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REVIEWS Impact of Product-Related Factors on Immunogenicity of Biotherapeutics
SATISH KUMAR SINGH Pzer, Inc., BioTherapeutics Pharmaceutical Sciences, Pharmaceutical Research and Development, Chestereld, Missouri 63017 Received 1 September 2009; revised 13 May 2010; accepted 24 May 2010 Published online 25 August 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22276 ABSTRACT: All protein therapeutics have the potential to be immunogenic. Several factors, including patient characteristics, disease state, and the therapy itself, inuence the generation of an immune response. Product-related factors such as the molecule design, the expression system, post-translational modications, impurities, contaminants, formulation and excipients, container, closure, as well as degradation products are all implicated. However, a critical examination of the available data shows that clear unequivocal evidence for the impact of these latter factors on clinical immunogenicity is lacking. No report could be found that clearly deconvolutes the clinical impact of the product attributes on patient susceptibility. Aggregation carries the greatest concern as a risk factor for immunogenicity, but the impact of aggregates is likely to depend on their structure as well as on the functionality (e.g., immunostimulatory or immunomodulatory) of the therapeutic. Preclinical studies are not yet capable of assessing the clinically relevant immunogenicity potential of these product-related factors. Simply addressing these risk factors as part of product development will not eliminate immunogenicity. Minimization of immunogenicity has to begin at the molecule design stage by reducing or eliminating antigenic epitopes and building in favorable physical and chemical properties. 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:354387, 2011 Keywords: biotechnology; protein formulation; protein aggregation; chemical stability; immunogenicity; antidrug antibodies
INTRODUCTION
All protein therapeutics have the potential to be immunogenic to some degree. The cause and impact of their immunogenicity vary widely. General immunologic or safety concerns with protein therapeutics include acute infusion or injection site reactions (anaphylactic or pseudo-allergic/anaphylactoid), serum sickness, effects related to the generation of antibodies against the therapeutic, as well as antibodies to the therapeutic that may cross-react with endogenous proteins. The latter aspect carries the greatest risk because of its potential to impact both safety and efcacy, whereas the others are managed through treatment protocols or, in the case of IgEmediated anaphylactic reactions, by completely stopping treatment.1,2
Correspondence to: Satish K. Singh (Telephone: +1-636-2479979; Fax: +1-860-686-7768; E-mail: satish.singh@pzer.com)
Journal of Pharmaceutical Sciences, Vol. 100, 354387 (2011) 2010 Wiley-Liss, Inc. and the American Pharmacists Association
From a survey of the literature and summaries of approved products, it becomes clear that antidrug antibodies (ADAs) in biotherapeutics are a rule rather than an exception. A review of the package inserts of (>100) approved biotherapeutics shows that ADAs are seen in almost all cases but, in very few cases, has there been a correlation made to impact on clinical efcacy and safety (although in many cases, this may reect paucity of data). Porter3 also concludes that no particular property of the protein can be identied as an obvious predictor of immunogenicity in humans. This lack of correlation even after many years of commercial use is a manifestation of the complexity of this subject. The literature on immunogenicity of biotherapeutics is large and daunting. Numerous reviews are available that summarize the incidence, describe the biology, examine the methods of detection and measurement, and dene strategies to screen, predict, assess, and minimize this very important component in the development of a biotherapeutic product.2,416 Apart from a cursory mention that product- and
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formulation-related factors in general and aggregation in particular are important, there are few reviews that examine the literature on the impact of product [(i.e., chemistry, manufacturing and control (CMC)] related factors on immunogenicity. Among these, Hermeling et al.,17 Rosenberg18 and, more recently, Cordoba-Rodriguez19 have taken a critical look at the impact of aggregation. Sharma2022 has provided a broad overview of product handling, manufacturing, and container closurerelated factors that have the potential to impact immunogenicity. The objective of this review was to provide the product development scientist a critical look at the current state of knowledge connecting CMC aspects and product-related clinical immunogenicity. Relevant cases from the literature are examined in some detail to shed light on often-repeated general statements about the impact of these factors. Because the factors determining immunogenicity are highly interdependent, other factors such as molecule design as well as patient characteristics and disease state are also briey covered for completeness. The emphasis is on clinical immunogenicity dened as generation of ADAs, although preclinical data are also assessed because often it is the only thing available. The discussion on excipients and container closure also covers their impact on the broader concern about immunogenicity and safety of protein therapeutics mentioned earlier. A couple of important caveats must be mentioned. First, the use of animal data, especially of normal animals, to study immunogenicity of human proteins has limited to no utility as a predictor of human response because of the protein being recognized as foreign by the animal immune system. Therefore, animal data, when reported in this review, are limited to assessing the relative impact of product-related factors. No extrapolation to the human clinical response is intended. A brief discussion about the value of animal models is provided in a later section of this paper. Secondly, the measurement of clinical immune response and the reported data is strongly dependent on the assay methodology and sensitivity, sampling technique, and denition of positive titer levels etc. Therefore, comparisons across products and sometimes within products cannot be easily made. Tabulations of immunological data provided in this review have therefore been annotated with the reference. Comparisons of immune responses across products are generally valid when data are taken from the same reference indicating a clinical trial in which the products have been directly compared by the same assay. Discussion about the immunogenicity assays are beyond the scope of this review, and the reader interested in this aspect is referred to the original references.
