. For the purpose of patient safety and to reduce the risk of viral contamination of the preparations, manufacturers of plasma-derived products are recommended to use a multiple step strategy, including: testing of blood donors, polymerase chain reaction (PCR) analyses of plasma pools, and virus removal and inactivating steps, in the production process [12]. For the intended use of thrombin as a fibrin glue the presented process was developed for large-scale preparation of thrombin from human plasma, based on two patent applications [13,14]. The preparation procedure may also be used for a wider range of clinical thrombin applications [15]. Materials and methods The chromatographic system, a BioProcess system, and all chromatographic gels (diethyl aminoethyl (DEAE) Sepharose Fast Flow (FF), Chelating Sepharose FF, Heparin Sepharose FF, and Butyl Sepharose FF) were obtained from GE Healthcare (Uppsala, Sweden). Thrombin that is included in the commercial fibrin glue products Quixil and Tisseel, was purchased from Omrix Biopharmaceuticals (Tel-Hashomer, Israel) and Baxter-Immuno (Vienna, Austria), respectively, and used for comparison. An outline of the prothrombin preparation, activation to thrombin, and final thrombin purification is presented in Fig. 1. The initial process steps are part of the regular manufacturing process for a commercial FIX product (Nanotiv) [16]. A waste fraction (flow-through) from the heparin affinity chromatography step is used for further preparation of thrombin. Table 1 shows the operative parameter details for all five chromatographic steps in the thrombin preparation. Preparation of prothrombin and a FX-rich fraction Frozen blood plasma (1200 L) was thawed and the resulting cryoprecipitate was removed by centrifugation. The resulting cryosupernatant was passed through a DEAE gel (DEAE No. 1). The eluate was concentrated by ultrafiltration and treated by the solvent/detergent method (S/D) to inactivate lipid-enveloped viruses [17]. Subsequently, the S/D chemicals were extracted and the aqueous phase was filtered, diluted, and applied to a Heparin Sepharose FF column, and the flow-through fraction (containing prothrombin and FX) was collected [13,16]. Purification of prothrombin The prothrombin fraction was passed through a DEAE Sepharose FF column (DEAE No. 2), to which prothrombin and FX became bound, while the remaining S/D chemicals passed through. Prothrombin and FX were then eluted and further purified by IMAC, using Chelating Sepharose FF, with iminodiacetic acid (IDA) as the functional ligand, which had been previously charged with cupric ions (Cu2+). The collected prothrombin and FX-containing fraction were concentrated and stored at −70 °C [13]. Activation of prothrombin and purification of thrombin The ultrafiltered prothrombin fraction was activated to thrombin by adding sodium citrate to a final concentration of 950 mM (pH 8.0) and incubated for 18 h at 44 °C. The activation of prothrombin was optimized, based on previous works [18–20], which included tests of different citrate and protein concentrations, different incubation times and temperatures, and additions of calcium and polyethylene glycol (PEG) 4000 (results not shown). The method presented here was selected based on the high yield obtained, and the use of relatively short incubation times [14]. Pasteurization was used to inactivate heat-labile viruses, which was followed by further purification of thrombin by HIC. Viral safety was further enhanced by virus filtration. The thrombin preparation was ultrafiltrated and concentrated in formulation buffer (5% (w/w) sucrose, 0.15 M NaCl, 0.15 M Arg, 0.15 M Lys and 0.15 M Gly, pH 7.0) to 250,000 IU/mL, and finally frozen at −70 °C [14]. Analytical methods used to characterize thrombin Novex 10% NuPAGE Bis–Tris 1 mm gels from Invitrogen (Carlsbad, CA, USA) were used for non-reducing SDS-PAGE [21], followed by silver staining [22]. The clotting activity of thrombin was determined according to Clauss [23]. FX activity was determined by the chromogenic substrate method [24]. Prothrombin activity was measured as thrombin activity, after activation of prothrombin to thrombin with snake venom (ecarin), by a chromogenic substrate method [25]. The purity of α-thrombin was determined by analytical HIC [26]. Protein concentration wassteps and, in the final step, measured according to Peterson [27], based on the Lowry method [28]. Results Prothrombin was harvested from plasma by DEAE-IEX (No. 1), together with several coagulation factors (including prothrombin, FIX, and FX), and heparin affinity chromatography was then performed, where prothrombin and FX were collected in the nonbinding (flow-through) fraction (Fig. 2). Both of these steps are included in the regular manufacturing process of commercial FIX. Two purification steps were added for further purification of prothrombin. The first involved IEX, DEAE (No. 2), which was similar to the previous DEAE step, but optimized for prothrombin/ FX purification and for removal of S/D chemicals. The concentrations of the remaining TNBP and Triton X-100 in the IEX eluate were b1 μg/mL and b2.5 μg/mL, respectively (results not shown). The second step was IMAC, using chelating Sepharose FF, where prothrombin and FX were separated from more highmolecular weight proteins and co-eluted in the flow-through fraction (Fig. 3), as demonstrated by the SDS-PAGE of collected fractions (results not shown). Following activation of prothrombin to thrombin, using a high concentration of citrate during incubation, HIC was used for the final purification of thrombin (Fig. 4). The results fromthe preparation of thrombin are summarized in Table 2,which shows an increasing specific activity of prothrombin, thrombin, and FX in the purification process. Analytical HIC, which separates α-thrombin from several other thrombin forms and preforms (β-thrombin, γ-thrombin, prothrombin, prethrombin 1, and prethrombin 2), is therefore useful for thrombin purity determination [26]. The results fromanalytical HIC (Fig. 5 and Table 2) showa clear increase in the α-thrombin concentration after the preparative HIC step in the production process. The SDS-PAGE results (Fig. 6) also indicate an increase in the α-thrombin content, and a