Biologicals xxx (2014) 1e11
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Review
Quantifying the thrombogenic potential of human plasma-derived immunoglobulin products W.A. Germishuizen a, D.C. Gyure a, D. Stubbings a, T. Burnouf b, * a b
National Bioproducts Institute, Pinetown, South Africa Graduate Institute of Biomedical Materials and Tissue Engineering, Taipei Medical University, 250 Wuxing St., Taipei City 110, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 January 2014 Received in revised form 24 April 2014 Accepted 29 April 2014 Available online xxx
Polyvalent immunoglobulin G (IgG) products obtained by fractionation of human plasma are used to treat a broad range of conditions, including immunodeficiency syndromes and autoimmune, inflammatory, and infectious diseases. Recent incidences of increased thromboembolic events (TEEs) associated with intravenous (IV) IgG (IVIG) led to recalls of some products and increased regulatory oversight of manufacturing processes in order to ensure that products are essentially free of procoagulant/thrombogenic plasma protein contaminants. Laboratory investigations have now identified activated factor XI (FXIa) as the likely causative agent of IVIG-related TEEs. Quantification of the thrombogenic potential is becoming a requirement made to fractionators (a) to validate the capacity of IVIG and subcutaneous IgG manufacturing processes to remove procoagulant contaminants and (b) to establish the safety of the final products. However, in the absence of a recommended test by the main regulatory authorities, several analytical approaches have been evaluated by fractionators, regulators, and university groups. This review focuses on the scientific rationale, merits, and applications of several analytical methods of quantifying the thrombogenic potential of IgG products and intermediates to meet the latest regulatory requirements. © 2014 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Keywords: Thrombosis IVIG SCIG FXIa Procoagulant
1. Introduction Human plasma-derived intravenous and subcutaneous polyvalent immunoglobulin G (IgG, IVIG/SCIG) medicinal products are immunomodulatory agents used to treat primary and secondary immunodeficiency syndromes and a variety of autoimmune, inflammatory, infectious, transplantation-related, and chronic diseases such as idiopathic thrombocytopenic purpura, chronic syninflammatory demyelinating polyneuropathy, Guillain-Barre drome, and Kawasaki disease [1,2]. IgG products are now the leading therapeutic products made from plasma and contribute to over 50% to sales of all fractionated plasma products. As they are prepared from plasma collected from a high number of healthy blood donors (1000e40,000), they contain a broad and diverse spectrum of polyclonal humoral antibodies resulting from the cumulative exposure of the donor population to the environment [3]. Therefore, IVIG and SCIG preparations recognize a broad range of bacterial, viral, and other infectious agent antigens, and also a large number of self-antigens [4]. In general, IVIG and SCIG products are
* Corresponding author. E-mail address: [email protected] (T. Burnouf).
considered safe and effective. Some common mild to moderate adverse events include a low-grade fever, headaches, malaise, nausea, and myalgia [1]. Less common but serious and potentially fatal adverse events include thromboembolic events (TEEs), acute renal failure, anaphylaxis, hemolysis, and aseptic meningitis [3,5]. Recently, the occurrence of transfusion-related acute lung injury (TRALI) following an IVIG infusion was described [6,7]. A process for the large-scale isolation of IgG from human plasma using cold-ethanol and pH fractionation steps was first developed by Cohn and co-workers in the 1940s [8], and later refined by Kistler and Nitschmann to improve the yield and purity while reducing the requirement for ethanol [9]. The modern multistep IVIG production process includes fractionation of plasma, protein purification by precipitation and/or chromatography, reduction (inactivation or removal) of blood-borne viruses, and stabilization of the IgG fraction [10]. Growing clinical demands for immunomodulatory therapies have prompted further process improvements in the last few years [3,10] to avoid some or all typical ethanol fractionation steps, increase IgG recovery, improve viral safety, replace the traditional low pH/pepsin used to reduce anticomplementary activity, and improve viral safety with more efficient procedures. However, these modifications may have caused unintentional co-purification of contaminant plasma proteins and
http://dx.doi.org/10.1016/j.biologicals.2014.04.002 1045-1056/© 2014 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
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Abbreviations Apo-H aPTT CAP CHMP ELISA FDA FXI FXIa IEC IgG IM IMIG IV IVIG
apolipoprotein H activated partial thromboplastin time caprylic acid Committee for Medicinal Products for Human Use enzyme-linked immunosorbent assay United States Food and Drug Administration factor XI activated factor XI ion exchange chromatography immunoglobulin G intramuscular intramuscular immunoglobulin G intravenous intravenous immunoglobulin G
inadvertent contact activation of hemostasis zymogen proteins such as coagulation factor XI (FXI), leading to increased TEEs, especially when combined with recent increases in infused doses of IVIG products for immunomodulatory therapies. In 2010, Octapharma Pharmazeutika (Vienna, Austria) altered the manufacturing process of Octagam® 5% in a way that unintentionally led to a higher-than-normal number of reports of serious TEEs. The company recalled 31 lots from the US market in August 2010, and all lots from the European market in September 2010 [11]. Following suspension of marketing authorization in Germany and Sweden, the US Food and Drug Administration (FDA) and the Committee for Medicinal Products for Human Use (CHMP) recommended suspension of all marketing authorization across the US [12] and European Union (EU) [13,14]. A detailed investigation of the process identified activated FXI (FXIa) as the likely cause of the unusually high rate of TEEs [11,15e18] with kallikrein (KK) considered to have played a minor role [11]. Critical steps in the manufacturing process could explain the presence of factors that triggered TEEs. Corrective measures in Octagam®'s manufacturing process were implemented to reduce FXIa-related procoagulant activity [11,13,14], including minimizing the FXIa level by adsorption upstream in the manufacturing process and performing quality control tests on final batches for the absence of FIXa prior to release on the market. The suspension of the marketing authorization for Octagam® was lifted in May 2011 [14,15]. Around the same time, Omr-IgG-am®, an IVIG product by Omrix Biopharmaceuticals (Kiryat-Ono, Israel) was recalled from the Israeli market due to a similar increase in TEE occurrence [1]. TEEs, including strokes and pulmonary emboli, were also observed after use of the subcutaneous immunoglobulin, Vivaglobin® (CSL Behring GmbH, Marburg, Germany), in 2011 [15,19]. Such product recalls from the EU and US and increased awareness of TEE-related risks of IVIG, prompted manufacturers and regulatory agencies to scrutinize existing IVIG manufacturing processes for their capacity to generate thrombogenic substances [11]. The US FDA and other regulatory agencies analyzed data associating IVIG/SCIG use and risks of thrombosis [20]. The European Pharmacopoeia (Ph. Eur.) publication, Human Normal Immunoglobulin for Intravenous Administration, Monograph 0918, was revised [15,21] to state that products should exhibit no thrombogenic (procoagulant) activity as follows: “The method of preparation also includes a step or steps that have been shown to remove thrombosis-generating agents. Emphasis is given to the identification of activated coagulation factors and their zymogens and process steps that may cause their activation. Consideration is also
KK NaPTT NIBSC PEG PEU Ph. Eur. PKA PTC SC SCIG S/D TEE TGA TRALI TTP
kallikrein non-activated partial thromboplastin time National Institute for Biological Standards and Control polyethylene glycol plasma equivalent units European Pharmacopoeia prekallikrein activator peak thrombin concentration subcutaneous subcutaneous immunoglobulin G solvent/detergent thromboembolic event(s) thrombin-generation assay transfusion-related acute lung injury time to peak
