Immunogenicity of biopharmaceuticals
1Department of Nephrology-Dialysis-Transplantation, University Hospital of Nancy, France, 2Renal Medicine and Transplantation Department, Guy's Hospital, London, UK and 3Department of Innovation Studies, Utrecht University, Utrecht, The Netherlands
Correspondence and offprint requests to: Dr Michele Kessler, Service de Néphrologie, Hôpitaux de Brabois, CHU de Nancy, 54500 Vandoeuvre les Nancy, France. Email: m.kessler{at}chu-nancy.fr
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Abstract |
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 Top Abstract IntroductionImmunogenicityFactors contributing to...Consequence of immunogenicity to...Measuring immunogenicitySummary and conclusionsReferences |
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The availability of biopharmaceuticals has been increasing over the past decade and as their patents expire, the emergence of biosimilar agents approaches. The primary issue of concern for the safety of these agents is the potential for immunogenicity. Both product- and host-related factors have documented impact on the immune response, but many factors are still unknown. Although in many cases the presence of antibodies may have little clinical consequence, the upsurge of pure red cell aplasia cases further increased concerns about potential clinical consequences of extensive use of biopharmaceuticals and biosimilars. Available laboratory measurement methods are insufficient to predict biological and clinical properties of biopharmaceuticals, or even to compare their bioequivalence. Comparison of results from different studies is complicated by the variability of assay measurements, presentation of data and lack of standardization.
Keywords: antigen; biopharmaceuticals; biosimilars; erythropoietin; immunogenicity; PRCA; risk factors
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Introduction |
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TopAbstractIntroductionImmunogenicityFactors contributing to...Consequence of immunogenicity to...Measuring immunogenicitySummary and conclusionsReferences |
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The availability of biopharmaceuticals has been increasing over the past decade and it is estimated that by 2010 around half of the newly approved pharmaceutical agents will be of such origin [1]. The first generation of such products were of non-human origin, such as bovine-insulin, streptokinase or staphylokinase. These were followed by natural products of human origin, such as growth hormone or factor VIII. The more recent preparations include human recombinant DNA products, such as interferon (IFN)
2a, IFNß, erythropoietin, insulin and growth hormone. As patent protection of many available biopharmaceuticals has recently expired or is close to that stage, the development of follow-on ‘generic’ and next generation biosimilars has been increasing in the past few years with further agents to follow.
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Immunogenicity |
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Most biopharmaceuticals induce immune responses (immunogenicity), which in many cases do not have clinically relevant consequences. However, in some cases the consequences can be severe and potentially lethal, causing a loss of efficacy of the drug or even worse, leading to autoimmunity to endogenous molecules.
In case of exogenous protein products (neo-antigens or non-self antigens), such as biopharmaceuticals derived from non-human origin (microbial, plant or animal), the immune response to the foreign protein leads to neutralizing antibodies. This immune response is mediated by T cells and occurs as a fast reaction after first meeting the antigen.
The immune response to endogenous proteins of human origin (self-antigen), such as human recombinant DNA products, leads to binding antibodies. This response is mediated by B cells through the breakdown of immune tolerance, and the reaction develops slowly and disappears after treatment withdrawal.
The theoretical basis for immunogenicity to biopharmaceuticals is based on their foreign nature, being of exogenous origin (neo-antigens or non-self antigens), or their similarity to self molecules (self antigens). In both cases, it is the activation of antibody-secreting B cells that leads to the clinical manifestation of immunogenicity.
There are two ways in which such immunogenicity can occur. First impurities, such as endotoxins or denatured proteins within a biopharmaceutical may provide a second, so-called ‘danger’ signal to T cells that may then send activating signals to B cells and hence, break B-cell tolerance. Second, B-cell tolerance can be broken via a T-cell independent response. If a biopharmaceutical for instance is not uniformly soluble it can form aggregates (Figure 1). The immune system may confuse these aggregates with viruses, and B cells are activated to proliferate and produce auto-reactive binding antibodies.
