Manufacturing Process of Intratect efficaciously ... · Manufacturing Process of Intratect...

Article ID: WMC004514 ISSN 2046-1690

Manufacturing Process of Intratect efficaciouslyeliminates thrombogenic potentialPeer review status:No

Corresponding Author:Dr. Regina Voges-Haas,Head of Laboratory, Development Plasmaproteins - Germany

Submitting Author:Dr. Steffen Kistner,Director, Research Hematology, Landsteinerstr. 5, 63303 - Germany

Other Authors:Dr. Veit Braun,Director, Research Bioanalytics - Germany

Dr. Rainer Schmeidl,Vice President, Corporate Drug Safety - Germany

Dr. Gerlinde Kuchta,Senior Medical Saftey Advisor, Medical Evaluations Plasmaproteins - Germany

Article ID: WMC004514

Article Type: Industry Sponsored Research

Submitted on:15-Jan-2014, 06:00:14 PM GMT Published on: 16-Jan-2014, 05:57:19 AM GMT

Article URL: http://www.webmedcentral.com/article_view/4514

Subject Categories:IMMUNOTHERAPY

Keywords:Immunoglobulins, IVIG, Thromboembolic events, Caprylic Acid, TGA, NaPTT, Clotting factors,Coagulation factors, Pharmacovigilance

How to cite the article:Voges-Haas R, Braun V, Schmeidl R, Kuchta G, Kistner S. Manufacturing Process ofIntratect efficaciously eliminates thrombogenic potential. WebmedCentral IMMUNOTHERAPY2014;5(1):WMC004514

Copyright: This is an open-access article distributed under the terms of the Creative Commons AttributionLicense(CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided theoriginal author and source are credited.

Source(s) of Funding:

None

Competing Interests:

All authors are employees of Biotest AG.

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Manufacturing Process of Intratect efficaciouslyeliminates thrombogenic potentialAuthor(s): Voges-Haas R, Braun V, Schmeidl R, Kuchta G, Kistner S

Abstract

In 2010, an unexpected increase in the reporting rateof serious thromboembolic adverse events associatedwith the application of immunoglobulin preparationsfrom one specific manufacturer caused increasedconcern regarding this topic. As a result, monographsfor immunoglobulin preparations were revised. Hence,plasma manufacturers were required to validate atleast one step in the manufacturing process for theeffective reduction of the thrombogenic potential inimmunoglobulin preparations. Biotest validated thestep in their manufacturing process of theirimmunoglobulin products which incorporates caprylicacid treatment and performed detailed analysesincluding non-activated partial thromboplastin time,thrombin generation assay, factor XIa levels, totalproteolytic activity and prekallikrein levels at variousstages in the production process.

Biotest demonstrated that treatment with caprylic acidduring manufacturing was effective in reducing thethrombogenic potential resulting in the generation ofan immunoglobulin product with an improved safetyprofile. Moreover, Biotest showed the absence ofvarious factors involved in the coagulation cascade inall of the Biotest immunoglobulin products. Throughproducing high quality immunoglobulin products,Biotest is committed to ensuring maximum patientsafety.

Introduction

Thromboembolic events (TEEs), in the context oftemporary increased viscosity, are an establishedclass-related risk associated with immunoglobulintreatment. TEEs are usually associated with certainrisk factors, including advanced age, infusion volumeand rate and patient's medical history of vasculardisease or thrombotic episodes (EMA Guidelines2012). In 2010, an unexpected increase in thereporting rate of serious TEEs with the use ofintravenous immunoglobulin (IVIG) preparations fromone specific manufacturer highlighted the riskassociated with immunoglobulins as a class.

The main factors contributing to TEEs are thepresence of procoagulant substances, particularlyfactor XIa which was identified as the probable primarycause of these incidents (Roemisch et al., 2011;Etscheid et al., 2012). Moreover, in a preparation forsubcutaneous administration, a high concentration ofprekallikrein/kallikrein was observed (Schulte, 2011).

As a result, the monographs on "Human normalimmunoglobulin for intravenous administration (0918)"and "Human normal immunglobulin (0338)" wererevised by regulatory authorities. Subsequently, allmanufacturers producing immunoglobulin productsfrom human plasma were required to demonstrate thatcertain manufacturing steps were in place capable ofremoving thrombosis-generating agents. It also had tobe shown that the final immunoglobulin product "doesnot exhibit thrombogenic (procoagulant) activity" (EMAmonograph 0918).