to be given to other procoagulant agents that could be introduced by the manufacturing process.” Manufacturers were given until 1 July 2012 to comply. The Ph. Eur. publication, Human Normal Immunoglobulin, Monograph 0338, was also updated following the revision of Monograph 0918 [22], with implementation on 1 January 2013. In September 2013, the US FDA instructed IVIG manufacturers to add information describing the risk of thrombosis and strategies to mitigate the risk, to the boxed warning. Similar instructions were given for SCIG and intramuscular (IM) IgG products [23]. This implies that various steps in the IVIG manufacturing process should be demonstrated to have effectively contributed to eliminating any thrombogenic potential leading to an inherently safe therapeutic product. For instance, the capacity for eliminating FXI/FXIa can be evaluated through spiking experiments using purified FXI. However, there are currently no officially approved methods for such assessments in revised monographs. While the US FDA promotes the use of a thrombin-generation assay (TGA), research papers have combined several analytical methods to quantify the thrombogenic potential associated with IVIG manufacturing processes and products, including the non-activated partial thromboplastin time (NaPTT), TGA, and FXIa chromogenic assays. Harmonization of test methods is needed to obtain congruent results, and a formal recommendation for batch release testing still needs to be included in the revised monographs. The objective of this review article is to describe a general approach to quantifying the thrombogenic risks associated with IVIG and SCIG products and manufacturing processes. The clinical context of TEEs associated with IVIG and recent changes to IVIG manufacturing technology that might have influenced the thrombogenic potential are discussed, as are measures taken by the fractionation industry in response to the revised regulatory requirements. Lastly, the merits and applications of various relevant analytical methods in quantifying the thrombogenic potential of IVIG products are evaluated in detail. 2. Background 2.1. TEE risks associated with IVIG products Despite their effectiveness and good safety record, the use of IVIG products is associated with adverse events including but not limited to aseptic meningitis, neutropenia, anaphylaxis, and less commonly observed TEEs, particularly in patients with other risk factors that contribute to thromboses. TEEs due to IgG products are
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likely to be acute, occurring either during administration or within 24 h of administration. A retrospective analysis of data from a large health claims-related database [1], and post-marketing surveillance over the period of 2008e2010 determined an average sameday TEE incidence rate of ~1%, (122 of 11,785 individuals exposed to IgG products), with rates reaching 2% for specific products. Venous same-day TEEs were more common than arterial TEEs, and TEE rates on the next day were much lower than on the same day as the infusion. Thus the first IgG dose may be the most critical with respect to developing TEEs. In a follow-up study of same-day TEE incidence over the period of 2008e2012, the authors found that 233 out of 14,944 individuals exposed to IgG products (~1.5%) experienced same day TEE's [24]. In a study by the Paul Ehrlich Institute [15], frequencies of TEEs associated with the use of 4 different IVIG products and 2 SCIGs in 2006e2011 were determined and compared. More than 50% of TEEs (79/156) occurred within 24 h of administration, with stroke being the most frequent (45), followed by myocardial infarction (24), pulmonary embolism (18), and venous thrombosis (17). In total, 100 of 156 TEEs were deemed to be drug-related and attributed to a specific IgG product [15]. Records of 46 patients given IVIG therapy in 2002e2004 indicated that 13% with autoimmune disorders developed thrombotic complications, with 50% of these occurring during the IVIG infusion [25]. Factors such as an increased age, familial or personal history of vascular disease, thrombosis or acquired/inherited thrombotic disorders, anti-phospholipid syndrome, hyperlipidemia, hypertension, diabetes mellitus, obesity, and prolonged periods of immobilization may place patients at high risk for an IVIG-related thrombosis. Four main mechanisms of IVIG-associated TEEs were identified. 2.1.1. Increased concentrations of procoagulant clotting factors IVIG products may contain increased concentrations of FXIa or other procoagulant clotting factors. A high-dose infusion of IVIG with FXIa to patients with inflammatory conditions and/or a preexisting vascular pathology might predispose them to thrombotic complications [15,16]. The predominant thrombogenic marker was identified to be FXIa, as even minute quantities can result in significant thrombin generation [16]. Elevated levels of FXIa activity was also identified in several commercial hyperimmune IgG products associated with increased same-day TEEs [26]. Hypotension was correlated with increased concentrations of the prekallikrein activator (PKA) in plasma protein fractions. PKA is an active 28-kDa form of FXIIa, resulting from proteolytic cleavage of FXII by KK, FXIa, plasmin, or trypsin. KK at a concentration of 50 ng/mL is sufficient to activate FXII in the presence of an activating surface [18]. Apolipoprotein H (Apo-H) plays an active role in the clotting cascade, and thus can potentially contribute to TEEs if present in IgG products. Procoagulant activity, as measured using TGA, increased with an increase in Apo-H concentration in spiking experiments. Recently, human Apo-H was identified in trace amounts in commercial IgG products, which may explain some of the side effects (including TEEs) associated with the use of these products [27]. 2.1.2. Increased blood viscosity High-dose IVIG and rapid infusion rates may increase the blood viscosity during infusion, possibly as a result of the high protein content and protein polymers, platelet activation, and arterial vasospasms. In turn, the increased blood viscosity can cause a hypercoagulable state [2,16,26,28e31]. A 4-fold abnormal increase in viscosity concomitant with high-dose IVIG administration (>2 g/kg/day) was reported in as many as 85% of patients [31]. As a result, the US FDA issued a warning on the occurrence of
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thromboses after IVIG use, in particular with 10% protein concentrated products [23]. 2.1.3. Anti-cardiolipin antibodies Certain antibodies (anti-cardiolipin) present in IVIG products could in theory induce thrombosis. IVIG is a pooled polyspecific IgG product that contains a wide spectrum of antibodies, including amounts of anti-cardiolipin antibodies. Detectable levels of antiphospholipid antibodies of 16e19 IU/mL may be present in IVIG preparations [28,32,33]. They can potentiate thromboses in situations of hypercoaguable states, such as hyperhomocysteinemia or anti-phospholipid syndrome. 2.1.4. Platelet and monocyte activation Arterial and multiple disseminated thrombi observed with FXI concentrates and TEE-associated IVIG products may indicate that blood cell activation could be an additional mechanism for adverse reactions from the use of IVIG [34]. A recent study of the effect of 20 commercial IVIG preparations on monocyte and platelet activation revealed that all IVIG products led to increased tissue factor expression in monocytes, independent of FXIa content. The IVIG preparations further led to increased formation of monocyteplatelet aggregates, mediated by CD154, a protein that was present in all the IVIG batches tested [34]. A high molecular weight anti-CD154-reactive species was found in an IVIG batch with known increased TEE risk, indicating that this protein could contribute to adverse events based on blood cell activation. 2.2. Process-related increases in the thrombogenic risk of IVIG products A typical IVIG production process is based on ethanol plasma fractionation as stated above. Manufacturing IVIGs from fractions II þ III (or fractions I þ II þ III) usually involves a precipitation step for initial purification, typically using ethanol, caprylate, or polyethylene glycol (PEG) as precipitating agents, followed by polishing steps such as ion exchange chromatography (IEC) [10]. This should yield at least 95% IgG purity to comply with the Ph. Eur. [21]. However, to meet growing clinical demands and ensure improved viral safety, IVIG manufacturers have implemented a number of process modifications with some processes yielding in excess of 4e4.5 g IgG/l plasma, or higher, depending upon the IgG content of the starting plasma. Major recent advances in production steps of IVIG products are summarized below [3,10,35,36]: a. Initiating IgG finishing purification steps from upstream ethanol fractionation fractions, thereby avoiding IgG losses typically associated with precipitation of Cohn fractions II and III. b. Subjecting the upstream fractions II þ III to modified ethanol⁄pH precipitation, solvent/detergent (S/D) treatment, and hydrophobic interaction or cation-exchange chromatography. However, to negate the 10% loss in IgG and the longer processing time, the process was substituted for 2 stages of octanoic acid (also called caprylic acid, CAP), treatment (to precipitate nonIgG contaminants) and 2 anion-exchange chromatographic adsorption steps. c. Purifying fractions I þ II þ III by CAP precipitation combined with low-pH incubation and anion exchange chromatography. d. Purifying fractions II þ III through a modified ethanol precipitation step followed by cation-exchange and anion-exchange chromatography. e. Preparing IVIG through a high-yielding chromatographic process, in which the first ethanol precipitate (fraction I supernatant) is subjected to incubation at pH 5.2 to precipitate euglobulins and then chromatography on an anion-exchanger
