Contamination of therapeutic human immunoglobulin preparations with apolipoprotein H...

Electrophoresis 2014, 35, 515–521 515

Friedrich Lackner1

Gerhard Beck1

Stephanie Eichmeir1

Manfred Gemeiner2

Karin Hummel3Dieter Pullirsch1

Ebrahim Razzazi-Fazeli3Alexandra Seifner1

Ingrid Miller2

1AGES Medizinmarktaufsicht,Vienna, Austria

2Institute of MedicalBiochemistry, Department ofBiomedical Sciences, Universityof Veterinary Medicine Vienna,Austria

3VetCore Facility, University ofVeterinary Medicine Vienna,Austria

Received July 11, 2013Revised September 10, 2013Accepted October 2, 2013

Research Article

Contamination of therapeutic humanimmunoglobulin preparations withapolipoprotein H (�2-glycoprotein I)

Polyclonal immunoglobulin (Ig) concentrates are important biological medicinal productsand the assurance of their quality and safety is crucial. In our present approach we usedproteomic methods to check the purity of commercial Ig products of different origin. Theexperimental setup included nonreducing 2DE or DIGE combined with MALDI-TOF andthe thrombin generation assay, a routine safety test for pharmaceutical Ig preparations,and was complemented by a specific immunoassay. 2DE patterns displayed contamina-tions with trace amounts of human apolipoprotein H (Apo-H), transferrin, albumin, andits fragments. In contrast to the latter, Apo-H is a protein that is active in the coagula-tion cascade, and thus a potential involvement in thromboembolic events in vivo cannotbe excluded. It was found by 2DE and MALDI-TOF to be a contaminant of several Igpreparations. Spiking experiments of Ig preparations with pure Apo-H demonstrated anApo-H concentration dependent increase in thrombin generation assay values. Traces ofApo-H are possibly also contributing to unwanted side effects, as already known for factorXIa. The significance of Apo-H contaminations for these side effects might be verified bydetailed analyses of pharmacovigilance data.

Keywords:

2DE / Human apolipoprotein H / Immunoglobulin concentrates / Thrombin gen-eration assay / Thromboembolic events DOI 10.1002/elps.201300319

� Additional supporting information may be found in the online version of thisarticle at the publisher’s web-site

1 Introduction

In recent years usage of immunoglobulin (Ig) concentrates,namely for intravenous application, has grown significantlyin a variety of medical areas. Presently, their main applica-tion is in replacement therapy in immune deficiencies, butthe indications are ever widening. Igs are increasingly im-portant for global health care, being used for the therapy oflife-threatening acute and chronic diseases. As this class of bi-ological medicinal products is purified from human plasma,the growing global demand for Igs poses a challenge for the

Correspondence: Ingrid Miller, Department fur BiomedizinischeWissenschaften, Institut fur Medizinische Biochemie, Veterinar-medizinische Universitat Wien, Veterinarplatz 1, A-1210 Vienna,AustriaE-mail: [email protected]: +431-25077-4290

Abbreviations: Apo-H, apolipoprotein H; EP, European Phar-macopoeia; i.m., intramuscular; i.v., intravenous; s.c., subcu-taneous; TEE, thromboembolic event; TGA, thrombin gener-ation assay

plasma fractionation industry [1]. It is unlikely that replace-ment therapy could be provided by recombinant technology,as Ig preparations must contain the full range of protectiveantibodies in order to provide prophylaxis against infections.The concern of regulators to minimize the transmission ofblood-borne diseases has also caused severe shortages of Igs[2]. If Ig products are intended to be marketed in Europe, theyhave to comply with the monographs of the European Phar-macopoeia (EP). Polyclonal Ig concentrates are manufacturedby initial processing of large pools of human plasma by coldethanol precipitation, followed by different methods of purifi-cation and virus inactivation. Apart from their main proteincomponent IgG, they consist of complex mixtures of pro-teins originating from plasma and of modified proteins anddegradation products from fractionation, purification and vi-ral inactivation steps. Igs are currently controlled using assaysdescribed in the monographs of the EP. Monographs requesta purity of at least 95% for intravenously (i.v.) and at least90% for intramuscularly (i.m.) and subcutaneously (s.c.) ap-plied Igs, as determined with zone electrophoresis. Another

Colour Online: See the article online to view Fig. 3 in colour.

C© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

516 F. Lackner et al. Electrophoresis 2014, 35, 515–521

Table 1. Results for commercial Ig products

Igproduct

Description and comments Proteinmg/mL

TGA(NP/DP) (nMThrombina))

Elisa(Apo-H)

2DE (Apo-Hspecific spottrain)b)

MS confirmations for:

A Ig sample from a collaborative study 27 580/85 nd +, × ndB Routine sample, i.v. Ig 50 589/552 ++ × Albumin, transferrinC Routine sample, i.v. Ig 50 247/nd nd - ndD i.v. Ig 50 707/647 ++ + Apo-HE s.c. Igc) 145 824/803 - × ndF Routine sample, i.m. Ig 160 756/794 - - ndG Ig sample from a collaborative study 35 769/814 nd - TransferrinH Ig sample from a collaborative study 34 812/843 + + Apo-HI Ig sample from a collaborative study 32 728/748 + + Apo-HJ Ig sample from a collaborative study 32 659/662 nd -, × Albumin, transferrin

nd: not done. TGA results for therapeutic Ig preparations are given for human normal and factor XI depleted plasma. Results above 350nmol thrombin/L indicate increased risk for TEEs after application. NP = human normal plasma, DP = factor XI depleted plasma. Igsamples A, G, H, I, and J were provided by NIBSC, samples D and E by ANSM.a) 350 nM is regarded as cut-off (high risk of TEEs).b) x, other spots in this gel area.c) Involved in TEEs; withdrawn by manufacturer, not available on the market.

requirement is a sum of peak areas of monomers and dimersof at least 90% of the total area of the chromatogram for i.v.Igs and of 85% for i.m. and s.c. Igs, respectively, as deter-mined with size exclusion chromatography. However, thereis a need for improved analytical methods to further assessthe safety, identity, and purity of these products. In 2011,there were reports about unexpectedly increasing numbersof thromboembolic events (TEEs) occurring in patients af-ter donations of i.v. Igs of a pharmaceutical manufacturer[3]. The thrombin generation assay (TGA) is a universal testfor measuring the kinetics of thrombin generation in vitro.Recently, the TGA was modified to measure thrombogenicimpurities in i.v. Igs [4]. It is currently used routinely assafety test in order to detect contaminations of Ig batcheswith procoagulant impurities [5], as TGA positive batcheswere found to be involved in the occurrence of deep TEEs. Anactivated coagulation factor, namely factor XIa, remaining inone product after plasma fractionation, was identified as theroot cause of TEEs. However, other impurities not detected bythe standard tests of the EP cannot be excluded as additionalcauses of TEEs. Thrombogenic impurities were found withthe TGA also in other i.v., i.m., and s.c. applied Igs [6]. Mono-graphs of the EP are amended to deal with this challenge.New monographs of the EP require the absence of thrombo-genic impurities, but suitable analytical methods are not yetdefined.

We describe orthogonal analytical methods to detect pos-sibly harmful protein impurities in Igs. This combinationmay be useful to evaluate the quality and safety of Igs alsoregarding other than the described trace proteins. We usedthe TGA, a highly sensitive fluorometric method recently es-tablished to detect thrombogenic impurities, and a modified2DE method, applied also as DIGE, to get a general view overcontaminating proteins, and MALDI-TOF MS for identifica-tion of contaminants.

2 Materials and methods

2.1 Samples

The apolipoprotein H (Apo-H) used as reference substancewas purchased from Fitzgerald Industries International (Ac-ton, MA). It is a highly purified glycoprotein from humanserum. According to the Certificate of Analysis AntigenDatasheet of the manufacturer, a contamination with humanIgs is not detectable (0.1% of total protein), and the manu-facturer claims that due to the purification method a possiblecontamination with coagulation factor XIa would have beeninactivated by plasma protease inhibitors (personal commu-nication).

