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Article ID: WMC001917 ISSN 20461690
Pasteurization Inactivates Clotting Enzymes During Flebogamma® and Flebogamma® DIF Production
Author(s):Dr. Marta Jose, Dr. Nuria Marzo, Dr. Mariona Bono, Dr. Marta Carretero, Ms. Maite Lopez, Mr. Pere Ristol, Dr. Juan I Jorquera
Corresponding Author: Dr. Marta Jose, Head, Bioscience Laboratory Department, Instituto Grifols S.A., Research and Development Area, 08150  Spain
Submitting Author: Dr. Marta Jose, Department Head, Bioscience Laboratory, Instituto Grifols S.A., Research and Development, 08150  Spain
Previous Article Reference:http://www.webmedcentral.com/article_view/1425 Article ID:WMC001917 Article Type:Research articles Submitted on:06May2011, 12:46:13 PM GMTPublished on:07May2011, 02:04:55 PM GMT Article URL:http://www.webmedcentral.com/article_view/1917 Subject Categories:IMMUNOTHERAPY Keywords:Intravenous immunoglobulin, IVIG, Clotting enzyme inactivation, Pasteurization, Flebogamma
How to cite the article:Jose M , Marzo N , Bono M , Carretero M , Lopez M , Ristol P , Jorquera J I. Pasteurization Inactivates Clotting Enzymes During Flebogamma® and Flebogamma® DIF Production . WebmedCentral IMMUNOTHERAPY 2011;2(5):WMC001917
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Pasteurization Inactivates Clotting Enzymes During Flebogamma® and Flebogamma® DIF Production
Abstract
Background: A recent thromboembolic adverse events o u t b r e a k l i n k e d t o a s p e c i f i c i n t r a v e n o u s immunoglobulin (IVIG) brand has raised serious safety concerns. Methods: Clotting (pro)enzyme activities in the starting fraction (Fr) II+III employed for ® ® Flebogamma and Flebogamma DIF (Grifols) production, capacity of purification steps to remove them, capacity of Pasteurisation for (pro)enzymes inactivation and presence of (pro)enzyme activities in final products from different IVIG manufacturers were assessed by well established techniques. Results: Activities in FrII+III ranged from 1533% (FX) to 7692% (FXI) and 121201% (FVII) versus the estimated plasma pool original content. FrII+III showed very short clotting times in NaPTT assay, high PKA and “kallikrein” content, and high thrombin generation capacity. After Pasteurisation, FXII, FXI, ® FIX, FX, FVII and FII activities in Flebogamma DIF were below the quantitation limit and all tests showed no clotting activity or coagulation activation markers. Two IVIG final products from other manufacturers showed increased thrombin generation capacity and notably shortened clotting times in NaPTT assay (using FXIdeficient plasma). When artificially activated FrII+III samples were spiked into the corresponding production process intermediate, Pasteurisation inactivated all clotting factor activities studied. Conclusion: Pasteurisation step of the ® ® Flebogamma and Flebogamma DIF production process inactivates clotting (pro)enzymes and coagulation factor activation markers. Introduction
Serious safety concerns recently arose worldwide regarding an apparent increased incidence of thromboembolic adverse events (TAEs) associated with the use of an intravenous immunoglobulin (IVIG) preparation from a specific manufacturer, which finally lead to the suspension of the corresponding marketing authorization by the European Commission [1]. Generally, TAEs may rarely occur with the use of
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these products. Causes are thought to be mainly of a physical nature, while such incidence is almost always associated with underlying diseases and known cardiovascular risk factors [2]. No increment in the incidence of TAEs has been reported so far in any other marketed IVIG. Different authors attribute activated coagulation factor (F) XI (FXIa) with a relevant role in the thrombogenic potential of IVIGs [35]. Although no clinical tests for FXIa are available, there are a number of in vitro tests that have long been used to assess the procoagulant activity of blood derivatives, particularly those with a significant risk of thrombogenicity [4, 68].
The development of pure and safe preparations is a major goal of plasma derivative manufacturers, including diminishing or virtually eliminating the risk of IVIGassociated TAEs. For this purpose, different ® manufacturers apply different strategies. Flebogamma ® and Flebogamma DIF are highly purified IVIGs from Grifols. Their production processes include a series of purification steps with the distinctive capacity of being able to eliminate accompanying proteins including proteolytic enzymes [3]: polyethylene glycol (PEG) precipitation, ion exchange chromatography (IEC) and Pasteurization. Remarkably, Pasteurization heatdenaturation capacity for proteins is commonly perceived exclusively as a virus inactivation step and its role in coagulation factor inactivation is often disregarded in spite of its relevance. In this study, a thorough characterization of the presence of procoagulant activities in the starting plasma fraction used for IVIG production and the capacity of common purification steps to remove them was assessed in the context of the production process ® ® of Flebogamma and Flebogamma DIF. Additionally, different commercial IVIG preparations were directly compared in tests aimed to characterize procoagulant activities. Methods
Clotting (pro)enzyme activities in the starting fraction ® ® (Fr) II+III used for Flebogamma and Flebogamma DIF (Instituto Grifols S.A., Barcelona, Spain) IVIG production, the capacity of purification steps to remove
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them, the capacity of Pasteurization for (pro)enzyme inactivation, and the presence of (pro)enzyme activities in final products from different manufacturers were assessed.