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that extracts albumin. The IgG-rich un-adsorbed fraction is further purified by an anion-exchange purification step. f. Implementing chromatographic procedures to remove IgA, a potential cause of severe anaphylactic shock in patients with an IgA deficiency [37]. g. Preparing liquid products to avoid production costs associated with the freeze-drying step and to facilitate administration to patients. Modifying the manufacturing steps can potentially have unintentional consequences such as co-purification of thrombogenic agents (activated coagulation factors and their zymogens) or the inadvertent activation of zymogens. Twenty-six of 29 IVIG lots from 8 different manufacturers shortened the clotting time of FXIdeficient plasma, and 14 of these had procoagulant FXI activity higher than that of a pool of plasma [16]. Traces of activated clotting factors (e.g., FXa, FIXa, FVIIa, and thrombin) or contact system factors (FXIa and FXIIa) may be present in cryo-poor plasma or intermediate fractions from which IVIG is isolated and purified [13]. This in turn may induce protein proteolytic degradation and activation during production, lead to loss of biological function and potential risks of TEEs during clinical applications as described previously [3,11]. Under non-physiological conditions, activated FXII can activate FXI, which subsequently activates FIX resulting in thrombin generation [38,39]. FXIa activity may increase in some IVIG product samples stored at 4 C [16], indicating that conversion of FXI to FXIa can occur during storage. The authors hypothesized that FXIa activity observed in some test samples may have resulted from contact activation during the IVIG purification process, and perhaps even during storage of the source plasma. In other cases, FXIa may remain stable or decrease over time. A possible mechanism for the co-purification of thrombogenic agents involves anion exchangers at slightly acidic pH (of 5.2e6.0), to allow IgG (positively charged, pI value: 6.3e7.3) to flow past the resin particles while the main residual impurities (which are mostly negatively charged, pI: 2.7e5.5) bind to the resin [40]. Anion-exchangers have a limited capacity to adsorb positively charged impurities such as thrombin (pI: 7.0e7.6), FXI, or FXIa (both with pI values of near 9.1) [37]. As fraction I is rich in coagulant proteins, an increase in FXIa, thrombin, or other procoagulant substances in the starting fraction for IVIG production can result from a switch from fractions II þ III to fractions I þ II þ III when fraction I is not removed. A modified process step may exceed the purification capacity of the combined precipitation plus IEC steps, thereby increasing concentrations of these proteases in the final product. Further, when S/D treatment is applied, cation-exchange chromatography used to remove the S/D can co-concentrate IgG and FXI [37]. Differences in the manufacturing and purification processes of immunoglobulin products from human plasma therefore affect the purity and tolerability of the final product [41]. Thus, underlying risk factors for TEEs include recipient predispositions (e.g., advanced age and previous TEEs) and product factors (e.g., protein concentration, infusion rates, route of administration, and manufacturing processes, i.e., the presence of elevated FXIa activity). 3. General approach to quantifying the thrombogenic potential of immunoglobulin products and production processes 3.1. Response by key IVIG manufacturers to the amended monographs Manufacturers now have to demonstrate the capacity of their IVIG production processes to reduce and ultimately eliminate the
thrombogenic potential of their products. These factors should be qualitatively and quantitatively determined in cryo-poor plasma, several intermediate fractions, and the final product. Some measures focused on validating processes so that routine testing of products would not be required. Representative intermediate products/samples from key manufacturing steps and final products were evaluated to see if they activated coagulation factors to demonstrate elimination of thrombogenic proteins and show the absence of procoagulant activity/contaminating proteins, in order to comply with the revised Ph. Eur. 0918. Octapharma evaluated the capacity of its Octagam® production process to remove activated FXI. They implemented an FXI adsorption step upstream in the process to remove FXI. The FXI content is routinely monitored by an enzyme-linked immunosorbent assay (ELISA) after the adsorption step, while the absence of procoagulant activity of the final product is confirmed by TGA as part of a batch release [11]. CSL Behring AG (Bern, Switzerland) evaluated and published the capacity of the Privigen® purification process to deplete thrombogenic FXIa, leading to a final product with procoagulant activity below the detection limit [42]. Grifols (Barcelona, Spain) demonstrated their Flebogamma® and Flebogamma® DIF 5% IVIG products to be free of clotting (pro)enzymes and coagulation factor activation markers, and showed that pasteurization was the key step in removing procoagulant contaminating proteins [38,40]. Baxter's (Vienna, Austria) approach to minimizing procoagulant impurities in their Gammagard® liquid IGIV product indicated that the II þ III paste resuspension and filtration steps removed >98% of FXI, with further removal of procoagulant impurities occurring during downstream purification processes [43]. 3.2. Evaluating the capacity of key process steps to remove FXIa and other procoagulant contaminants from the final product In most cases, the prothrombotic activity of IVIG batches suspected of inducing TEE is studied in the final product, either as a result of increased regulatory oversight of these products or on a voluntary basis. However, clotting factor activities can be quantified after various key IVIG purification steps to evaluate their capacity to reduce or remove protein procoagulant contaminants [40,42]. Modern IVIG production processes include a series of purification steps with distinctive capacities to eliminate contaminating proteins including proteolytic enzymes. Table 1 provides a summary of studies to evaluate the capacity of various IVIG manufacturing steps to remove or reduce activated coagulation factors. Spiking studies either with various concentrations of FXIa, ranging from 0.3 to 100 ng/mL [37,42] or Fraction II þ III, which is rich in coagulation factors, were done to evaluate the ability of these process steps to remove activated coagulation factors [40]. Activities in Fraction II þ III ranged from 15 to 33% (FX) to 76e92% (FXI) and 121e201% (FVII) versus the estimated original plasma pool content. FVII activity detected in Fraction II þ III by clotting factor assays indicated some FVII activation. Except for FXI, all other factor activities studied (FXII, FXI, FIX, FX, FVII and FII) were below the quantitation limit after PEG precipitation, DEAE chromatography purification and concentration by ultrafiltration. After treatment at pH 4.0 for 4.5 h at 37 C, KK activities and thrombin generation capacity were still detectable, illustrating limited capacity to inactivate clotting enzymes. Pasteurization reduced FXI activity below the quantitation limit, even in the most concentrated samples. Pasteurization, typically perceived exclusively as a virus inactivation step, is now appearing as important for inactivation of coagulation enzymes in IgG solution [40]. A FXIa spiking study showed that CAP used in the production of Privigen® can remove coagulation factors and their zymogens (FXI and FXIa) [42]. CAP
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Table 1 A summary of IVIG manufacturing process steps that have been shown to reduce or remove FXI/FXIa. Process step
Comments
Refs.