The Ig samples used in this study were obtained from amanufacturer (Octapharma, Vienna, Austria) and from twoOMCLs (Official Medicines Control Laboratories), the ANSM(Agence Nationale de Securite du Medicament et des Produitsde Sante, Paris, France), and the NIBSC (National Institutefor Biological Standards and Control, London, UK), and arelisted in Table 1.

2.2 Electrophoresis

2.2.1 Nonreducing 2DE with silver staining

The here-applied 2DE is based on widely used protocols asdetailed [7]. In brief, the method is based on IPGs on home-made 10–11 cm strips of pH 4–10 NL and for the second di-mension on 140 × 140 × 1.5 mm SDS-PAGE gels of 10–15%T, 2.7% C, run in the classical Laemmli system (in an SE600chamber, Hoefer Scientific Instruments, San Francisco, CA).The only necessary modification was exclusion of reducingand alkylation agents in all steps [8, 9], to avoid spreading of


Electrophoresis 2014, 35, 515–521 Proteomics and 2DE 517

Ig chains over the whole gel pattern. An MS-compatible mod-ified Heukeshoven silver stain was performed for detection[7]. Gels were scanned with an ImageScanner III under thecontrol of the software LabScan v6.0 (both GE Healthcare LifeSciences, Munich, Germany). Although gels were run understandardized conditions, 2DE was performed for qualitativereasons only, due to the presence of the huge saturated Igspot.

2.2.2 2D-DIGE

The above-described 2DE system was also used for DIGE.Labeling of the samples prior to electrophoresis with fluores-cent minimal CyDyes R© (GE Healthcare) was performed asrecommended by the manufacturer and as described earlier[7]. Images were scanned on a Typhoon 9400 (GE Health-care). In the present case, the DIGE concept was only appliedto qualitatively compare protein spot patterns of differentsamples and to confirm co-migration with a commerciallyavailable pure substance.

2.3 Mass spectrometry

2.3.1 Sample preparation

Spots of interest were excised, washed, destained [10], re-duced with DTT, and alkylated with iodoacetamide [11]. In-gel digestion was performed with trypsin (Trypsin Gold, MassSpectrometry Grade, Promega, Madison, WI) [12] with a finaltrypsin concentration of 20 ng/�L.

2.3.2 Data acquisition by MALDI TOF/TOF

Dried peptides were concentrated and desalted using mi-crobed C18 Zip-Tips (Millipore, Bedford, MA) according tothe manufacturer’s instructions. Desalted peptides (0.5 �L)were spotted onto a disposable AnchorChip MALDI targetplate prespotted with alpha-cyano-4-hydroxycinnamic acid(PAC target, Bruker Daltonics, Bremen, Germany). Data wereacquired on a MALDI-TOF/TOF mass spectrometer (Ultra-flex II, Bruker Daltonics) in MS and MS/MS modes. Spectraprocessing and peak annotation were carried out using Flex-Analysis and Biotools (Bruker Daltonics).

2.3.3 Data acquisition by nano-LC-ESI MS/MS

Dried peptides were reconstituted in 4 �L of 0.1% TFA. Nano-HPLC separation was performed on an Ultimate 3000 RSLCsystem (Dionex, Amsterdam, the Netherlands). Sample pre-concentration and desalting was accomplished with a 1 mmAcclaim PepMap �-precolumn (Dionex). The separation wasperformed on a 15 cm Acclaim PepMap C18 column (75 �mid, 3 �m particle size, and 100 A pore size) with a flow rate of

300 nL/min. The gradient starts with 4% B (80% ACN with0.1% formic acid) and increases to 55% B in 30 min. It is fol-lowed by a washing step with 90% B for 5 min. Mobile Phase Aconsists of mQH2O with 0.1% formic acid. Injection volumewas 1 �L partial loop injection mode. For mass spectrometricanalysis an HCT Esquire mass spectrometer (Bruker Dalton-ics) was used. Data were directly analyzed using HyStar Soft-ware 3.2 (Bruker Daltonics) combining Esquire Control 6.1(Bruker Daltonics) and the Chromeleon DCMS link (Dionex)as well as ProteinScape 2.0 (Bruker Daltonics).