Therefore, Biotest carried out a thorough investigationof its manufacturing processes of IVIGs andsubcutaneous immunoglobulins with regard to thethrombogenic potential of these products. Specifically,Biotest invest igated non-act ivated part ialthromboplastin time (NaPTT), thrombin generationassay (TGA), factor XIa activity, total proteolyticactivity (PA) and prekallikrein (PKK) levels at variousproduction steps, and a variety of additionalcoagulation factors in the final drug product. In order tocomply with the regulatory request for validation of onemanufacturing step for the reduction of thrombogenicsubstances, Biotest choose to validate the mostcapable process step which incorporates caprylic acidtreatment in order to reduce thrombogenic substances.Furthermore, Biotest tested for the absence of variouscomponents of the coagulation cascade (factors VIII,IX, X, XI, XII, and the activated factors IIa, IXa, Xa) inall Biotest immunoglobulin products in order todetermine the safety of these products.

In general, Biotest incorporates several approaches toensure production of efficacious products withfavorable safety profiles, in turn providing maximalpatient safety. As for all products from Biotest, qualityof their immunoglobulin products is based on anumber of parameters. These include aspectsregarding process understanding, thoroughperformance of production and quality, and continuouspharmacov ig i lance dur ing the pre- and



post-authorization phase of any product (illustration 1).

Through full understanding of the process, themanufacturing steps were designed so that quality canbe permanently assured. As a result of the revision ofthe monograph, a detailed risk analysis was performedand every process step was evaluated with regard tothe potential generation or reduction of thrombogenicsubstances by activation/concentration of clottingfactors, or inactivation/removal of clotting factors.

With two major production steps (fractionation offraction I/III and treatment with caprylic acid) for thereduction of thrombogenic substances, and continuousprocess controls, Biotest has incorporated stringentmeasures for manufacturing a safe immunoglobulinproduct. The incorporation of these two steps foreffective reduction of thrombogenic substancesillustrates the commitment of Biotest to the quality ofits products and hence patient's safety which is ofmajor importance to the company.

As well as reducing the thrombogenic potential atcertain steps in the manufacturing process, to test thesafety of Biotest ’s IVIG products, furthermeasurements regarding the levels of thrombogenicsubstances in the final drug product were performed.

Finally, Biotest's pharmacovigilance activities ensurethat the safety of the therapeutic use of the product iscontinuously and thoroughly monitored so that anunexpected signal, even if very rare, would be reliablydetected and managed. Monitoring includes collection,documentation, evaluation and assessment ofsuspected adverse drug reactions and qualitycomplaints originating from all sources worldwide andthroughout the life-cycle of a product.

Methods

Materials

For reference purposes a serum pool was preparedfrom at least 20 donors to eliminate individual assayvariability which could occur, as has been describedpreviously (Roemisch et al., 2012). This serum poolserved as a positive control for the NaPTT, TGA andFXIa assays and was stored at ≤ -70°C within 8 hoursof blood sampling. For each batch of serum pool,assay specific target tolerance ranges weredetermined from at least 6 mutually independentmeasurements.

Analytical Methods

Non-activated partial thromboplastin time (NaPTT)measurement

The NaPTT of immunoglobulins was performed on the

basis of European Pharmacopoeia 2.6.22 with anadaptation to 1:4 dilution, in place of 1:10 and 1:100dilutions, to increase sensitivity for thrombogenicsubstances possibly contained in immunoglobulins. Inaddition to the coagulation time in seconds, thenormalized coagulation time (as related to thecoagulation time of the test buffer) was reported as apercentage.

Thrombin generation assay (TGA)

Thrombin generation in samples was measured usinga POLARstar Omega multidetection fluorescencepolarization microplate reader (BMG LABTECH) usingTGA reagents and calibrator manufactured byTechnoclone GmbH. In brief, 60 µl substrate-RCHmixture and 30 µl deficient plasma were used per well.For the calibration series no deficient plasma wasused. 40 µl of the corresponding calibrator or 10 µl ofthe corresponding sample were analyzed. If necessary(e.g. thrombin peak above calibration), a dilution of thesample in Tris-NaCl-Buffer with 0.5% BSA wasperformed. Each measurement was determined atleast twice. Deficient plasma, e.g. FXI-deficient plasmafor immunoglobulins, was used as the basis fordetermining the thromboembolic potential ofimmunoglobulins.