PEG precipitation, DEAE chromatography purification, concentration by ultrafiltration
Spiking with FrII þ III was used to study the capacity of various process steps in the purification of Flebogamma® and Flebogamma® DIF to remove FXIa. After PEG precipitation, DEAE chromatography purification and concentration by ultrafiltration, all clotting factor activities studied (FXII, FIX, FX, FVII and FII) were below the quantitation limit with the exception of exception of FXI. However, there was some reduction in FXI clotting activity, dropping from 71 to 88 IU/g of total protein, to 0.4e1.1 IU FXI/g protein. In the purification of Flebogamma® and Flebogamma® DIF, the acid pH treatment step showed a limited capacity for reducing clotting factor activities even in the lowest spike proportion, with the exception of PKA. The FXI clotting activity dropped from 0.4 to 1.1 IU FXI/g protein to 0e0.8 IU FXI/g protein. In the evaluation of the Privigen® manufacturing process, the capacity of a module pH 4 process (including membrane filtration and incubation at pH 4 steps) to reduce spiked FXIa (50 ng/mL) was demonstrated to reduce the FXIa specific activity by 47.8% (the actual FXIa concentration was reduced by 29.3%). In the purification of Flebogamma® and Flebogamma® DIF, the pasteurization step showed a significant capacity to reduce clotting factor activities to below the quantitation limit, even when spiking with high concentrations of FrII þ III (up to a 20% v/v). The capacities of the S HyperCel and CM Ceramic HyperD F cation exchangers and HyperCel STAR AX and Q HyperCel anion exchangers to remove spiked FXI at a concentration of 1 IU/mL (corresponding to 5 mg/mL FXI antigen content) were evaluated. 35% of the initial FXI was removed after a two-step process consisting of S HyperCel and HyperCel STAR AX columns. FXI and FXIa bind to some extend to cation exchangers due to the high isoelectric points of 8.9e9.1, respectively. Removal of spiked FXI at a final concentration of 1 IU/mL was evaluated using Mustang S chromatographic membranes. A 26-fold FXI removal was achieved by processing the purified IgG fraction with spiked FXI through Mustang S cationexchanger membranes at pH 6.0 and 12.7 mS/cm. The FXI concentration decreased from 121 ng/mL to 4.7 ng/mL in the filtrate. Overall, the S HyperCel/HyperCel STAR AX/Mustang S purification process reduced the spiked FXI by more than 97%. As part of an evaluation of the Privigen® manufacturing process, the capacity of CAP treatment to reduce FXIa was studied. A kinetic study confirmed the CAP treatment of a 50 ng/mL FXIa-spiked solution in IgG led to a 99.9% reduction in concentration to below the detection limit of 0.14 ng/mL after of 40 min of treatment. A modular CAP process step was demonstrated to reduce FXIa specific activity was reduced by 81.4% specific activity by 81.4%. The CAP module consisted of Module OA included octanoic acid fractionation, calcium-phosphate incubation, liquid/solid separation, and ultra/ diafiltration.
[40]
Acid pH step
Pasteurization
Ion exchange chromatography
Membrane cation exchange chromatography
CAP/Octanoic acid treatment
treatment in combination with depth filtration is effective to precipitate or adsorb, respectively, FXI/FXIa from cryo-poor plasma prior to processing into IgG [42,44]. Spiking experiments also showed that a cation-exchange chromatographic membrane at carefully selected pH and conductivity can be used for dedicated removal of FXI without significantly affecting IgG recovery [37]. Removal of FXI and/or FXIa from solution can also be achieved through absorption on a heparin-linked affinity chromatographic gel [44,45]. In summary, the key IgG process steps with the potential capacity for removing or inactivating coagulation factors are depth filtration, CAP precipitation, cation exchange membrane chromatography, and pasteurization, albeit at specific processing conditions. Analytical techniques to quantify clotting factor activities should be able to detect traces of FXI and/or its activated form, FXIa, in both routine production and spiking experiments [42]. The influence of the sample matrix on the accuracy, quality, and integrity of the analytical results is a significant issue because assays were not originally designed for this purpose, as they are all based on testing plasma samples. 3.3. Proposed analytical tests to quantify FXIa contamination levels A number of publications promoted the implementation of clotting factor tests as routine release criteria for IVIG products, especially in the context of a lack of a formal recommendation for batch release testing in the revised monographs. A number of tests seem suitable for quantifying the prothrombotic potential of IVIG, and their merit and specific applications in IVIG products and intermediates are discussed in more detail below and summarized in Table 2. Analytical assays included here are the TGA, aPTT, NaPTT, a chromogenic FXIa assay, FXI measurement with an ELISA, and an in vivo assay, the Wessler stasis model.
[40]
[42]
[40]
[37,44]
[37]
[42,44]
3.3.1. TGA The TGA is increasingly being explored as a global assay for detecting thrombogenic agents in plasma-derived products. Typically, the test is performed by mixing solutions being investigated with citrated or FXI-deficient plasma, adding phospholipids, minute amounts of tissue factor, and a specific, slow-reacting fluorogenic peptide substrate for thrombin. Thrombin formation is then initiated by the addition of calcium and monitored with a fluorescence photometer [46]. FXIa in the sample triggers initiation of the intrinsic coagulation pathway leading to thrombin generation [46]. Recently, a limit of detection as low as 0.3 pM FXIa, or ~0.1 ng/mL, was demonstrated [47]. By following changes in the fluorescence over time, the peak concentration of thrombin in a sample can be calculated using a thrombin calibration curve. Fig. 1 shows a typical TGA pattern. The assay has three read-out parameters (peak height, time to the peak, and lag time), and has the advantage of giving information on the amount of thrombin formed and the time to reach peak levels of thrombin [38,46]. To calibrate the assay, an external standard series consisting of purified FXIa spiked into a TGA-negative product matrix can be used [42]. Commercial suppliers of TGA reagents and analytical software include Technothrombin® TGA (Technoclone, Vienna, Austria) and a TGA test system from Thrombinoscope® (Maastricht, the Netherlands). In a conventional clotting experiment such as the NaPTT, the time taken for a sample to clot is measured. This is only part of the relevant information, as several events triggered by initiation of the clotting cascade still follow. Thus, when using the TGA, information on the coagulation process is gathered even after clotting has occurred, allowing the detection of modulators and activators of the coagulation cascade. To obtain maximum sensitivity, FXIa activation should be minimized as this factor plays an important role in amplifying the clotting cascade. Corn trypsin inhibitor added to blood during collection inactivates FXIIa, which in turn prevents early FXIa activation and results in FXIa-free or at least FXIa-poor
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Table 2 A summary of typical intravenous immunoglobulin G (IVIG) analytical test results, showing ranges of batches with normal and increased thromboembolic event (TEE) risks. Test a. TGA assay results TGA peak thrombin concentration (PTC)
TGA readout parameter (time to peak; TTP)
Results associated with the TEE risk
Normal result (no TEE risk)
Comments
Refs.
~170 nM thrombin
2e20 nM thrombin
Evaluation of procoagulation factors in Flebogamma® and Flebogamma® DIF final products, including product intermediates after spiking with fractions II þ III, using a Technothrombin® TGA assay (Technoclone, Vienna, Austria). FXI clotting activity was found in fractions II þ III to be 71e88 mU/mg of total protein. This level dropped to below the detection limit after key production steps. The Flebogamma® and Flebogamma® DIF final products, and several commercially-available IVIG products, had thrombin concentrations ~2e20 nM, while a 5% IVIG product associated with TEEs had a thrombin level of ~170 nM.
[38,40]
In order to obtain a standard thrombin-generation assay (TGA), TGA analysis of samples containing 0e1 mU/mL of FXIa was done. PTC increased from 50 nM PTC (baseline) to ~350 nM. An internal acceptance limit for FXIa was set at 500 nM PTC, and lowered to 350 nM to increase the safety margin. For comparison, PTC of FFP ranged 195e437 nM.
[11]
580e623 nM thrombin
68 nM thrombin
TEE-associated batches from 4 IVIG and 2 SCIG products studied showed peak thrombin concentrations of >500 nM. This corresponds to FXIa concentrations of >17 mU/mL.
