CMYKP
`
`Reply
`
`REPLY (cid:132)
`
`Reply to the letter to the editor by Johannes Ring and Rudi Valenta on the article “Assessment of dextran
`antigenicity of intravenous iron products by an immunodiffusion assay”
`
`Susann Neiser, Katrin Schwarz, Maria Wilhelm, Felix Funk, Peter Geisser, Susanna Burckhardt
`
`Vifor (International) Inc. St. Gallen, Switzerland
`
`Received for publication:
`Accepted:
`
`20/08/2012
`27/08/2012
`
`In response to the letter by Johannes Ring and Rudi Valenta, we want to clarify that, in our original
`paper1, we did not “postulate that antibodies […] can be used to predict the risk of clinical allergic reactions
`in patients”. We agree with Ring and Valenta that in vitro tests of possible antigens with monoclonal anti-
`bodies can generally neither assess the risk of anaphylaxis in an individual patient, nor can they predict
`the numerical risk of such anaphylaxis in the clinical setting. We did, however, find a correlation between
`our results and clinical findings with the tested intravenous iron preparations and thus concluded that
`“immunoassay data agree well with clinical observations and thus represent a possible approach for the
`evaluation of the risk of dextran-induced anaphylactic reaction (DIAR)”.
`
`Iron sucrose (Venofer®) was introduced in the 1950s by Laboratorien Hausmann AG, the predecessor of
`Vifor (International) Inc., as the first dextran-free intravenous iron preparation in Europe. In connection with
`the registration of Venofer® in the US, the reverse single radial immunodiffusion assay used in our study1
`was developed in 1998 by Dr. H. Hedin (Pharmacia & Upjohn AB, Uppsala, Sweden) for Vifor to fully exclude
`the presence of dextran, which might occur as an impurity of sucrose. Although no such test is required by
`the regulatory authorities, it is still used today as an additional safety measure in Vifor’s routine quality
`control analyses. Over the years, we have tested with this assay not only various types of dextrans but
`also different intravenous iron dextran preparations, which all showed a positive result. In contrast, carbo-
`hydrates and intravenous iron products that do not contain dextran always gave a negative result.
`When the two new dextran-based iron preparations Feraheme® and MonoFer® came on the market, we
`tested them in this assay. Ferumoxytol (Feraheme®, Rienso®) contains a carboxymethylated dextran2 and
`was marketed as non-immunogenic, but caused an anaphylactic reaction after application to a patient with
`a known history of adverse reaction to iron dextran3. Iron isomaltoside 1000 (MonoFer®) contains reduced
`Dextran 1 as a ligand4, which does not react in the immunoassay and probably acts as a hapten like Dextran
`15. Thus, both intravenous iron preparations were expected to give a negative result. Surprisingly, we found
`that Feraheme® and MonoFer® gave a positive reaction in the immunodiffusion assay.
`
`Because we were running out of the antibody used for quality control, we recently outsourced the devel-
`opment of a new antidextran antibody and of an enzyme-linked immune-sorbent assay (ELISA) based on
`this new antibody to an independent, external laboratory (GenScript, Piscalaway, NJ, USA). The antibody
`was developed by immunisation of BALB/c mice with dextran 50’000–KLH conjugate. Monoclonal antibodies
`(mouse IgG1-isotype) were produced in hybridoma cells.
`
`Reverse single radial immunodiffusion assays with this new antidextran antibody confirmed the published
`results1, i.e. the positive reactions for Feraheme®, MonoFer®, and Dextran 5, as well as the negative result
`for the reduced Dextran 1 isolated from MonoFer®. Moreover, ELISAs were performed by GenScript on blinded
`
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`04-12-2012 12:36:2104-12-2012 12:36:21
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`Port J Nephrol Hypert 2012; 26(4): 308-312 311
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`Pharmacosmos, Exh. 1051, p. 1
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`

`
`Susann Neiser, Katrin Schwarz, Maria Wilhelm, Felix Funk, Peter Geisser, Susanna Burckhardt
`
`CMYKP
`
`samples, and the results were fully aligned with the previously reported immunodiffusion assay data1: posi-
`tive reactions were observed with CosmoFer®, Feraheme®, MonoFer®, and Dextran 5, and negative results
`with Ferinject®, Venofer®, Ferrlecit®, the reduced Dextran 1 isolated from MonoFer®, and Dextran 1 (manu-
`script in preparation). Taken together, these additional data confirm our published results1 and address the
`criticisms of insufficient characterisation of the antibody as well as that of the test method used.
`
`Although this additional evidence strongly corroborates our previous data, we would like to address a
`few of the other criticisms raised by Ring and Valenta. The area of the precipitate in immunodiffusion assays
`depends on the relative concentrations of antigen and antibody. For the inverse technique we used, the
`size of the precipitate area has even been shown to be inversely proportional to the concentration of a
`given antigen6. The quantification of the antigens was beyond the scope of our work, and no conclusion
`can be drawn in this regard. However, the observed precipitates definitely reflect positive reactions of the
`antidextran antibodies with the respective antigen.
