Erythropoietic proteins and antibody-mediated pure red cell aplasia: a potential role for micelles

Sir,

In a recent edition of NDT, Locatelli et al. [1] provided a useful review of the chronology and potential causes of anti-erythropoietin antibody-mediated pure red cell aplasia (PRCA) with erythropoiesis-stimulating agents, together with current actions and recommendations. The authors note the potential causal link between a change in the formulation of Eprex® (epoetin alfa), where the stabilizer human serum albumin was replaced with polysorbate 80 and glycine, and the recent upsurge in PRCA cases associated with its use [2]. However, a mechanism for how this formulation change breaks the immune tolerance to erythropoietin was not given. In principle, two main mechanisms exist by which B cell tolerance to self antigens can be broken. One way of breaking tolerance is to present the self antigens in combination with a danger signal, such as denatured protein or endotoxins. The most potent way to induce antibodies to self antigens is to present the self antigens in highly structured arrays that resemble viral capsid-like structures. Several potential causes of antibody induction with Eprex have been put forward, including the presence of leached contaminants from the rubber stoppers of pre-filled syringes or the silicon oil used as a lubricant, which may act as ‘danger signals’.

We have shown recently the presence of surfactant molecule aggregations (otherwise known as micelles) in the Eprex formulation, which are caused by the high concentration of polysorbate 80. We have also shown that epoetin alfa is associated with these micelles, which may lead to the formation of structures with repeated epoetin antigens. These, in turn, may be presented at the surface of the micelles and break B cell tolerance. We employed gel permeation chromatography (GPC) and enzyme-linked immunosorbent assay (ELISA) to determine whether samples of Eprex (epoetin alfa) and NeoRecormon® (epoetin beta) formulations contained micelles and how much epoetin was micelle-associated [3]. The critical micelle concentration (CMC) is the concentration of a surfactant at which an appreciable number of micelles are formed, and CMCs for polysorbates 20 and 80 have been calculated previously [4]. Notably, Eprex contains 0.03% (w/v) Tween (polysorbate) 80, around 20 times its CMC; in contrast, NeoRecormon contains 0.01% (w/v) Tween 20, only approximately 1.5 times its CMC.

GPC analysis confirmed that the Eprex formulation contained micelles of polysorbate 80, while no micelles were detected with NeoRecormon. Subsequent analysis of the GPC fractions by ELISA demonstrated that with the Eprex samples, small amounts of epoetin were co-eluted with the polysorbate 80 micelles. No such co-elution was seen with NeoRecormon. The findings suggest that not only does the Eprex formulation contain polysorbate 80 micelles, but also that epoetin alfa molecules are solubilized in or attached to these micelles.

Micelle-associated epoetin is, therefore, a potential risk factor for immunogenicity in anaemic patients, and further investigation utilizing animal models may provide additional relevant data.

Conflict of interest statement. H.S. has participated in meetings and publications sponsored by Amgen, the makers of darbepoetin (Aranesp®); Johnson and Johnson, the makers of epoetin-alpha (Eprex®); and F. Hoffmann-La Roche, the makers of epoetin-beta (NeoRecormon®).

Huub Schellekens

Central Laboratory Animal Institute and Department of Innovation Studies Utrecht University Utrecht, the Netherlands Email: huubs{at}xs4all.nl

References

  1. Locatelli F, Aljama P, Barany P et al. Erythropoiesis stimulating agents and antibody-mediated pure red cell aplasia: where are we now and where do we go from here? Nephrol Dial Transplant 2004; 19: 288–293[Free Full Text]
  2. Macdougall IC. Pure red cell aplasia with anti-erythropoietin antibodies occurs more commonly with one formulation of epoetin than another. Curr Med Res Opin 2004; 20: 83–86[CrossRef][ISI][Medline]
  3. Hermeling S, Schellekens H, Crommelin DJA, Jiskoot W. Micelle-associated protein in epoetin formulations: a risk factor for immunogenicity? Pharmaceut Res 2003; 20: 1903–1907[CrossRef][ISI][Medline]
  4. Wan LS, Lee PF. CMC of polysorbates. J Pharm Sci 1974; 63: 136–137[ISI][Medline]




This Article
Extract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (1)
Disclaimer
Request Permissions
Google Scholar
Articles by Schellekens, H.
PubMed
PubMed Citation
Articles by Schellekens, H.