[15]
>350 nM
0.002 U/mL
NaPTT assay
250 s, while those products with a TEE risk showed NaPTTs of 200 s, while the NaPTT ratio was >0.8. In cases associated with a high TEE risk, the NaPTT ratio was 0.82
The analysis of Flebogamma® and Flebogamma® DIF final products, and several commercially available IVIG products (including 2 products associated with high TEEs), showed NaPTT ratios of >0.82 for most products, and ratios of 0.62e0.78 for those associated with a high TEE risk.
c. Activated FXI chromogenic assay, FXIa ELISA, in vivo Wessler rabbit and KPA assay results >3.1 pM FXIa N/A Polylysine-based chromogenic assay for FIXa generation to quantify amount of FXIa. Activated FXI Samples with an activity of >3.1 pM (~0.6 ng/mL) of FXIa were regarded as positive. Four chromogenic of the samples were positive to activate FIX to FIXa. assay
FXIa ELISA
In vivo thrombosis model using rabbits
PKA concentration (mg/mL)
300 ng/mL
300 ng/mL FXIa, corresponding to a dose of 450 ng/kg FXIa
>5 mg⁄mL
9e15 ng/mL
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Review
Quantifying the thrombogenic potential of human plasma-derived immunoglobulin products W.A. Germishuizen a, D.C. Gyure a, D. Stubbings a, T. Burnouf b, * a b
National Bioproducts Institute, Pinetown, South Africa Graduate Institute of Biomedical Materials and Tissue Engineering, Taipei Medical University, 250 Wuxing St., Taipei City 110, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 January 2014 Received in revised form 24 April 2014 Accepted 29 April 2014 Available online xxx
Polyvalent immunoglobulin G (IgG) products obtained by fractionation of human plasma are used to treat a broad range of conditions, including immunodeficiency syndromes and autoimmune, inflammatory, and infectious diseases. Recent incidences of increased thromboembolic events (TEEs) associated with intravenous (IV) IgG (IVIG) led to recalls of some products and increased regulatory oversight of manufacturing processes in order to ensure that products are essentially free of procoagulant/thrombogenic plasma protein contaminants. Laboratory investigations have now identified activated factor XI (FXIa) as the likely causative agent of IVIG-related TEEs. Quantification of the thrombogenic potential is becoming a requirement made to fractionators (a) to validate the capacity of IVIG and subcutaneous IgG manufacturing processes to remove procoagulant contaminants and (b) to establish the safety of the final products. However, in the absence of a recommended test by the main regulatory authorities, several analytical approaches have been evaluated by fractionators, regulators, and university groups. This review focuses on the scientific rationale, merits, and applications of several analytical methods of quantifying the thrombogenic potential of IgG products and intermediates to meet the latest regulatory requirements. © 2014 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Keywords: Thrombosis IVIG SCIG FXIa Procoagulant
1. Introduction Human plasma-derived intravenous and subcutaneous polyvalent immunoglobulin G (IgG, IVIG/SCIG) medicinal products are immunomodulatory agents used to treat primary and secondary immunodeficiency syndromes and a variety of autoimmune, inflammatory, infectious, transplantation-related, and chronic diseases such as idiopathic thrombocytopenic purpura, chronic syninflammatory demyelinating polyneuropathy, Guillain-Barre drome, and Kawasaki disease [1,2]. IgG products are now the leading therapeutic products made from plasma and contribute to over 50% to sales of all fractionated plasma products. As they are prepared from plasma collected from a high number of healthy blood donors (1000e40,000), they contain a broad and diverse spectrum of polyclonal humoral antibodies resulting from the cumulative exposure of the donor population to the environment [3]. Therefore, IVIG and SCIG preparations recognize a broad range of bacterial, viral, and other infectious agent antigens, and also a large number of self-antigens [4]. In general, IVIG and SCIG products are
* Corresponding author. E-mail address: [email protected] (T. Burnouf).
considered safe and effective. Some common mild to moderate adverse events include a low-grade fever, headaches, malaise, nausea, and myalgia [1]. Less common but serious and potentially fatal adverse events include thromboembolic events (TEEs), acute renal failure, anaphylaxis, hemolysis, and aseptic meningitis [3,5]. Recently, the occurrence of transfusion-related acute lung injury (TRALI) following an IVIG infusion was described [6,7]. A process for the large-scale isolation of IgG from human plasma using cold-ethanol and pH fractionation steps was first developed by Cohn and co-workers in the 1940s [8], and later refined by Kistler and Nitschmann to improve the yield and purity while reducing the requirement for ethanol [9]. The modern multistep IVIG production process includes fractionation of plasma, protein purification by precipitation and/or chromatography, reduction (inactivation or removal) of blood-borne viruses, and stabilization of the IgG fraction [10]. Growing clinical demands for immunomodulatory therapies have prompted further process improvements in the last few years [3,10] to avoid some or all typical ethanol fractionation steps, increase IgG recovery, improve viral safety, replace the traditional low pH/pepsin used to reduce anticomplementary activity, and improve viral safety with more efficient procedures. However, these modifications may have caused unintentional co-purification of contaminant plasma proteins and
http://dx.doi.org/10.1016/j.biologicals.2014.04.002 1045-1056/© 2014 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
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Abbreviations Apo-H aPTT CAP CHMP ELISA FDA FXI FXIa IEC IgG IM IMIG IV IVIG
apolipoprotein H activated partial thromboplastin time caprylic acid Committee for Medicinal Products for Human Use enzyme-linked immunosorbent assay United States Food and Drug Administration factor XI activated factor XI ion exchange chromatography immunoglobulin G intramuscular intramuscular immunoglobulin G intravenous intravenous immunoglobulin G
inadvertent contact activation of hemostasis zymogen proteins such as coagulation factor XI (FXI), leading to increased TEEs, especially when combined with recent increases in infused doses of IVIG products for immunomodulatory therapies. In 2010, Octapharma Pharmazeutika (Vienna, Austria) altered the manufacturing process of Octagam® 5% in a way that unintentionally led to a higher-than-normal number of reports of serious TEEs. The company recalled 31 lots from the US market in August 2010, and all lots from the European market in September 2010 [11]. Following suspension of marketing authorization in Germany and Sweden, the US Food and Drug Administration (FDA) and the Committee for Medicinal Products for Human Use (CHMP) recommended suspension of all marketing authorization across the US [12] and European Union (EU) [13,14]. A detailed investigation of the process identified activated FXI (FXIa) as the likely cause of the unusually high rate of TEEs [11,15e18] with kallikrein (KK) considered to have played a minor role [11]. Critical steps in the manufacturing process could explain the presence of factors that triggered TEEs. Corrective measures in Octagam®'s manufacturing process were implemented to reduce FXIa-related procoagulant activity [11,13,14], including minimizing the FXIa level by adsorption upstream in the manufacturing process and performing quality control tests on final batches for the absence of FIXa prior to release on the market. The suspension of the marketing authorization for Octagam® was lifted in May 2011 [14,15]. Around the same time, Omr-IgG-am®, an IVIG product by Omrix Biopharmaceuticals (Kiryat-Ono, Israel) was recalled from the Israeli market due to a similar increase in TEE occurrence [1]. TEEs, including strokes and pulmonary emboli, were also observed after use of the subcutaneous immunoglobulin, Vivaglobin® (CSL Behring GmbH, Marburg, Germany), in 2011 [15,19]. Such product recalls from the EU and US and increased awareness of TEE-related risks of IVIG, prompted manufacturers and regulatory agencies to scrutinize existing IVIG manufacturing processes for their capacity to generate thrombogenic substances [11]. The US FDA and other regulatory agencies analyzed data associating IVIG/SCIG use and risks of thrombosis [20]. The European Pharmacopoeia (Ph. Eur.) publication, Human Normal Immunoglobulin for Intravenous Administration, Monograph 0918, was revised [15,21] to state that products should exhibit no thrombogenic (procoagulant) activity as follows: “The method of preparation also includes a step or steps that have been shown to remove thrombosis-generating agents. Emphasis is given to the identification of activated coagulation factors and their zymogens and process steps that may cause their activation. Consideration is also
KK NaPTT NIBSC PEG PEU Ph. Eur. PKA PTC SC SCIG S/D TEE TGA TRALI TTP