Purification processes
F r I I + I I I w a s i n d u s t r i a l l y o b t a i n e d f r o m cryoprecipitatedepleted human plasma (obtained from healthy donors) using Cohn’s method with minor proprietary modifications [9]. The purification ® ® processes of Flebogamma and Flebogamma DIF have been published elsewhere [10, 11]. Samples of FrII+III industrial extraction as well as samples from intermediate purification stages were analyzed with the methods described below. Determination of coagulation (pro)enzymes by one stage clotting assays Clotting assays [8] were performed in a Q Hemostasis Analyzer (Diagnostic Grifols, Barcelona, Spain) using the corresponding prothrombin time (DGPT) and APTT (DGAPTT) reagents as well as the factor deficient plasmas from the same manufacturer. FXII, FXI, FIX, FX, FVII and FII were determined in the industrial extraction of FrII+III, as well as in several process intermediates and final products.
Determination of activated factors (NaPTT)
The nonactivated partial thromboplastin time (NaPTT) was performed as described in the European Pharmacopoeia (Ph. Eur.) monograph [4] on samples tested either neat or after dilution in the assay buffer. I n s o m e e x p e r i m e n t s t h e s t a n d a r d m e t h o d (NaPTTPPP) was modified using FXI deficient plasma (NaPTTFXIDef) in place of the platelet poor plasma (PPP; both types of plasmas from Diagnostic Grifols S.A., Barcelona, Spain). In order to discard the possibility of the inhibitory effects at high IVIG concentrations, the NaPTT was tested on samples either neat or after 1/5 and 1/10 dilutions (FrII+III suspensions were additionally tested at 1/100 dilution) without evidence of inhibitory effects at high IVIG concentrations. For simplicity, illustrations 1 and 3 show only the NaPTT results from neat samples but illustrations 4 and 5 show NaPTT results from samples assayed neat and after 1/5 dilution.
NaPTTFXIDef test was also applied to different lots (n= 1 to n= 4) of six IVIG products from five different manufacturers and concentrations identified as A, B1, B2, C, D and E (all liquid excepting B1, which was lyophilized). The lot from Product A identified as “A 5% (2007)” was manufactured in 2007 and was analyzed one year after its expiration date. The remaining lots were analyzed within their shelf life.
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Presence of thrombin
Thrombin presence was determined following the Ph. Eur. monograph [6]. Samples were assayed after incubation for 3 h and 6 h at 37ºC and for 24 h at 25 ºC.
Determination of prekallikrein activator (PKA)
Prekallikrein activator (PKA) was determined according to Ph. Eur. monograph [7] using S2302 (Chromogenix, Milano, Italy) as substrate. Determination of Kallikrein and/or “Kallikreinlike substances”. Kallikrein and/or “kallikreinlike” dependent S2302 hydrolytic activity (“kallikrein”) was determined using the PKA assay.
Thrombin generation test (TGT) Thrombin generation was determined using the Technothrombin® TGA (Technoclone, Vienna, Austria), with a fluorescence reader and specially adapted software according to the manufacture instructions. Briefly, thrombin generation is initiated in a mixture of platelet poor plasma (PPP) or FXI deficient plasma and sample (32 µl of PPP/FXI deficient plasma and 8 µl of sample) by the addition of 10 µl of a reagent containing a low concentration of phospholipid micelles with 0.5 pM recombinant human tissue factor in Tris Hepes buffer and 50 µl of TGA fluorogenic substrate (0.5 mM ZGGRAMC, 15 mM CaCl2). For the assay control (vehicle) the test sample is replaced with 8 µl of 0.15 M NaCl. The fluorimetric m e a s u r e m e n t s a t 3 6 0 n m a n d 4 6 0 n m (excitation/emission) are conducted at 37ºC. From the changes in fluorescence over time, the peak concentration of thrombin (nM) in the sample was calculated using a thrombin calibration curve. The value of the vehicle in the same microplate was subtracted from the result of each test sample. TGTFXIDef test was also applied to different lots (n= 1 to n= 4) of products of manufacturers A, B, C, D and E. Laboratory preparation of FrII+III spiking suspensions.