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Factors contributing to immunogenicity |
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Product-related factors
There are documented product- and host-related factors leading to immunogenicity of biopharmaceuticals [2]. Product-related factors include structural properties, such as protein sequence, the presence of exogenous or endogenous epitopes and the degree of glycosylation influencing protein degradation, exposure of antigenic sites and solubility. The higher immunogenicity of an Escherichia coli-derived IFNß product has been linked to the lack of glycosylation compared with other Chinese hamster ovary (CHO) cell-derived products [3]. Other product-related factors influencing immunogenicity are formulation and storage, downstream processing and the level of impurity or presence of contaminants. Evidence for the importance of these factors can be found in the reported variation in antigenicity of IFNß products produced at different manufacturing sites [4]. Changing the formulation and storage of IFN
2a has been shown to lower immunogenicity [4]. Further documented examples include the effect of downstream processing on the immunogenicity of factor VIII and the induction of antibodies against insulin and growth hormone due to product impurity [5, 6].
Host-related factors
Several host-related factors affect the immunogenicity of a biopharmaceutical. The genetic predisposition of a patient may influence the production of neutralizing antibodies. For example, the major histocompatibility complex (MHC) allele affects host recognition of antigen in T-cell mediated responses. Alternatively, the genetic sequence encoding the endogenous equivalent of the therapeutic protein may play a role. Haemophilia A patients treated with factor VIII have been shown to have different probabilities of developing immunogenicity depending on their endogenous expression of the protein [7, 8]. Patients with genetic deletion of factor VIII recognized it as foreign and produced neutralizing antibodies against it. In contrast, this occurs much less frequently among patients with genetic mutations in the protein itself.
Concomitant illnesses, particularly of the kidney and liver, may also influence immunogenicity. Autoimmune diseases predispose patients to producing antibodies against therapeutic proteins.
Dose and route of administration are important determinants. Higher doses or prolonged duration of treatment increase exposure and thereby heighten the risk of developing immunogenicity. Immunogenicity appears to be greater if the biopharmaceutical is administered subcutaneously (SC) or intramuscularly and has decreasing severity with intravenous and local administration [9].
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Consequence of immunogenicity to biopharmaceuticals |
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In many cases, the presence of antibodies has little or no biological and clinical consequence. However, even in case of well-established innovator biopharmaceutical products, biological and clinical consequences of immunogenicity have been observed.
The most common biological effect is the loss of efficacy, as has been described for IFN and ß [6]. The loss of efficacy may be restored with increasing dose such as Factor VIII for haemophilia A patients [6]. Clinical consequences may be manifested in general immune effects, such as anaphylaxis, allergic reactions or serum sickness. These have been relatively common historically but have become less common with the increasing availability of highly purified products and more stringent regulation of established biopharmaceuticals.
Major clinical impact is seen, however, if a natural protein with essential biological activity is neutralized. Such consequences have been described in the case of megakaryocyte-derived growth factor (MDGF), where antibodies against the biopharmaceutical also neutralized endogenous thrombopoietin leading to severe thrombocytopenia [10].
An upsurge in the incidence of antibody-mediated pure red cell aplasia (PRCA) observed outside the US between 2000 and 2002 revealed that a small change in the formulation of a well-established innovator product with extensive patient years experience may have significant clinical consequences [11, 12].
The PRCA cases were associated with a breakdown of immune tolerance to erythropoietin treatment resulting in neutralizing antibody formation not only against the recombinant protein, but also the native erythropoietin [13]. The sharp increase in incidence occurred primarily among those on SC epoetin (EPO) therapy (marketed as Eprex®/Erypo® by Johnson & Johnson), and coincided with replacement of human serum albumin as stabilizer by glycine and polysorbate 80 in 1998. Subsequent withdrawal of the SC formulation of EPO led to a considerable decrease in the incidence of PRCA cases.