2.3.4 Data analysis

Processed spectra were searched via an in-house Mas-cot server (Matrix Science, Boston, MA) in the Swiss-Protdatabase using the following search parameters: taxonomy:mammals; global modifications carbamidomethylation oncysteine; variable modifications: Oxidation (M); MS tolerance100 ppm; MS/MS tolerance 1 Da; one missed cleavage al-lowed. Identifications were considered statistically significantwhere p < 0.05.

2.4 Thrombin generation assay

The TGA is based on monitoring the fluorescence generatedby thrombin cleavage of a fluorigenic substrate over timeupon activation of the coagulation cascade by the presence oftissue factor and negatively charged phospholipids in plasma.From the changes in fluorescence over time, the concentra-tion of thrombin (nmol/L) in the sample can be calculatedusing the respective thrombin calibration curve.

In our laboratory, the TGA for the determinationof thrombin generating potential in IgG samples wasperformed as reported in detail [6]. The “TGA Cal Set”consisting of a “thrombin calibrator” and a dilution buffer,the fluorogenic substrate “TGA SUB2” (1 mM Z-GlyGly-Arg-AMC in 15 mM CaCl2) and the activator “TGA RC High”(high concentration of phospholipid micelles, ∼5 pM rhTFin Tris-HEPES-NaCl) were purchased from Technoclone(Vienna, Austria), Precision Biologic FXI deficient plasmaas well as pooled normal plasma from Coachrom (Vienna,Austria). The fluorescence microtiter plate reader was aBiotek FLx800 and operated with the following parameters:Sample Vortex—5 s, measuring interval—60 s, measuringtime—60 min. The excitation wavelength was set to 360 nm,the emission wavelength was 460 nm. The parameter PeakThrombin accounts for the peak height in the first derivativeof the original result. The IgG sample is diluted in plasma toa concentration of about 1%, thereby mimicking the situationin the patient. Samples are analyzed twice and the average isgiven as the result. Intraassay variation corresponds to lessthan 10% and interassay variation to less than 15%.

In the TGA used to assess thrombogenic impurities inIg products [13], 350 nM thrombin are defined as threshold



Figure 1. Silver stained 2DEs of different commercial Ig products. Samples from different manufacturers: (A) product A, (B) product C,(C) product D; for further product details see Table 1. Apo-H specific spots are only seen in product D. Separation of 150 �g protein innonreducing 2DE in a nonlinear pH gradient 4–10 followed by SDS-PAGE (T = 10–15%, C = 2.7%) and MS-compatible silver stain. Markedprotein spots were subjected to MS analysis for protein identification (Table 2, Supporting Information Table 1). Numbering according toTable 2, L- are Ig light chains (�-, �-) [8].

based on preclinical and clinical data. Igs exceeding the cut-off could bear the risk of causing TEEs.

The use of factor XI deficient plasma accounts for the factthat increased factor XIa levels in i.v. Igs have been identifiedas a root cause for TEEs. The use of pooled normal plasma ad-dresses the remaining risk that procoagulants different fromfactor XIa could be present that would have been overseen infactor XI deficient plasma. The TGA with factor XI deficientplasma is presently a standard method for safety testing ofi.v. Igs, as the use of plasma deficient for other coagulationfactors did not result in increased sensitivity.

2.5 Apolipoprotein H ELISA

Apo-H was assayed in Ig preparations using a commerciallyavailable ELISA from Uscn Life Science (Wuhan, China).The test is performed on microplates precoated with anti-Apo-H antibodies. Apo-H antigen in the samples is detectedusing a biotin-conjugated antibody specific for Apo-H, andavidin conjugated to horseradish peroxidase. Colour devel-opment is measured at 450 nm with a microplate reader(Thermo/Labsystems Multiskan, Ascent 2.6 software) andApo-H concentrations calculated based on a standard curve.