Factor XIa (FXIa) activity

FXIa activity in samples was determined using achromogenic assay (BIOPHEN Factor XIa, A220412).The assay was performed as described by themanufacturer using a kinetic method in a coagulationanalyzer. Control and samples were measured at leasttwice.

Prekallikrein (PKK) measurements

Plasma kallikrein activator (Unicorn 0071, containingellagic acid, phospholipid and a plasma fraction withFXII and high molecular weight kininogen) convertsprekallikrein to kallikrein. The resultant kallikreinactivity can then be determined kinetically viaproteo ly t ic c leavage of the chromophorpara-nitroaniline from the chromogenic substrateS-2302 (Chromogenix). The concentration increase ofcleaved para-nitroaniline was determined at 405 nmover time which correlates with prekallikrein activity.The assay was performed using an automated kineticmethod in a coagulation analyzer. Control andsamples were measured at least twice. NormalReference Plasma was used for calibration andReference Control Normal as the positive control (bothfrom Precision Biologics).

Total proteolytic activity (PA)

For the assessment of total proteolytic activity,



samples were analyzed using a kinetic method withthe chromogenic substrate S-2288 (Chromogenix).The change in absorption per minute is directlyconverted into U/l as described by the manufacturer ofthe chromogenic substrate (Chromogenix).

Factor IIa (FIIa), Factor Xa (FXa) and Factor IXa (FIXa)activity

Activity of activated coagulation factors IIa and Xa wasdetermined using an automated kinetic chromogenicmethod with S-2238 (FIIa) or S-2765 (FXa) as thechromogenic substrate in a coagulation analyzer.Purified factors IIa (Enzyme Research) or Xa(Chromogenix) were used for calibration. Activity offactor IXa was determined using the BIOPHEN FactorIXa test kit (Hyphen BioMed) adapted to an automatedkinetic method.

Factor VIII (FVIII), Factor IX (FIX), Factor X (FX),Factor XI (FXI) and Factor XII (FXII) activity

The activity of coagulation factors VIII, IX, X, XI andXII was determined using an automated kinetic clottingmethod with Actin FSL (FVIII, FIX, FXI, FXII) orInnovin (FX) as an activator (Siemens) and therespective deficient plasmas in a coagulation analyzer.Standard human plasma (Siemens) was used forcalibration.

Pharmacovigilance

At Biotest, product safety is of central importance.High product qual i ty is complemented bycomprehensive pharmacovigilance proceduresthroughout the development of a medicinal product,from early-phase cl inical tr ials through topost-authorization and until the end of a product's lifecycle. During the latter, safety data spontaneouslyreported from sources such as healthcareprofessionals and/or pat ients, as wel l ascomprehensive real- l i fe safety data f romnon-interventional studies are collected.

Following the observed issue on thromboembolicevents (TEEs), EU marketing authorization holders ofimmunoglobulin preparations were requested toperform an evaluation of the TEE status for the timeperiod January 2001 to December 31st, 2011.

At Biotest, the evaluation was performed for theintravenous (iv) immunoglobulin Intratect and thesubsequent iv "FH" family, including Cytotect CP (FH),Hepatect CP (FH), Varitect CP (FH), and theimmunoglobulins for subcutaneous (sc) administration,Zutectra and Fovepta.

The Biotest global pharmacovigilance database wassearched for TEEs from all sources (all reporters), byevery category (spontaneous reporting, clinical trials

and non-interventional studies) using the StandardMedDRA Query (SMQ) 'Embolic and thromboticevents'. Cases identified as a result of this searchwere re-evaluated manually by expert health careprofessionals experienced in pharmacovigilance.

Results

Validation of the process step "Treatment with caprylicacid"

A major requirement of the regulatory authorities wasthe validation of one production step capable ofreducing thrombogenic substances during themanufacture of immunoglobulin products. For thisvalidation, the production step using treatment withcaprylic acid was chosen since this is the mosteffective step for the reduction of thrombogenicsubstances. During this manufacturing step, theeffectiveness of different amounts of caprylic acid(standard, low and very low) and different pHs(standard, low or high) in reducing thrombogenicsubstances, were tested. Samples were analyzedaccording to the above described methods (TGA,NaPTT, Factor XIa, PA and PKK) before and aftertreatment with caprylic acid. Results are shown inillustrations 2-5.