`
`As highlighted by Ring and Valenta, the mechanism of severe DIAR is an immune complex anaphylaxis.
`This mechanism obviously requires the presence of specific antibodies, which have been suggested to play
`a causal role in the development of DIAR7. The antibody titre, especially that of IgG, has been correlated
`to the severity of DIAR in several publications8-11. The statement cited by Ring and Valenta, that the anti-
`bodies per se are of no pathogenic importance, is misleading. Correctly, Richter and Hedin (1982)12 state
`that “all patients with severe reactions have high titers”. However, “[…] only a small proportion of those
`with high titers develop anaphylactic reactions.”
`
`In conclusion, since monoclonal as well as human antidextran antibodies react to the repetitive structure
`of dextran, a positive in vitro reaction between an intravenous iron preparation and a monoclonal antidex-
`tran antibody suggests that a similar reaction can occur in vivo – even if its likelihood to provoke a DIAR
`and thus its clinical relevance cannot be assessed. Working with human antisera would yield little additional
`value since it also would not allow for the assessment of the numerical risk of DIAR in a clinical setting.
`
`Conflict of interest statement.
`The authors are employed by Vifor (International) Inc. St. Gallen, Switzerland.
`
`References
`
` 1. Neiser S, Wilhelm M, Schwarz K, Funk F, Geisser P, Burckhardt S. Assessment of
`dextran antigenicity of intravenous iron products by an immunodiffusion assay. Port
`J Nephrol Hypert 2011;25(3):219-224
`
` 2. Groman EV, Paul KG, Frigo TB, Bengele H, Lewis JM. Heat stable colloidal iron oxides
`coated with reduced carbohydrates and carbohdrate derivatives. US Patent 2003;No.
`6,599,498
`
` 8. Kraft D, Hedin H, Richter W, Scheiner O, Rumpold H, Devey ME. Immunoglobulin class
`and subclass distribution on dextran-reactive antibodies in human reactors and non
`reactors to clinical dextran. Allergy 1982;37:481-489
`
` 9. Hedin H, Richter W. Pathomechanisms of dextran-induced anaphylactiod/anaphylactic
`rections in man. Int Archs Allergy appl Immun 1982;68:122-126
`
` 10. Richter W, Hedin H, Ring J. Immunologische Befunde bei der Infusion kolloidaler
`Lösungen. Med Welt 1977;28(42):1717-1719
`
` 3. Santosh S, Podaralla P, Miller B. Anaphylaxis with elevated serum tryptase after admin-
`istration of intravenous ferumoxytol. Nephrol Dial Transplant Plus 2010;3:341-342
`
` 11. Hedin H, Richter W, Ring J. Dextran-induced anaphylactoid reactions in man. Role of
`dextran reactive antibodies. Int Archs Allergy Apply Immun 1976;52:145-159
`
` 4. Public Assessment Report, Scientific discussion, MonoFer® 100 mg/ml solution for
`injection/infusion (iron(III)isomaltoside 1000). SE/H/734/01/DC, 2009. http://www.
`lakemedelsverket.se/SPC_PIL/Pdf/par/Monofer%20solution%20for%20infusion-injec-
`tion.pdf. Accessed July 2nd, 2012.
`
` 5. Ljungström KG. Safety of dextran in relation to other colloids – ten years experience
`with hapten inhibition. Infusionsther Transfusionsmed 1993;20:206-210
`
` 6. Vaerman JP, Lebacq-Verheyden AM, Scolari L, Heremans JF. Further studies on single
`radial immunodiffusion. II. The reversed system: diffusion of antibodies in antigen-
`containing gels. Immunochemistry 1969;6:287-293
`
` 7. Richter W, Hedin H, Ring J, Kraft D, Messmer K. Anaphylaktoide Reaktionen nach Dextran.
`I. Immunologische Grundlagen und klinische Befunde. Allergologie 1980;3:51-58
`
` 12. Richter AW, Hedin H. Dextran hypersensitivity. Immunology Today 1982;3:132-138
`
`Correspondence to:
`Dr Susanna Burckhardt
`Head of Chemical and Preclinical Research & Development
`Vifor Pharma · Vifor (International) Ltd.
`Rechenstrasse 37 · P.O. Box · CH-9001 St. Gallen, Switzerland.
`E-mail:susanna.burckhardt@viforpharma.com
`
`312 Port J Nephrol Hypert 2012; 26(4): 308-312
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`04-12-2012 12:36:2304-12-2012 12:36:23
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`Pharmacosmos, Exh. 1051, p. 2