3 Results

Commercially available Ig concentrates have been investi-gated with the TGA for their thrombogenic potential, sometypical results are presented in Table 1. Samples with high,intermediate and low thrombogenic potential were analyzedwith 2DE for further characterization. In the chosen nonre-ducing 2DE system IgG is found as a large spot at the upperright end of the 2DE gel (Fig. 1). Thus, the rest of the gel is

available for the separation of minor or trace proteins, whichcan be well detected in silver stained patterns at a high over-all protein load. Figure 1A–C represent three commercialproducts, differing in trace component patterns. The mostfrequent and most abundant contaminants were identified(Table 2) as serum albumin and its fragments, transferrin(Fig. 1B) and Apo-H (Fig. 1C), besides traces of Ig light chains.The position of the latter had already been investigated in aprevious study [8]. The products analysed in Fig. 1 variednot only in composition of trace proteins, but also in TGAlevels. In product C (Fig. 1B) low values for TGA were deter-mined (247 nM thrombin), in contrast to products shown inFig. 1A and C (products A and D, 580 and 707 nM thrombin).MS identification of Apo-H was performed on three differentsamples from independent gels (Table 1). Moreover, furthervalidation of MS identification was sought by DIGE, to con-firm comigration of a commercial Apo-H preparation withthe spot chain identified as Apo-H. Results are given in Fig. 2(DIGE gel after channel separation): Fig. 2A shows a samplewith high TGA value (707 nM thrombin), Apo-H and free Iglight chains as main by-products; Fig. 2B displays a prepara-tion for i.m. use. The Apo-H standard clearly comigrates withthe previously identified spot chain (Fig. 2A and C).

Different Ig concentrates were analysed with nonreduc-ing 2DE, and several of them showed the typical spot chain ofApo-H. Screening the same samples with the Apo-H ELISAsupported these findings, and products with a more markedApo-H spot chain showed higher reactivity in the immunoas-say (Supporting Information Fig. 1). The commercial ELISAkit is designed for human serum containing approximately200 �g/mL Apo-H. The ratio Apo-H/IgG is very unfavor-able in the Ig concentrates, therefore the ELISA values areslightly biased due to matrix effects and may be regarded assemiquantitative only. Nevertheless, we could clearly estimatethat in different Ig products different amounts of Apo-H arepresent (Table 1).



Table 2. Mass spectrometric analysis of protein impurities after 2DE

Spot no. Protein identified (accession number) Mascot score Mr theor. (kD) pI theor. No. of peptides Modifications

1a) Serotransferrin precursor 1423 77.0 7.0 26 Carbamidomethyl: 2TRFE_HUMAN

2b) Serotransferrin precursor 110 77.0 7.0 2TRFE_HUMAN Oxidation: 1

3b) Serotransferrin precursor 164 77.0 7.0 3TRFE_HUMAN Oxidation: 1

4a) Serotransferrin precursor 786 77.0 7.0 17 Carbamidomethyl: 12TRFE_HUMAN Carbamidomethyl: 6

5b) Serotransferrin precursor 254 77.0 7.0 4TRFE_HUMAN Carbamidomethyl: 9

6b) Serum albumin precursor 365 69.3 5.9 7ALBU_HUMAN

7b) Serum albumin precursor 184 69.3 5.9 7ALBU_HUMAN

8b) Beta-2-glycoprotein 1 precursor 184 38.3 9.5 3 Carbamidomethyl: 2APOH_HUMAN Carbamidomethyl: 13



a) Analyzed by nano-LC-ESI MS/MS.b) Analyzed by MALDI-TOF/TOF.