Laboratory FrII+III suspensions were used as spikes for the industrial materials before acid pH treatment or Pasteurization, in order to investigate the capacity of these steps in the inactivation of clotting (pro)enzymes. Samples of FrII+III were suspended in a ratio 0.3g FrII+III/ml of water and diluted 1: 4 in 0.15 M NaCl. After pH adjustment (7.37.4 with 0.1 N NaOH), the suspension was incubated for 60 min. at 30ºC under gentle stirring, further diluted 1: 1 in 0.15 M NaCl , incubated again for 90 min (under gentle stirring) at 30ºC and filtered through a 0.45mm pore filter.
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These activated FrII+III derived samples were spiked (v/v) 1/5, 1/10 and 1/20 in samples taken from the manufacturing intermediates (containing about 99% pure IgG) before acid pH (4.5 hours, pH 4.0, 37 ºC) treatment or before Pasteurization (10h, 60ºC) were applied to the corresponding mixtures in the laboratory. Results
Coagulation (pro)enzyme content and activation status of industrially resuspended FrII+III. Assuming 100% from the expected content of all coagulation proenzymes in the normal human plasma pool used for the preparation of FrII+III, the total recovery of activities in the industrially resuspended FrII+III (onestage clotting assays on frozen samples with an average OD280of 56.6±10.4, n=2) ranged from 1533% for FX to 121201% for FVII, with intermediate values for the remaining activities tested: 3540% for FXII, 6168% for FII, 6770% for FIX and 7692% for FXI. Resuspended FrII+III showed very short clotting times in the NaPTTPPP assay, high PKA and “kallikrein” content, as well as high thrombin generation capacity (Illustration 1). Followup of coagulation activities during the purification process With the only exception of FXI (Illustration 1), all remaining clotting factor activities studied were below the quantitation limit of the corresponding onestage ® assays in all of the Flebogamma DIF process intermediates studied, including the first gross purification step after industrial extraction (PEG precipitation).
FXI clotting activity in FrII+III dropped from 7188 IU/g of total protein, to 0.41.1 IU FXI/g protein after PEG precipitation, DEAE chromatography purification and concentration by ultrafiltration. After acid pH treatment, FXI was still detectable in two of the three processes analyzed. However, after Pasteurization FXI activity was always below the quantitation limit, even in the most concentrated samples (10 % final bulk product before dosage in vials, see Illustration 1).
“Kallikrein” activities and thrombin generation capacity could be observed in some samples even after DEAE chromatography purification, including the acid pH treated intermediate. However, after Pasteurization all tests showed negative results for clotting activity and for coagulation activation indicators (Illustration 1).
Final product results ® One to four lots of Flebogamma 5% DIF were
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analyzed by onestage clotting assays and using clotting factor activation tests (thrombin presence, NaPTTPPP, NaPTTFXIDef, PKA, TGTPPP or TGTFXIDef) with negative results. In addition, ® ® Flebogamma 10% DIF and Flebogamma 5% were tested by TGTFXIDef (Illustration 2) while ® Flebogamma 5% was also tested by NaPTTFXIDef (Illustration 3) with no indication of clotting factor activation in any test.
Also in Illustration 2, products A and B1 from two other manufacturers showed markedly increased thrombin generation capacity (TGTFXIDef) compared to the vehicle or to the rest of products. As seen in Illustration 3, the same products (A and B1) showed notably shorter clotting times in the NaPTTFXIDef assay, in contrast to the majority of products, that showed results comparable to the assay control.