A number of possible mechanisms have been proposed to explain the observed upsurge of PRCA. The modification in the drug formulation probably played a major role [13]. The role of leachates has been investigated [14], although results failed to show their significant effect in immune responses. This direction of investigation was based on the observation that among patients receiving SC EPO from syringes with Teflon-coated stoppers, the incidence of PRCA was lower. The role of micelles (polysorbate 80 plus EPO) is currently under investigation [15]. It is nevertheless likely that the immunogenicity of this particular product has been enhanced by the way the product was stored, handled and administered. Most certainly a combination of factors contributed to the reduced incidence of PRCA by 2003.
The case of PRCA has two-fold relevance with regard to quality and safety of biopharmaceutical and biosimilar products. Although the innovator product has been in use for years, it took time until the link between the relatively small modification in the product formulation and the upsurge of PRCA cases was established. The picture becomes even more complex when biosimilar products are considered. Even when biosimilars are produced from the same genetic construct, using the same technique, formulation and packaging as the innovator product, there is no guarantee that they are comparable with the reference product. Bioassays of follow-up EPO preparations manufactured in India, Asia and South America show their dissimilarities compared with the innovator EPO product, despite their claimed substitutability and bioequivalence.
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Measuring immunogenicity |
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Assessing the immunogenicity of biopharmaceuticals is becoming increasingly important with the emergence of biosimilar agents. Essentially there are two main types of assays: the radioimmunoprecipitation assays (RIPA) and enzyme-linked immunosorbent assays (ELISA) which determine binding antibodies, while bioassays identify the presence of neutralizing antibodies [16]. These assays are usually used in conjunction. Patient sera are first screened for the presence of binding antibodies and, if positive, the presence of neutralizing antibodies is tested for with the more cumbersome bioassay.
When a biopharmaceutical has unique determinants, assays become highly product specific. One problem is that quality assurance assays for biopharmaceuticals are less sensitive and precise than are tests for small molecules, hence it is difficult to analyse impurities [2]. The timing of sample collection may also influence assay results—immunogenicity typically develops after prolonged treatment. No single technique is available to predict immunogenicity of a biopharmaceutical product [17]. Methods used in different laboratories undertaking bioassays vary not only according to how antibody levels are determined but also the way results are reported. Comparison of assay results between studies, and laboratory sites face immense difficulties without international standardization of the assay procedures and data presentation [18].
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Summary and conclusions |
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TopAbstractIntroductionImmunogenicityFactors contributing to...Consequence of immunogenicity to...Measuring immunogenicitySummary and conclusionsReferences |
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Immunogenicity of biopharmaceuticals is of concern from clinical and safety aspects, and this issue is particularly important with the increasing number of biosimilar agents. Both product and host-related factors have documented impact on immunogenicity, but many factors are still unknown. In many cases, the presence of antibodies has little clinical consequence. The upsurge of PRCA cases, however, further increased concerns within the scientific community about potential clinical consequences of extensive use of biopharmaceuticals and biosimilars. It has initiated extensive research towards explaining the underlying immunological mechanisms. The picture is further complicated as available laboratory measurement methods are insufficient to predict biological and clinical properties of biopharmaceuticals, or even to compare their bioequivalence. Comparison of results from different studies is complicated by the variability of assay measurements, data presentation and lack of standardization.
Conflict of interest statements. M.K. is currently conducting research sponsored by Roche. D.G. has received honoraria for speaking from Fujisawa, Novartis, Merck, Roche, Amgen, Abbott, Shire, Genzyme and Nabi, is a member of advisory boards for Nabi, Roche, Amgen, Abbott, Genzyme and Shire and holds unrestricted educational grants from Astra-Zeneca and Roche. H.S. has participated as a speaker at meetings and as author in publications sponsored by Roche, Amgen and Johnson & Johnson.
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