Figure 2. 2D-DIGE of Ig preparations under nonreducing conditions. Samples: (A) product D (Cy3, high TGA), (B) product F (Cy5, highTGA), (C) commercial Apo-H from human serum (Cy2). The train of spots characteristic for Apo-H (C) is easily seen in product D (A), butnot in product F (B). For the Ig preparations, 12.5 �g protein per sample was labeled with Cy3 and Cy5, respectively (8 nmol dye/mgprotein), for Apo-H 2.5 �g protein was coupled with Cy2 (6.4 nmol dye/mg protein). Separation in nonreducing 2DE in a nonlinear pHgradient 4–10 followed by SDS-PAGE (T = 10–15%, C = 2.7%). The scanned image was split into single channels and turned into greylevels.

Additionally, the reactivity of Apo-H substance in theTGA was evaluated. The results of spiking an i.v. Ig withlow TGA activity with Apo-H purified from human plasmaare shown in Fig. 3. We found a linear relationship betweenApo-H concentration and thrombin generated in the TGAfrom 5 to 160 �g/mL both with factor XI depleted and nor-mal human plasma. TGA activity was heat sensitive (65�C,30 min). Endogenous TGA-activity was not quenched or trig-gered by Apo-H. Lag Time and Time to Peak correspondedwith Thrombin generation (data not shown).

4 Discussion

Current requirements for the purity of human normal Ig andhuman normal Ig for i.v. administration are established in therespective monographs of the EP [14, 15]. In our approach,we characterized plasma proteins in Ig preparations with theTGA, 2DE, and MALDI-TOF MS in order to gain a globaloverview over a broad spectrum of minor proteins presentin final product batches. Some abundant proteins as albu-min and transferrin present in large amounts in the source



0

100

200

300

400

500

600

0 20 40 60 80 100 120 140 160 180

Thro

mbi

n (n

M)

µµg Apo-H/ml Ig

Figure 3. Thrombin generated in the TGA. A commercial i.v. Igproduct (Octagam) with low activity in the TGA was spiked withhuman Apo-H. Ten microliters Apo-H solutions were added to 50�L Ig solution each to obtain the indicated Apo-H concentration.TGA was done in factor XI depleted human plasma, as described.Each data point represents the mean of six measurements (threeindependent assays, each measurement in duplicate). TGA activ-ity caused by Apo-H was quenched by heat (65�C, 30 min). LagTime and Time to Peak as described for the TGA were modifiedas expected (data not shown).

material and consequently also found as major contaminantsin final Ig products seem to cause no major safety concernsregarding unwanted side effects in patients according to clini-cal experience. However, other protein contaminants presentonly in trace amounts may cause major adverse reactions, asdemonstrated for the activated coagulation factor XIa. Thisactivated factor, which is highly reactive in the TGA, wasidentified as root cause of TEEs after administration of ani.v. Ig [13]. This impurity was recognized as significant forthe safety of therapeutic Igs [16]. The TGA with factor XI-depleted plasma is now one of the standard tests to detectthrombogenic impurities in therapeutic i.v. Igs.

2DE under reducing conditions has been applied success-fully for the last 35 years for qualitative and quantitative eval-uation of protein patterns of complex mixtures, among themserum/plasma [7]. This approach has recently been foundhelpful in quality control of blood products and monoclonalantibodies [17–19]. We have also used this method previouslyto analyze trace components in human albumin productsfrom different manufacturers and to identify a nongenuineproduct due to its altered “fingerprint protein” pattern [20].

In the present approach, we relied on 2DE under non-reducing conditions as this allowed a better separation of thedominant protein IgG from proteinaceous impurities. As inother applications, this method has been able to visualize oth-erwise overlapping spot chains or patterns [9, 21, 22]. In thepresent investigation, nonreducing 2DE proved useful for thestudy of protein impurities that differ between Ig products,as shown in Figs. 1 and 2. 2DE protein patterns (either insimple silver stain or as DIGE) are “fingerprints” characteris-tic for Ig products, as type and amount of impurities are notonly specific for the purification process, but also for produc-tion batches of the same final product and may be used asindicators for the efficiency of purification.