kallikrein non-activated partial thromboplastin time National Institute for Biological Standards and Control polyethylene glycol plasma equivalent units European Pharmacopoeia prekallikrein activator peak thrombin concentration subcutaneous subcutaneous immunoglobulin G solvent/detergent thromboembolic event(s) thrombin-generation assay transfusion-related acute lung injury time to peak
to be given to other procoagulant agents that could be introduced by the manufacturing process.” Manufacturers were given until 1 July 2012 to comply. The Ph. Eur. publication, Human Normal Immunoglobulin, Monograph 0338, was also updated following the revision of Monograph 0918 [22], with implementation on 1 January 2013. In September 2013, the US FDA instructed IVIG manufacturers to add information describing the risk of thrombosis and strategies to mitigate the risk, to the boxed warning. Similar instructions were given for SCIG and intramuscular (IM) IgG products [23]. This implies that various steps in the IVIG manufacturing process should be demonstrated to have effectively contributed to eliminating any thrombogenic potential leading to an inherently safe therapeutic product. For instance, the capacity for eliminating FXI/FXIa can be evaluated through spiking experiments using purified FXI. However, there are currently no officially approved methods for such assessments in revised monographs. While the US FDA promotes the use of a thrombin-generation assay (TGA), research papers have combined several analytical methods to quantify the thrombogenic potential associated with IVIG manufacturing processes and products, including the non-activated partial thromboplastin time (NaPTT), TGA, and FXIa chromogenic assays. Harmonization of test methods is needed to obtain congruent results, and a formal recommendation for batch release testing still needs to be included in the revised monographs. The objective of this review article is to describe a general approach to quantifying the thrombogenic risks associated with IVIG and SCIG products and manufacturing processes. The clinical context of TEEs associated with IVIG and recent changes to IVIG manufacturing technology that might have influenced the thrombogenic potential are discussed, as are measures taken by the fractionation industry in response to the revised regulatory requirements. Lastly, the merits and applications of various relevant analytical methods in quantifying the thrombogenic potential of IVIG products are evaluated in detail. 2. Background 2.1. TEE risks associated with IVIG products Despite their effectiveness and good safety record, the use of IVIG products is associated with adverse events including but not limited to aseptic meningitis, neutropenia, anaphylaxis, and less commonly observed TEEs, particularly in patients with other risk factors that contribute to thromboses. TEEs due to IgG products are
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likely to be acute, occurring either during administration or within 24 h of administration. A retrospective analysis of data from a large health claims-related database [1], and post-marketing surveillance over the period of 2008e2010 determined an average sameday TEE incidence rate of ~1%, (122 of 11,785 individuals exposed to IgG products), with rates reaching 2% for specific products. Venous same-day TEEs were more common than arterial TEEs, and TEE rates on the next day were much lower than on the same day as the infusion. Thus the first IgG dose may be the most critical with respect to developing TEEs. In a follow-up study of same-day TEE incidence over the period of 2008e2012, the authors found that 233 out of 14,944 individuals exposed to IgG products (~1.5%) experienced same day TEE's [24]. In a study by the Paul Ehrlich Institute [15], frequencies of TEEs associated with the use of 4 different IVIG products and 2 SCIGs in 2006e2011 were determined and compared. More than 50% of TEEs (79/156) occurred within 24 h of administration, with stroke being the most frequent (45), followed by myocardial infarction (24), pulmonary embolism (18), and venous thrombosis (17). In total, 100 of 156 TEEs were deemed to be drug-related and attributed to a specific IgG product [15]. Records of 46 patients given IVIG therapy in 2002e2004 indicated that 13% with autoimmune disorders developed thrombotic complications, with 50% of these occurring during the IVIG infusion [25]. Factors such as an increased age, familial or personal history of vascular disease, thrombosis or acquired/inherited thrombotic disorders, anti-phospholipid syndrome, hyperlipidemia, hypertension, diabetes mellitus, obesity, and prolonged periods of immobilization may place patients at high risk for an IVIG-related thrombosis. Four main mechanisms of IVIG-associated TEEs were identified. 2.1.1. Increased concentrations of procoagulant clotting factors IVIG products may contain increased concentrations of FXIa or other procoagulant clotting factors. A high-dose infusion of IVIG with FXIa to patients with inflammatory conditions and/or a preexisting vascular pathology might predispose them to thrombotic complications [15,16]. The predominant thrombogenic marker was identified to be FXIa, as even minute quantities can result in significant thrombin generation [16]. Elevated levels of FXIa activity was also identified in several commercial hyperimmune IgG products associated with increased same-day TEEs [26]. Hypotension was correlated with increased concentrations of the prekallikrein activator (PKA) in plasma protein fractions. PKA is an active 28-kDa form of FXIIa, resulting from proteolytic cleavage of FXII by KK, FXIa, plasmin, or trypsin. KK at a concentration of 50 ng/mL is sufficient to activate FXII in the presence of an activating surface [18]. Apolipoprotein H (Apo-H) plays an active role in the clotting cascade, and thus can potentially contribute to TEEs if present in IgG products. Procoagulant activity, as measured using TGA, increased with an increase in Apo-H concentration in spiking experiments. Recently, human Apo-H was identified in trace amounts in commercial IgG products, which may explain some of the side effects (including TEEs) associated with the use of these products [27]. 2.1.2. Increased blood viscosity High-dose IVIG and rapid infusion rates may increase the blood viscosity during infusion, possibly as a result of the high protein content and protein polymers, platelet activation, and arterial vasospasms. In turn, the increased blood viscosity can cause a hypercoagulable state [2,16,26,28e31]. A 4-fold abnormal increase in viscosity concomitant with high-dose IVIG administration (>2 g/kg/day) was reported in as many as 85% of patients [31]. As a result, the US FDA issued a warning on the occurrence of
3
thromboses after IVIG use, in particular with 10% protein concentrated products [23]. 2.1.3. Anti-cardiolipin antibodies Certain antibodies (anti-cardiolipin) present in IVIG products could in theory induce thrombosis. IVIG is a pooled polyspecific IgG product that contains a wide spectrum of antibodies, including amounts of anti-cardiolipin antibodies. Detectable levels of antiphospholipid antibodies of 16e19 IU/mL may be present in IVIG preparations [28,32,33]. They can potentiate thromboses in situations of hypercoaguable states, such as hyperhomocysteinemia or anti-phospholipid syndrome. 2.1.4. Platelet and monocyte activation Arterial and multiple disseminated thrombi observed with FXI concentrates and TEE-associated IVIG products may indicate that blood cell activation could be an additional mechanism for adverse reactions from the use of IVIG [34]. A recent study of the effect of 20 commercial IVIG preparations on monocyte and platelet activation revealed that all IVIG products led to increased tissue factor expression in monocytes, independent of FXIa content. The IVIG preparations further led to increased formation of monocyteplatelet aggregates, mediated by CD154, a protein that was present in all the IVIG batches tested [34]. A high molecular weight anti-CD154-reactive species was found in an IVIG batch with known increased TEE risk, indicating that this protein could contribute to adverse events based on blood cell activation. 2.2. Process-related increases in the thrombogenic risk of IVIG products A typical IVIG production process is based on ethanol plasma fractionation as stated above. Manufacturing IVIGs from fractions II þ III (or fractions I þ II þ III) usually involves a precipitation step for initial purification, typically using ethanol, caprylate, or polyethylene glycol (PEG) as precipitating agents, followed by polishing steps such as ion exchange chromatography (IEC) [10]. This should yield at least 95% IgG purity to comply with the Ph. Eur. [21]. However, to meet growing clinical demands and ensure improved viral safety, IVIG manufacturers have implemented a number of process modifications with some processes yielding in excess of 4e4.5 g IgG/l plasma, or higher, depending upon the IgG content of the starting plasma. Major recent advances in production steps of IVIG products are summarized below [3,10,35,36]: a. Initiating IgG finishing purification steps from upstream ethanol fractionation fractions, thereby avoiding IgG losses typically associated with precipitation of Cohn fractions II and III. b. Subjecting the upstream fractions II þ III to modified ethanol⁄pH precipitation, solvent/detergent (S/D) treatment, and hydrophobic interaction or cation-exchange chromatography. However, to negate the 10% loss in IgG and the longer processing time, the process was substituted for 2 stages of octanoic acid (also called caprylic acid, CAP), treatment (to precipitate nonIgG contaminants) and 2 anion-exchange chromatographic adsorption steps. c. Purifying fractions I þ II þ III by CAP precipitation combined with low-pH incubation and anion exchange chromatography. d. Purifying fractions II þ III through a modified ethanol precipitation step followed by cation-exchange and anion-exchange chromatography. e. Preparing IVIG through a high-yielding chromatographic process, in which the first ethanol precipitate (fraction I supernatant) is subjected to incubation at pH 5.2 to precipitate euglobulins and then chromatography on an anion-exchanger