Measurement of Biotest drug product: Intratect

Further evidence regarding the safety of the productIntratect was gained by detailed measurement ofvarious parameters with thrombogenic potential in thisproduct. It could be shown, that all evaluatedparameters were below the limit of quantification forIntratect (illustrations 6 and 7). Thus a reduction ofthrombogenic substances throughout the productionprocess was achieved, conferring an improved safetyprofile for this product.

Measurement of Biotest drug products: Allimmunoglobulin products

In order to assess the safety of all of Biotest’simmunoglobulin products, these were evaluated usingstandard test methods and the NaPTT and TGAanalyses. It could be shown that in all preparations, areduction of thrombogenic substances was achieved(illustrations 8 and 9).

Pharmacovigilance

For Cytotect CP (FH), Hepatect CP (FH), Varitect CP



(FH), Zutectra and Fovepta no TEE reports have beenreceived during the reporting period (2001 to 2011).

For Intratect, a total of 21 reports on TEEs have beenreceived during that time period; whereas 14 out ofthese 21 reports originated from a non-interventionalobservational study (NIS), one (1) report originatedfrom a clinical trial, and 6 reports were obtained fromspontaneous reporting.

Notably, during the Biotest NIS, an advanced adverseevent reporting scheme, comparable to that used ininterventional clinical trials, was performed. Alladverse events (AEs) were collected and documentedindependent from a causal relationship and on thisbasis adverse drug reactions (ADRs) were identifiedbased on medical assessment: 9 out of the 14 AEsfrom the NIS were assessed as "not related" and 2 (of14) were assessed as "unlikely related" to Intratect bythe reporting physician and also by the company. Theremaining 3 (of 14) AEs were regarded as suspectedADRs (at least possibly related).

The 1 report received from a clinical trial (studynumber 960) was assessed as causally 'not related' toIntratect by the investigator and also by the company.

For the 6 unsolicited spontaneous reports, theprinciple of implied causality was applied. Thesereports correspond cumulatively to approximately340,000 Defined Single Standard Doses of Intratect(30 g protein) respective infusions.

Of the 6 spontaneous TEE reports for Intratect, 2events occurred in 2008, 1 event in 2010, and 3events in 2011. In all other calendar years, no TEEreports were received for Intratect. The reporting rateper year relative to exposure, derived from thespontaneous reporting is as follows:

1. 2008 (2 spontaneous reports): 1 report per 739,360 g protein distributed, or 1 report per 24,645 Defined Single Standard Doses2. 2010 (1 spontaneous reports): 1 report per 1,960,635 g protein distributed, or 1 report per 65,355 Defined Single Standard Doses3. 2011 ( 3 spontaneous reports): 1 report per 803,362 g protein distributed, or 1 report per 26,779 Defined Single Standard Doses4. 2001 – 2007; 2009:: Reporting rate= 0

In conclusion, for the period evaluated (January 2001to 31 December 2011) there were no findings

indicating an unexpected increase in thromboembolicevents (TEEs) with the Biotest immunoglobulin FHproduct family, either in terms of severity, nature,frequency or patient population at risk.

Discussion

Particularly when unexpected in terms of nature,frequency or severity, suspected adverse drugreactions (adverse events considered causally relatedto medicinal products) are always of concern to allstakeholders in the healthcare system, and especiallyfor patients and prescribers. As a result of aninvestigation into an increased frequency ofthromboembol ic adverse events wi th animmunoglobulin preparation from one manufacturer,root cause analysis revealed occasionally increasedconcentrations of activated FXI, correlating withelevated levels measured in the TGA (Roemisch et al.,2011) and increased levels of prekallikrein/ kallikrein(Etscheid et al., 2012). As a result, regulatoryauthor i t ies rev ised the monographs forimmunoglobulin products (EMA monographs 0918"Human normal immunoglobulin for intravenousadministrat ion" and 0338 "Human normalimmunoglobulin") insofar that all manufacturers ofplasma derived products were required to investigateboth end-product and production processes carefullyin relation to thrombogenic substances.