We identified a contamination of several final productbatches of Igs (D, H, I) with the plasmatic glycoprotein Apo-H(Figs. 1 and 2, Table 1). Its characteristic spot chain insilver-stained 2DE and in DIGE gels was only detectableunder nonreducing conditions, as it is otherwise hiddenby the prominent IgG heavy chain. In addition, final prod-uct batches from different manufacturers were tested witha specific ELISA (Table 1, Supporting Information Fig. 1).Although this ELISA showed some matrix effects, our pre-liminary ELISA data as well as 2DE and MS data demon-strate that this impurity is present in Ig products in greatlyvarying amounts. The structure of Apo-H, also known as�2-glycoprotein I, and the implications for phospholipid bind-ing had been solved from the crystal form [23]. Apo-H isinvolved in a variety of physiological pathways, includingblood coagulation and the immune response [24]. Complexesformed by Apo-H and acidic phospholipids are suggestedto act as antigen for autoimmune phospholipid antibodiesassociated with clinical events such as antiphospholipid syn-drome, lupus erythematosus, thrombosis, and recurrent fetalloss [25]. It is known that Apo-H exhibits procoagulant as wellas anti-coagulant activity in vitro [26]. We demonstrate herethat Apo-H is reactive in the TGA by spiking a commerciallyavailable Ig preparation with low TGA reactivity with a com-mercially available human plasma-derived Apo-H (Fig. 3).We found TGA reactivities (Thrombin generation, Lag Time,Time to Peak), which corresponded to the Apo-H concentra-tions in the Ig preparations. Apo-H was also found to bind tofactor XI and to inhibit its activation by thrombin and factorXIIa [27]. The TGA activity seen in the reference prepara-tion (Fig. 3) is most likely related to Apo-H itself, as factorXIa is inactivated during purification by plasma protease in-hibitors [28]. This will be further investigated in future testswith recombinant human Apo-H or neutralizing antibodies,if available. In addition, we plan to set up a validated ELISAas a future screening method to quantify Apo-H in Igs.

Our data demonstrate the presence of Apo-H in commer-cially available Ig preparations. We found small and variableamounts of contaminating Apo-H in those preparations, butthis needs to be investigated in future studies in a largernumber of different preparations and batches of Igs fromdifferent as well as from identical producers together withimplications for the clinical situation and possible impact onproduct safety. Not all Ig preparations with high TGA activi-ties contained detectable amounts of Apo-H (Fig. 2B), but asthis protein plays an active, not yet fully understood role inthe coagulation system, it may be one risk factor besides thealready known activated factor XIa or other yet undetectedthrombogenic impurities. In addition, this study underpinsthe usefulness of the presented combination of methodsfor the study of biological medicines, to ensure their qual-ity and to improve the already high safety of these medicinalproducts.

Samples A and G-J of Ig therapeutic products were kindlyprovided by Elaine Gray, NIBSC, London, UK; samples D andE were kindly provided by Laurent Fleury and Valerie Lievre,



ANSM (former AFSSAPS), Paris, France. We acknowledge thecontribution of Patrick Bayer, Austrian OMCL, AGES, Viennafor fruitful discussions and support for this project.

The authors have declared no conflict of interest.

5 References

[1] Robert, P., Pharmaceuticals Policy Law 2009, 11, 359–367.

[2] Chapel, H. M., Clin. Exp. Immunol. 1999, 118(S1), 29–34.

[3] FDA, U.S. Food and Drug Administration, Public Work-shop; Rockville, MD, USA 2011, 280–293.

[4] Grundmann, C., Kusch, M., Keitel, S., Hunfeld, A.,Breitner-Ruddock, S., Seitz, R., Konig, H., Webmed. Cen-tral Immunother. 2010, 1, WMC001116.

[5] Roemisch, J., Zapfl, C., Zochling, A., Pock, K., Webmed.Central Immunother. 2012, 3, WMC003247.

[6] Seifner, A., Beck, G., Bayer, P., Eichmeir, S., Lackner,F., Rogelsperger, O., Weber, K., Wollein, G., Transfusion2013, doi 10.1111/trf.12280, in press.