Capacity of Pasteurization and acid pH treatment to inactivate procoagulant activities. Samples of industrial materials before acid pH treatment or before Pasteurization spiked with activated FrII+III (see methods) showed positive results when assayed for coagulation activation markers. The acid pH treatment showed a very limited effect on reducing the markers even in the lowest spike proportion, with the exception of PKA (Illustration 4), whereas Pasteurization rendered the mixtures negative for all tests performed, even with the highest spike volume (Illustration 5). References
1. European Comission. COMMISSION DECISION of 4.10.2010 concerning, in the framework of Article 107 of Directive 2001/83/EC of the European Parliament and of the Council, the marketing authorisations of medicinal products for human use "Octagam and a s s o c i a t e d n a m e s " . A v a i l a b l e a t : http://ec.europa.eu/health/documents/communityregis ter/html/ho17601.htm#EndOfPage. Last accessed: 12 Nov 2010. 2. Katz U, Achiron A, Sherer Y, Shoenfeld Y: Safety of intravenous immunoglobulin (IVIG) therapy. Autoimmun Rev 2007;6:257259. 3. Alving BM, Tankersley DL, Mason BL, Rossi F, Aronson DL, Finlayson JS: Contactactivated factors: contaminants of immunoglobulins preparations with coagulant and vasoactive properties. J Lab Clin Med 1980;96:334346. 4. European Pharmacopoeia Commission  Council of Europe European Department for the Quality of Medicines: Activated coagulation factors monograph
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(2.6.22); in: European Pharmacopoeia Strasbourg (France), Council of Europe, 2009. 5. Wolberg AS, Kon RH, Monroe DM, Hoffman M: Coagulation factor XI is a contaminant in intravenous immunoglobulin preparations. Am J Hematol 2000;65:3034. 6. European Pharmacopoeia Commission  Council of Europe European Department for the Quality of Medicines: Human prothrombin complex monograph (554); in: European Pharmacopoeia Strasbourg (France), Council of Europe, 2009. 7. European Pharmacopoeia Commission  Council of Europe European Department for the Quality of Medicines: Prekallikrein activator monograph (2.6.15); in: European Pharmacopoeia Strasbourg (France), Council of Europe, 2009. 8. Rizza CR, Rhymes IL: Coagulation assay of VIIIC and IXC; in: Bloom A L (ed): The Haemophilias Edinburgh, Churchill Livingstone, 1982, pp 1838. 9. Cohn EJ, Strong LE, Hughes WL, Armstrong SH: Preparation and properties of serum and plasma proteins; a system for the separation into fractions of the protein and lipoprotein components of biological tissues and fluids. J Am Chem Soc 1946;68:459475. 10. Jorquera JI: Flebogamma 5% DIF development: r a t i o n a l e f o r a n e w o p t i o n i n i n t r a v e n o u s immunoglobulin therapy. Clin Exp Immunol 2009;157 Suppl 1:1721. 11. Marzo N, Belda F, Ristol P, López M, Gajardo R, Jorquera JI: Hyperimmune immunoglobulins: Manufacturing technologies and safety procedures. Digest Liver Dis Suppl 2009;3:112118. 12. Salomon O, Steinberg DM, Zucker M, Varon D, Zivelin A, Seligsohn U: Patients with severe factor XI deficiency have a reduced incidence of deepvein thrombosis. Thromb Haemost 2011;105:269273. 13. Kistler P, Nitschmann H: Large scale production of human plasma fractions. Eight years experience with the alcohol fractionation procedure of Nitschmann, Kistler and Lergier. Vox Sang 1962;7:414424. 1 4 . C u r l i n g J M : M e t h o d s o f p l a s m a p r o t e i n fractionation. London, ACademic Press, 1980. 15. Lebing W, Remington KM, Schreiner C, Paul HI: Properties of a new intravenous immunoglobulin (IGIVC, 10%) produced by virus inactivation with caprylate and column chromatography. Vox Sang 2003;84:193201. 16. Uemura Y, Uriyu K, Hirao Y, Takechi K, Ishikawa H, Nakajima T, Kagitani Y, Yokoyama K, Funakoshi S, Nishida M,.: Inactivation and elimination of viruses d u r i n g t h e f r a c t i o n a t i o n o f a n i n t r a v e n o u s immunoglobulin preparation: liquid heat treatment and polyethylene glycol fractionation. Vox Sang 1989;56:155161.
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17. European Pharmacopoeia Commission  Council of Europe European Department for the Quality of Medicines: Human normal immunoglobulin for intravenous administration monograph (6.7.5675); in: European Pharmacopoeia Strasbourg (France), Council of Europe, 2009. 18. Smith EL, Hill RL, Lehman IR, Lefkowitz RJ, Handler PH, White A: Principles of Biochemistry: Mammalian Biochemistry. New York, McGrawHill, 1983. 19. Bouma BN, Griffin JH: Human blood coagulation factor XI. Purification, properties, and mechanism of activation by activated factor XII. J Biol Chem 1977;252:64326437. 20. Heck LW, Kaplan AP: Substrates of Hageman factor. I. Isolation and characterization of human factor XI (PTA) and inhibition of the activated enzyme by alpha 1antitrypsin. J Exp Med 1974;140:16151630. 21. Fenton JW, Fasco MJ, Stackrow AB: Human thrombins. Production, evaluation, and properties of alphathrombin. J Biol Chem 1977;252:35873598.
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Illustrations
Illustration 1
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Flebogamma® DIF purification process followup
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Illustration 2
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Thrombin generation test (TGT; mean±SD) assays with FXI deficient plasma, in products from different manufacturers (A, B1, B2, C, D, E and F). The lot identified as "A 5% (2007)" was manufactured in 2007.
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Illustration 3
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Nonactivated partial thromboplastin time (NaPTT; mean ± SD) assays with FXI deficient plasma in products from different manufacturers (A, B1, B2, C, D, E and F). The lot identified as "A 5% (2007)" was manufactured in 2007.
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Illustration 4
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Effect of acid pH treatment on activated clotting factors
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Illustration 5
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Effect of Pasteurization on activated clotting factors
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