To comply with implementation of the revisedmonographs, Biotest conducted a thorough anddetailed analysis of its overall production process,including validation of one production step in relationto the reduction of thrombogenic substances. Caprylicacid treatment is well known for eliminating impuritiesand also potentially thrombogenic substances (Moraisand Massaldi, 2012) and on the basis of this, Biotestchose this process step for validation. In the laboratorythe process was validated using both standardparameters and sub-optimal conditions, identified aslow amount of caprylic acid added per kg solution anda high pH value. Fraction II from an intermediatesupplier was also included in the investigations forcomparison and experimental control.

Analyses included the TGA, NaPTT and PA tests forgeneral assessment of the occurence of thrombogenicsubstances both in samples before and after caprylicacid treatment. Factor XIa and prekallikrein weretested to specifically address the root cause ofbiochemical parameters leading to TEEs in samplesbefore and after caprylic acid treatment and batches ofimmunoglobulin product (Intratect).



In this study it could be shown that all parametersinvestigated were substantially reduced in terms ofthrombgenic activity at the production step usingcaprylic acid. A reduction of all thrombogenicsubstances measured, was accomplished by thetreatment with caprylic acid, even when usingsub-optimal conditions (low amount of caprylicacid/high pH). Therefore this step proved to beeffective in generating a safer immunoglobulin product.Hence it could be shown in detail that thrombogenicsubstances are reduced by this validated productionstep, as required by authorities in the fulfillment of thechange to the monographs for immunoglobulinproducts.

These data were further supported by measurementsof the various coagulation and activated coagulationfactors (factors VIII, IX, X, XI, XII, and the activatedfactors IIa, IXa, Xa) in the drug product Intratect atdifferent concentrations (50 g/l and 100 g/l). At bothconcentrations, all measurements were below the limitof quantification (illustrations 6 and 7), confirming thequality of the immunoglobulin product in terms ofthrombogenic substances.

Investigations of other products in the Intratect familyand other Biotest immunogloblin products, CytotectCP, Hepatect CP, Varitect CP and Zutectra, showedthat no critical levels of thrombogenic substances werepresent as measured using the global tests of NaPTTand TGA.

A comprehensive and detai led analysis ofpharmacovigilance data did not indicate an increasedrisk of TEEs with the Biotest FH IVIG product family.No batch-related or time-dependent cluster has beenidentified, nor any new information exceeding existingclass related warnings and precautions on TEEs withregard to frequency, patient population and risk factors.

Overall, the Biotest "FH" product family has awell-established benefit-risk profile. The presenteddata do not provide any further evidence of previouslyunknown risks or safety issues with regard to TEEs.Therefore, Biotest has demonstrated that an effectiveproduction process is in place for the manufacture oftheir immunoglobulin products with a validated step forreducing thrombogenic potential resulting in saferproducts. Together, with an overall understanding ofthe processes involved from production, continuousquality control, in-depth analytical knowledge and postmarketing surveillance, Biotest is able to ensure thecontinuous high quali ty and eff icacy of i tsimmunoglobulin products for optimal treatment ofpatients.