[7] Miller, I., in: Cramer, R., Westermeier, R. (Eds.), MethodsMol. Biol., Vol. 854, Humana Press, New York 2012, pp.373–396.

[8] Miller, I., Goldfarb, M., in: Smejkal, G. B., Lazarev, A.(Eds.), Separation Methods in Proteomics, CRC Taylor &Francis, Boca Raton 2006, pp. 235–267.

[9] Miller, I., Teinfalt, M., Leschnik, M., Wait, R., Gemeiner,M., Proteomics 2004, 4, 257–260.

[10] Gharahdaghi, F., Weinberg, C.R, Meagher, D.A, Imai, B.S., Mische, S. M., Electrophoresis 1999, 20, 601–605.

[11] Jimenez, C. R., Huang, L., Qiu, Y., Burlingame, A. L., Cur-rent Protocols in Protein Science 2001, Chapter 16, Unit16.4.

[12] Shevchenko, A., Wilm, M., Vorm, O., Mann, M., Anal.Chem. 1996, 68, 850–858.

[13] Roemisch, J., 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., Webmed. Central Immunother. 2011, 2, WMC002002.

[14] European Pharmacopoeia Online 7.8. Human normalimmunoglobulin. Monograph 0338. Council of Europe,Strasbourg, France 2013.

[15] European Pharmacopoeia Online 7.8. Human normal im-munoglobulin for intravenous application. Monograph0918. Council of Europe, Strasbourg, France 2013.

[16] Etscheid, M., Breitner-Ruddock, S., Gross, S., Hunfeld,A., Seitz, R., Dodt, J., Vox Sang. 2012, 102, 40–46.

[17] Gay, M., Carrascal, M., Gorga, M., Pares, A., Abian, J.,Proteomics 2010, 10, 172–181.

[18] EMA COMMITTEE FOR MEDICINAL PRODUCTS FORHUMAN USE (CHMP), Guideline on development, pro-duction, characterization and specifications for mono-clonal antibodies and related products, London, 18 De-cember 2008, EMEA/CHMP/BWP/157653/2007.

[19] Kukrer, B., Filipe, V., van Duijn, E., Kasper, P. T., Vreeken,R. J., Heck, A. J. R., Jiskoot, W., Pharm. Res. 2010, 27,2197–2204.

[20] Miller, I., Gemeiner, M., Gostl, G., Weber, A., Unkelbach,U., Beck, G., Lackner, F., Proteomics 2011, 11, 2120–2123.

[21] Wait, R., Begum, S., Brambilla, D., Carabelli, A. M.,Conserva, F., Rocco Guerini, A., Eberini, I., Ballerio, R.,Gemeiner, M., Miller, I., Gianazza, E., Amino Acids 2005,28, 239–272.

[22] Miller, I., Eberini, I., Gianazza, E., Proteomics 2010, 10,586–610.

[23] Schwarzenbacher, R., Zeth, K., Diederichs, K., Gries, A.,Kostner, G. M., Laggner, P., Prassl, R., EMBO J. 1999, 18,6228–6239.

[24] De Groot, P. G., Meijers, J. C. M., J. ThrombosisHaemostasis 2011, 9, 1275–1284.

[25] McNeil, H. P., Simpson, R. J., Proc. Natl. Acad. Sci. 1990,87, 4120–4124.

[26] Ieko, M., Ichikawa, K., Triplett, D. A., Matsuura, E.,Atsumi, T., Sawada, K., Koike, T., Arthritis Rheumatism1999, 42, 167–174.

[27] Shi, T., Iverson, G. M., Qi, J. C., Cockerill, K. A., Linnik, M.D., Konecny, P., Krilis, S. A., Proc. Natl. Acad. Sci. 2004,101, 3939–3944.

[28] Scott, C. F., Schapira, M., James, H. L., Cohen, A. B.,Colman, R. W., J. Clin. Invest. 1982, 69, 844–852.


Contamination of therapeutic human immunoglobulin preparations with apolipoprotein H...

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