decrease in β-thrombin after the HIC step. An almost identical SDSPAGE migration pattern was obtained before and after the pasteurization step, indicating the absence of protein aggregation. Figure 3 Purification of prothrombin (batch Thromb 4). A) Ion-exchange chromatography (DEAE No. 2). Prothrombin and FX-rich fraction was collected in the eluate. B) Immobilized metal-affinity chromatography (IMAC). Prothrombin and FX-rich fraction were collected in the flow-through. Maximum absorbance was reached in A during the elution. Figure 2 Initial preparation of prothrombin (batch Thromb 4). A) Ion-exchange chromatography (DEAE No. 1) of the cryoprecipitation supernatant. Prothrombin was collected in the eluate together with several other coagulation factors. B) Heparin affinity chromatography. The prothrombin and FX-rich fraction was collected in the flow-through. Maximum absorbance was reached in both A and B. Figure 4 Purification of thrombin (batch Thromb 4) by hydrophobic interaction chromatography (HIC). Thrombin was collected in the eluate. The bands below the reference α-thrombin main band (Fig. 6) are assumed to be β- and γ-thrombin, based on known molecular weights of the non-reduced chains of β-thrombin (27 and 9 kDa), and γ-thrombin (three chains, all less than 13 kDa) [29,30]. An enzyme-linked immunoabsorbent assay (ELISA) of the purified thrombin, showed an activity–antigen ratio close to 1.0, indicating that almost all of the thrombin was biologically active. Moreover, the activity of the thrombin preparation was maintained after storage for six months at −70 °C (results not shown). Two thrombin preparations, included in commercial fibrin glue products, were compared with two batches prepared with the new process (Thromb 3 and 4). The calculated specific activities of Thromb 3 and 4 were substantially higher than that of the commercial thrombin samples, though this is explained by the addition of human serum albumin (HSA) to the commercial products, which significantly decreases the specific activity (Table 3). The final specific activity of the thrombin preparation was approximately 2400 IU/mg protein (Table 3), which corresponds to approximately 75% purity [1], and the total thrombin recovery was approximately 20% (Table 2). The thrombin purity, as determined by analytical HIC, was higher than that of the Tisseel thrombin sample. Quixil gave an unexpected high purity of thrombin (100%), as determined by analytical HIC (Table 3 and Fig. 5). Therefore, HSA alone was analyzed by analytical HIC and found to elute in the void (results not shown). The large void peak, which was excluded from the total integrated area in the HIC analysis of the Quixil thrombin sample, thus contained HSA, which was also confirmed in the Tisseel thrombin sample. Discussion A manufacturing process for thrombin has been developed. The initial steps in the process are part of the regular manufacturing process for a commercial FIX product (Nanotiv, Octapharma AB, Stockholm) with a waste fraction (flow-through) from the heparin affinity chromatography step being used for preparing thrombin. This approach, that extracts several different proteins from the same starting material (human plasma) during the preparation of biopharmaceuticals, is a cost-effective way of developing new products. By using a waste fraction, the manufacturing process for the original product does not need to be altered. Moreover, the original process does not need to be revalidated, or reapproved by the regulatory authorities. The process has one minor disadvantage; however, since the total number of purification steps is relatively high (five steps), due to the first two steps being optimized for FIX purification, with the additional purification steps for purification of prothrombin and thrombin. In the described process, by the IMAC step, many of the high-molecular weight proteins are removed, which otherwise would have prolonged the thrombin activation time. From the initial steps in this process, prothrombin and FX are co-purified, which facilitates the subsequent activation to thrombin, since FX is required in the activation process. Other thrombin forms, such as β- and γ-thrombin, generated by autolysis of α-thrombin in the activation process [1,2,29,30], and pre-forms of α-thrombin, such as prothrombin, prethrombin 1, and prethrombin 2, are normally separated fromα-thrombin in the following steps. By introducing HIC in the thrombin purification process, effective purification was possible without needing to remove the high citrate content that emanated from the activation and pasteurization. The analytical separation of different thrombin forms by HIC has been described [26], and this method was used as a tool in developing the described process. The obtained thrombin purity was approximately 75%, which is sufficient for use in fibrin glue and is similar to that used in commercial fibrin glue preparations [7]. Three virus-reducing techniques, based on different principles, were employed to ensure a high degree of viral safety. Two of the most widely used methods for virus inactivation were included: S/D treatment and pasteurization. In the S/D procedure, developed by Horowitz et al. [17], enveloped viruses, such as human immunodeficiency virus, hepatitis B, and hepatitis C viruses, are inactivated. With pasteurization, several viruses, which are not lipid-enveloped, are inactivated [12]. In addition, virus filtration (nanofiltration) was included as a complementary procedure to increase the viral safety. Both enveloped and non-enveloped viruses can be removed/reduced by virus filtration, which is based on size exclusion. In contrast, S/D treatment is ineffective against non-enveloped viruses, and some viruses, which may be either enveloped or non-enveloped, are relatively heat-resistant. Virus filtration was originally introduced to remove nonlipid-enveloped viruses (e.g. parvovirus B19 and hepatitis A virus) and to provide protection from unknown infectious agents [31]. Currently, plasma-derived products are usually subjected to one virus inactivation step (S/D or pasteurization) coupled to an additional virus reduction step (dry heat or nanofiltration) [12]. 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