Abbreviations

ADR: Adverse drug reaction

AE: Adverse event

EMA: European Medicines Agency

F: Coagulation factor

IVIG: Intravenous immunoglobulin

NaPTT: Non-activated partial thromboplastin time

NIS: Non-interventional observational study

PA: Total proteolytic activity

PKK: Prekallikrein

TEE: Thromboembolic event

TGA: Thrombin generation assay

References

1. EMA (European Medicines Agency) Guidelines2012. Guideline on core summary of productcharacteristics for human normal immunoglobulin forintravenous administration (IVIg) revision 4. Referenceno: EMA/CHMP/BPWP/94038/2007 Rev. 4. Availableat: http://www.ema.europa.eu2. EMA (European Medicines Agency) Monograph0338: Human normal immunoglobulin. Available at:www.ema.europa.eu3. EMA (European Medicines Agency) Monograph0918: Human normal immunoglobulin for intravenousadministration. Available at: www.ema.europa.eu4. Etscheid M, Breitner-Ruddock S, Hunfeld A, Seitz R,Dodt J. Identification of kallikrein and FXIa asimpurities in therapeutic immunoglobulins: implicationsfor the safety and control of intravenous bloodproducts. Vox Sang, 2012; 102 (1): 40-6. 5. Funk MB, Gross N, Gross S, Hunfeld A, Lohmann A,Guenay S, Hanschmann KM. Thromboembolic eventsassociated with immunoglobulin treatment. Vox Sang,2013; 105 (1): 54-64. 6. Morais V, Massaldi H. A model mechanism forprotein precipitation by caprylic acid: Application toplasma purification. Biotechnology and AppliedBiochemistry, 2012; 59 (1): 50-54.7. Roemisch JR Zapfl C, Zoechling A, Pock K.Comparison of Thrombin Generation Assay (TGA) andNon-Activated Partial Thromboplastin Time (NaPTT)for the Assessment of Enhanced Procoagulant Activityin Immunoglobulin Solutions. WebmedCentralImmunotherapy 2012; 3 (4): WMC003247.8. Roemisch JR, Kaar W, Zoechling A, Kannicht C,



Putz M, Kohla G, Schulz P, Pock K, Huber S, Fuchs B,Buchacher A, Krause D, Weinberger J, Rempeters G.Identification of Activated FXI as the MajorBiochemical Root Cause in IVIG Batches Associatedwith Thromboembolic Events. Analytical andExperimental Approaches Resulting in Corrective andPreventive Measures Implemented into the OctagamManufactur ing Process. WebmedCentra lImmunotherapy 2011; 2 (6): WMC002002.9. Schulte S. Characterization of proteolytical Activityin IgG. Vivaglobin. Risk Mitigation Strategies toAddress Potential Procoagulant Activity in ImmuneGlobulin Products Presentations. FDA, U.S. Food andDrug Administration. Department of Health & HumanS e r v i c e s 2 0 1 1 .http://www.fda.gov/downloads/BiologicsBloodVaccines/NewsEvents/WorkshopsMeetingsConferences/UCM260780.pdf



Illustrations

Illustration 1

Quality by design driven approach to gain high quality and safe IgG products.



Illustration 2

TGA before and after treatment with caprylic acid (CA). Peak thrombin levels (%) for samples, compared to Normal ReferencePlasma, are shown. 1, 2: Standard amount of CA, standard pH; 3: Low amount of CA, high pH; 4 Standard amount of CA, standardpH; 5: Standard amount of CA, high pH; 6: Low amount of CA, standard pH; 7: Low amount of CA, high pH; 8: Very low amount ofCA, standard pH; 9: Very low amount of CA, high pH.



Startingmaterial

Experimental parameter

Amount caprylic acid [g/kg]and pH

pH-valueafter

caprylicacid

treatment

TGAThrombin [nM]

Beforecaprylic

acidtreatment

Aftercaprylic

acidtreatment

Fraction IIfrom

supplier

Standard amount CA/kg solution;standard pH 5.5

Average 572.5 116.7SD 7.65 18.44


Average 553.2 93.7SD 17.44 6.75

Low amount CA/kg solution;high pH 5.6

Average 392.8 306.5SD 1.00 17.67

Fraction IIfrom

Biotest


Average 345.5 61.4SD 17.23 17.49

Standard amount CA/kg solution;high pH 5.6

Average 349.9 75.5SD 32.21 3.44

Low amount CA/kg solution;standard pH 5.5

Average 328.0 71.1SD 8.01 4.35

Low amount CA/kg solution;high pH 5.6

Average 346.8 83.8SD 15.13 11.07

Very low amount CA/kg solution;standard pH 5.5

Average 364.0 125.4SD 17.72 19.84

Very low amount CA/kg solution;high pH 5.6

Average 327.7 120.1SD 8.60 29.13

Illustration 3

Measurement of samples with the TGA before and after treatment with caprylic acid (CA). Peak thrombin levels (nM) are shown.Starting material from different sources (intermediate supplier; Biotest) and various combinations of relevant parameters (amountcaprylic acid and pH) were evaluated. SD, ± standard deviation.



Illustration 4

NaPTT before and after treatment with caprylic acid (CA). The percentage of the value compared to the buffer blank is shown. 1, 2:Standard amount CA, standard pH; 3: Low amount CA, high pH; 4 Standard amount CA, standard pH; 5: Standard amount CA,high pH; 6: Low amount CA, standard pH; 7: Low amount CA, high pH; 8: Very low amount CA, standard pH; 9: Very low amountCA, high pH.