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that extracts albumin. The IgG-rich un-adsorbed fraction is further purified by an anion-exchange purification step. f. Implementing chromatographic procedures to remove IgA, a potential cause of severe anaphylactic shock in patients with an IgA deficiency [37]. g. Preparing liquid products to avoid production costs associated with the freeze-drying step and to facilitate administration to patients. Modifying the manufacturing steps can potentially have unintentional consequences such as co-purification of thrombogenic agents (activated coagulation factors and their zymogens) or the inadvertent activation of zymogens. Twenty-six of 29 IVIG lots from 8 different manufacturers shortened the clotting time of FXIdeficient plasma, and 14 of these had procoagulant FXI activity higher than that of a pool of plasma [16]. Traces of activated clotting factors (e.g., FXa, FIXa, FVIIa, and thrombin) or contact system factors (FXIa and FXIIa) may be present in cryo-poor plasma or intermediate fractions from which IVIG is isolated and purified [13]. This in turn may induce protein proteolytic degradation and activation during production, lead to loss of biological function and potential risks of TEEs during clinical applications as described previously [3,11]. Under non-physiological conditions, activated FXII can activate FXI, which subsequently activates FIX resulting in thrombin generation [38,39]. FXIa activity may increase in some IVIG product samples stored at 4 C [16], indicating that conversion of FXI to FXIa can occur during storage. The authors hypothesized that FXIa activity observed in some test samples may have resulted from contact activation during the IVIG purification process, and perhaps even during storage of the source plasma. In other cases, FXIa may remain stable or decrease over time. A possible mechanism for the co-purification of thrombogenic agents involves anion exchangers at slightly acidic pH (of 5.2e6.0), to allow IgG (positively charged, pI value: 6.3e7.3) to flow past the resin particles while the main residual impurities (which are mostly negatively charged, pI: 2.7e5.5) bind to the resin [40]. Anion-exchangers have a limited capacity to adsorb positively charged impurities such as thrombin (pI: 7.0e7.6), FXI, or FXIa (both with pI values of near 9.1) [37]. As fraction I is rich in coagulant proteins, an increase in FXIa, thrombin, or other procoagulant substances in the starting fraction for IVIG production can result from a switch from fractions II þ III to fractions I þ II þ III when fraction I is not removed. A modified process step may exceed the purification capacity of the combined precipitation plus IEC steps, thereby increasing concentrations of these proteases in the final product. Further, when S/D treatment is applied, cation-exchange chromatography used to remove the S/D can co-concentrate IgG and FXI [37]. Differences in the manufacturing and purification processes of immunoglobulin products from human plasma therefore affect the purity and tolerability of the final product [41]. Thus, underlying risk factors for TEEs include recipient predispositions (e.g., advanced age and previous TEEs) and product factors (e.g., protein concentration, infusion rates, route of administration, and manufacturing processes, i.e., the presence of elevated FXIa activity). 3. General approach to quantifying the thrombogenic potential of immunoglobulin products and production processes 3.1. Response by key IVIG manufacturers to the amended monographs Manufacturers now have to demonstrate the capacity of their IVIG production processes to reduce and ultimately eliminate the
thrombogenic potential of their products. These factors should be qualitatively and quantitatively determined in cryo-poor plasma, several intermediate fractions, and the final product. Some measures focused on validating processes so that routine testing of products would not be required. Representative intermediate products/samples from key manufacturing steps and final products were evaluated to see if they activated coagulation factors to demonstrate elimination of thrombogenic proteins and show the absence of procoagulant activity/contaminating proteins, in order to comply with the revised Ph. Eur. 0918. Octapharma evaluated the capacity of its Octagam® production process to remove activated FXI. They implemented an FXI adsorption step upstream in the process to remove FXI. The FXI content is routinely monitored by an enzyme-linked immunosorbent assay (ELISA) after the adsorption step, while the absence of procoagulant activity of the final product is confirmed by TGA as part of a batch release [11]. CSL Behring AG (Bern, Switzerland) evaluated and published the capacity of the Privigen® purification process to deplete thrombogenic FXIa, leading to a final product with procoagulant activity below the detection limit [42]. Grifols (Barcelona, Spain) demonstrated their Flebogamma® and Flebogamma® DIF 5% IVIG products to be free of clotting (pro)enzymes and coagulation factor activation markers, and showed that pasteurization was the key step in removing procoagulant contaminating proteins [38,40]. Baxter's (Vienna, Austria) approach to minimizing procoagulant impurities in their Gammagard® liquid IGIV product indicated that the II þ III paste resuspension and filtration steps removed >98% of FXI, with further removal of procoagulant impurities occurring during downstream purification processes [43]. 3.2. Evaluating the capacity of key process steps to remove FXIa and other procoagulant contaminants from the final product In most cases, the prothrombotic activity of IVIG batches suspected of inducing TEE is studied in the final product, either as a result of increased regulatory oversight of these products or on a voluntary basis. However, clotting factor activities can be quantified after various key IVIG purification steps to evaluate their capacity to reduce or remove protein procoagulant contaminants [40,42]. Modern IVIG production processes include a series of purification steps with distinctive capacities to eliminate contaminating proteins including proteolytic enzymes. Table 1 provides a summary of studies to evaluate the capacity of various IVIG manufacturing steps to remove or reduce activated coagulation factors. Spiking studies either with various concentrations of FXIa, ranging from 0.3 to 100 ng/mL [37,42] or Fraction II þ III, which is rich in coagulation factors, were done to evaluate the ability of these process steps to remove activated coagulation factors [40]. Activities in Fraction II þ III ranged from 15 to 33% (FX) to 76e92% (FXI) and 121e201% (FVII) versus the estimated original plasma pool content. FVII activity detected in Fraction II þ III by clotting factor assays indicated some FVII activation. Except for FXI, all other factor activities studied (FXII, FXI, FIX, FX, FVII and FII) were below the quantitation limit after PEG precipitation, DEAE chromatography purification and concentration by ultrafiltration. After treatment at pH 4.0 for 4.5 h at 37 C, KK activities and thrombin generation capacity were still detectable, illustrating limited capacity to inactivate clotting enzymes. Pasteurization reduced FXI activity below the quantitation limit, even in the most concentrated samples. Pasteurization, typically perceived exclusively as a virus inactivation step, is now appearing as important for inactivation of coagulation enzymes in IgG solution [40]. A FXIa spiking study showed that CAP used in the production of Privigen® can remove coagulation factors and their zymogens (FXI and FXIa) [42]. CAP
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Table 1 A summary of IVIG manufacturing process steps that have been shown to reduce or remove FXI/FXIa. Process step
Comments
Refs.