Startingmaterial

Experimental parameterAmount caprylic acid [g/Kg]

and pH

FXIa PKK PAng/ml % of Norm U/l

beforecaprylic

acidtreat-ment

aftercaprylic

acidtreat-ment

beforecaprylic

acidtreat-ment

aftercaprylic

acidtreat-ment

beforecaprylic

acidtreat-ment

aftercaprylic

acidtreat-ment

Fraction IIfrom supplier

Standard amount CA/kg solution;standard pH

Average 161.9 0.3 34.2 <2.5 994.0 4.3SD 14.14 0.07 0.28 n. d.* 11.31 0.33


Average 142.5 0.3 35.8 <2.5 814.0 3.2SD 8.06 0.02 1.63 n. d.* 243.24 0.35

Low amount CA/kg solution;high pH

Average 211.6 1.0 26.4 <2.5 845.0 9.9SD 4.81 0.18 1.63 n. d.* 44.55 0.99

Fraction IIfrom Biotest


Average 0.4 0.2 2.9 <2.5 74.2 <3.0SD 0.49 0.04 0.00 n. d.* 6.65 n. d.*

Standard amount CA/kg solution; highpH

Average 0.4 0.2 3.1 <2.5 81.2 3.5SD 0.44 0.01 0.21 n. d.* 8.06 0.37

Low amount CA/kg solution; standardpH

Average 0.8 0.3 3.3 <2.5 82.2 4.9SD 0.18 0.06 0.07 n. d.* 1.13 0.45

Low amount CA/kg solution;high pH

Average 0.5 0.3 <2.5 <2.5 67.6 4.2SD 0.57 0.07 n. d. n. d.* 2.69 1.75

Very low amount CA/kg solution;standard pH

Average 0.9 0.5 <2.5 <2.5 77.5 6.7SD 0.26 0.10 n. d. n. d.* 4.35 0.92

Very low amount CA/kg solution; highpH

Average 0.4 0.4 3.3 <2.5 101.4 13.1SD 0.44 0.14 0.07 n. d.* 12.73 1.51

*n. d. – not determinable

Illustration 5

Measurement of samples for factor XIa (FXIa), prekallikrein (PKK) and proteolytic activity (PA) before and after treatment withcaprylic acid (CA). Peak thrombin levels (nM) are shown. Starting material from different origin and various combinations ofrelevant parameters (amount caprylic acid and pH) were evaluated. SD, ± standard deviation.



Parameter Unit Intratect(50 g/l)

Intratect (50 g/l)with fraction IIfrom supplier

Intratect(100 g/l)

Mean /Median

Maxi-mum

Factor VIII U/ml < 0.05 < 0.05 < 0.05 < 0.05 < 0.05Factor IX U/ml < 0.05 < 0.05 < 0.05 < 0.05 < 0.05Factor X U/ml < 0.10 < 0.10 < 0.10 < 0.10 < 0.10Factor XI U/ml < 0.05 < 0.05 < 0.05 < 0.05 < 0.05Factor XII U/ml < 0.05 < 0.05 < 0.05 < 0.05 < 0.05

Illustration 6

Analysis of coagulation factors present in: Intratect (50 g/l), Intratect (50 g/l) produced from fraction II of an intermediate supplierand Intratect (100 g/l).



Parameter Unit Intratect(50 g/l)

Intratect (50 g/l)with fraction IIfrom supplier

Intratect(100 g/l)

Mean /Median

Maxi-mum

Factor IIa IU/ml < 0.063 < 0.063 < 0.063 < 0.063 < 0.063Factor IXa mU/ml < 0.2 < 0.2 < 0.2 < 0.2 < 0.2Factor Xa IU/ml < 0.05 < 0.05 < 0.05 < 0.05 < 0.05

Illustration 7

Analysis of activated coagulation factors in: Intratect (50 g/l), Intratect (50 g/l) produced from fraction II of an intermediate supplierand Intratect (100 g/l).



Illustration 8

Measurement of Biotest immunoglobulin (IVIG) products with the NaPTT assay. The critical level was set to 150 seconds.



Illustration 9

TGA measurement of Biotest immunoglobulin (IVIG) products. The critical level was set at 350 nM peak thrombin.


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