PEG precipitation, DEAE chromatography purification, concentration by ultrafiltration
Spiking with FrII þ III was used to study the capacity of various process steps in the purification of Flebogamma® and Flebogamma® DIF to remove FXIa. After PEG precipitation, DEAE chromatography purification and concentration by ultrafiltration, all clotting factor activities studied (FXII, FIX, FX, FVII and FII) were below the quantitation limit with the exception of exception of FXI. However, there was some reduction in FXI clotting activity, dropping from 71 to 88 IU/g of total protein, to 0.4e1.1 IU FXI/g protein. In the purification of Flebogamma® and Flebogamma® DIF, the acid pH treatment step showed a limited capacity for reducing clotting factor activities even in the lowest spike proportion, with the exception of PKA. The FXI clotting activity dropped from 0.4 to 1.1 IU FXI/g protein to 0e0.8 IU FXI/g protein. In the evaluation of the Privigen® manufacturing process, the capacity of a module pH 4 process (including membrane filtration and incubation at pH 4 steps) to reduce spiked FXIa (50 ng/mL) was demonstrated to reduce the FXIa specific activity by 47.8% (the actual FXIa concentration was reduced by 29.3%). In the purification of Flebogamma® and Flebogamma® DIF, the pasteurization step showed a significant capacity to reduce clotting factor activities to below the quantitation limit, even when spiking with high concentrations of FrII þ III (up to a 20% v/v). The capacities of the S HyperCel and CM Ceramic HyperD F cation exchangers and HyperCel STAR AX and Q HyperCel anion exchangers to remove spiked FXI at a concentration of 1 IU/mL (corresponding to 5 mg/mL FXI antigen content) were evaluated. 35% of the initial FXI was removed after a two-step process consisting of S HyperCel and HyperCel STAR AX columns. FXI and FXIa bind to some extend to cation exchangers due to the high isoelectric points of 8.9e9.1, respectively. Removal of spiked FXI at a final concentration of 1 IU/mL was evaluated using Mustang S chromatographic membranes. A 26-fold FXI removal was achieved by processing the purified IgG fraction with spiked FXI through Mustang S cationexchanger membranes at pH 6.0 and 12.7 mS/cm. The FXI concentration decreased from 121 ng/mL to 4.7 ng/mL in the filtrate. Overall, the S HyperCel/HyperCel STAR AX/Mustang S purification process reduced the spiked FXI by more than 97%. As part of an evaluation of the Privigen® manufacturing process, the capacity of CAP treatment to reduce FXIa was studied. A kinetic study confirmed the CAP treatment of a 50 ng/mL FXIa-spiked solution in IgG led to a 99.9% reduction in concentration to below the detection limit of 0.14 ng/mL after of 40 min of treatment. A modular CAP process step was demonstrated to reduce FXIa specific activity was reduced by 81.4% specific activity by 81.4%. The CAP module consisted of Module OA included octanoic acid fractionation, calcium-phosphate incubation, liquid/solid separation, and ultra/ diafiltration.
[40]
Acid pH step
Pasteurization
Ion exchange chromatography
Membrane cation exchange chromatography
CAP/Octanoic acid treatment
treatment in combination with depth filtration is effective to precipitate or adsorb, respectively, FXI/FXIa from cryo-poor plasma prior to processing into IgG [42,44]. Spiking experiments also showed that a cation-exchange chromatographic membrane at carefully selected pH and conductivity can be used for dedicated removal of FXI without significantly affecting IgG recovery [37]. Removal of FXI and/or FXIa from solution can also be achieved through absorption on a heparin-linked affinity chromatographic gel [44,45]. In summary, the key IgG process steps with the potential capacity for removing or inactivating coagulation factors are depth filtration, CAP precipitation, cation exchange membrane chromatography, and pasteurization, albeit at specific processing conditions. Analytical techniques to quantify clotting factor activities should be able to detect traces of FXI and/or its activated form, FXIa, in both routine production and spiking experiments [42]. The influence of the sample matrix on the accuracy, quality, and integrity of the analytical results is a significant issue because assays were not originally designed for this purpose, as they are all based on testing plasma samples. 3.3. Proposed analytical tests to quantify FXIa contamination levels A number of publications promoted the implementation of clotting factor tests as routine release criteria for IVIG products, especially in the context of a lack of a formal recommendation for batch release testing in the revised monographs. A number of tests seem suitable for quantifying the prothrombotic potential of IVIG, and their merit and specific applications in IVIG products and intermediates are discussed in more detail below and summarized in Table 2. Analytical assays included here are the TGA, aPTT, NaPTT, a chromogenic FXIa assay, FXI measurement with an ELISA, and an in vivo assay, the Wessler stasis model.
[40]
[42]
[40]
[37,44]
[37]
[42,44]
3.3.1. TGA The TGA is increasingly being explored as a global assay for detecting thrombogenic agents in plasma-derived products. Typically, the test is performed by mixing solutions being investigated with citrated or FXI-deficient plasma, adding phospholipids, minute amounts of tissue factor, and a specific, slow-reacting fluorogenic peptide substrate for thrombin. Thrombin formation is then initiated by the addition of calcium and monitored with a fluorescence photometer [46]. FXIa in the sample triggers initiation of the intrinsic coagulation pathway leading to thrombin generation [46]. Recently, a limit of detection as low as 0.3 pM FXIa, or ~0.1 ng/mL, was demonstrated [47]. By following changes in the fluorescence over time, the peak concentration of thrombin in a sample can be calculated using a thrombin calibration curve. Fig. 1 shows a typical TGA pattern. The assay has three read-out parameters (peak height, time to the peak, and lag time), and has the advantage of giving information on the amount of thrombin formed and the time to reach peak levels of thrombin [38,46]. To calibrate the assay, an external standard series consisting of purified FXIa spiked into a TGA-negative product matrix can be used [42]. Commercial suppliers of TGA reagents and analytical software include Technothrombin® TGA (Technoclone, Vienna, Austria) and a TGA test system from Thrombinoscope® (Maastricht, the Netherlands). In a conventional clotting experiment such as the NaPTT, the time taken for a sample to clot is measured. This is only part of the relevant information, as several events triggered by initiation of the clotting cascade still follow. Thus, when using the TGA, information on the coagulation process is gathered even after clotting has occurred, allowing the detection of modulators and activators of the coagulation cascade. To obtain maximum sensitivity, FXIa activation should be minimized as this factor plays an important role in amplifying the clotting cascade. Corn trypsin inhibitor added to blood during collection inactivates FXIIa, which in turn prevents early FXIa activation and results in FXIa-free or at least FXIa-poor
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Table 2 A summary of typical intravenous immunoglobulin G (IVIG) analytical test results, showing ranges of batches with normal and increased thromboembolic event (TEE) risks. Test a. TGA assay results TGA peak thrombin concentration (PTC)
TGA readout parameter (time to peak; TTP)
Results associated with the TEE risk
Normal result (no TEE risk)
Comments
Refs.
~170 nM thrombin
2e20 nM thrombin
Evaluation of procoagulation factors in Flebogamma® and Flebogamma® DIF final products, including product intermediates after spiking with fractions II þ III, using a Technothrombin® TGA assay (Technoclone, Vienna, Austria). FXI clotting activity was found in fractions II þ III to be 71e88 mU/mg of total protein. This level dropped to below the detection limit after key production steps. The Flebogamma® and Flebogamma® DIF final products, and several commercially-available IVIG products, had thrombin concentrations ~2e20 nM, while a 5% IVIG product associated with TEEs had a thrombin level of ~170 nM.
[38,40]
In order to obtain a standard thrombin-generation assay (TGA), TGA analysis of samples containing 0e1 mU/mL of FXIa was done. PTC increased from 50 nM PTC (baseline) to ~350 nM. An internal acceptance limit for FXIa was set at 500 nM PTC, and lowered to 350 nM to increase the safety margin. For comparison, PTC of FFP ranged 195e437 nM.
[11]
580e623 nM thrombin
68 nM thrombin
TEE-associated batches from 4 IVIG and 2 SCIG products studied showed peak thrombin concentrations of >500 nM. This corresponds to FXIa concentrations of >17 mU/mL.
[15]
>350 nM
0.002 U/mL
NaPTT assay
250 s, while those products with a TEE risk showed NaPTTs of 200 s, while the NaPTT ratio was >0.8. In cases associated with a high TEE risk, the NaPTT ratio was 0.82
The analysis of Flebogamma® and Flebogamma® DIF final products, and several commercially available IVIG products (including 2 products associated with high TEEs), showed NaPTT ratios of >0.82 for most products, and ratios of 0.62e0.78 for those associated with a high TEE risk.
c. Activated FXI chromogenic assay, FXIa ELISA, in vivo Wessler rabbit and KPA assay results >3.1 pM FXIa N/A Polylysine-based chromogenic assay for FIXa generation to quantify amount of FXIa. Activated FXI Samples with an activity of >3.1 pM (~0.6 ng/mL) of FXIa were regarded as positive. Four chromogenic of the samples were positive to activate FIX to FIXa. assay
FXIa ELISA
In vivo thrombosis model using rabbits
PKA concentration (mg/mL)
300 ng/mL
300 ng/mL FXIa, corresponding to a dose of 450 ng/kg FXIa
>5 mg⁄mL
9e15 ng/mL