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Antigenicity and Immunogenicity The terms antigenicity and immunogenicity are often used interchangeably but actually have specic meanings. Antigenicity refers to the ability of the molecule to be recognized by existing antibodies or a T-cell receptor. A given molecule may be recognized by a variety of antibodies with different specicities, thus dening the antigenic epitopes of the molecule. Immunogenicity refers to the characteristic that endows a substance with the ability to provoke an immune response. An antigen can be highly immunogenic, weakly immunogenic, nonimmunogenic, or even tolerogenic. For example, high doses of deaggregated human immunoglobulin G (IgG) administered intravenously to a mouse can be tolerogenic, but the same antigen given in aggregated form subcutaneously can be highly immunogenic. From a therapeutic perspective, it is not the antigenicity but the immunogenicity that is important.23,24
IMMUNOGENICITY OF BIOTHERAPEUTICS
Therapeutic proteins can lead to antibody induction via two pathways: a T-cell-dependent and a T-cellindependent pathway.12,2529 Analysis of antibodies from clinical studies suggests that serious side effects are mainly driven by high levels of IgG antibodies, suggesting a T-cell-dependent pathway. IgG antibodies make up the majority of the ADA responses.30 The protein is taken up by antigenpresenting cells (APCs), digested, and the generated peptides (T-cell epitopes) presented via appropriate major histocompatibility complex (MHC) Class II molecules on their surface. Recognition of these peptides by T cells and subsequent activation of B cells generate antibody-secreting (short- and long-lived) plasma B cells. Some of the activated B cells also become memory cells, which maintain the pool of long-lived plasma cells and react rapidly to rechallenge by producing short-lived plasma cells. This T cell-dependent immune response is thus usually long lasting and of high titer, particularly for foreign or exogenous proteins. B cells can also be activated without cognate Tcell help by the so-called T-cellindependent pathway. For this purpose, the antigen has to be engulfed by specialized bloodborne peripheral dendritic cells (DCs), which then migrate to the spleen and present the antigen to B cells in the splenic marginal zone. This pathway is typically ascribed to particulate antigens displaying repetitive epitopes termed pathogenassociated molecular patterns, usually of bacterial or viral origin, and is also invoked when discussing potential immunogenic risk from aggregates in biotherapeutics. Due to lack of afnity maturation, this pathway typically results in a response of the IgM type,
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which is transient, of low titer, and poor specicity. However, delivery of a second signal by helper T cells or via pathways mediated by Toll-like receptors would allow for afnity maturation, class switching, and, thereby, a more efcient IgG response.27,3133 Immunogenicity of Foreign Proteins Immune response to foreign (exogenous) proteins, also called the classical immune response, arises via the T-celldependent pathway, as described earlier. The likelihood of an immunological reaction is higher because of the higher probability of a foreign epitope being recognized by a T cell. The result of a rst exposure is the generation of antibodies and induction of memory cells, and this priming leads to an immune response on re-exposure. The most serious immune response to a biotherapeutic generated by this mechanism is an acute hypersensitivity reaction in conjunction with administration, typically arising with re-exposure.34,35 Frequency of incidence and severity of response depend on duration between exposuresthe longer the delay, the lesser the severity. Other factors that inuence the outcome include protein antigenicity measured by the T-cell epitope content of the sequence, as well as the presence of adjuvant-like contaminants and/or impurities [host cell proteins (HCPs), endotoxins, leachates, etc.]. The latter impurities increase the probability of an immune response by stimulating the innate immune system.12,36 Immunogenicity of Proteins of Human Origin The human immune system is usually tolerant or anergic to autologous or proteins of human origin. The T-cell population, as a whole, has been selected to not respond to peptides for self-proteins. In the absence of a neoantigen, an immune response against a human protein, though not impossible, is highly unlikely unless the protein is presented to the immune system in a fashion that can reverse tolerance or T- and B-cell anergy by the earlier mentioned Tcelldependent pathway. The likelihood of breakage of tolerance to proteins of human origin or recombinant autologous proteins is considered a function of the abundance of the endogenous soluble protein. For proteins of low abundance, the immunologic tolerance is not complete. T and B cells specic for lowabundance proteins (autoantigens) may not be completely eliminated during early development. Under sufcient provocation (e.g., presence of molecules with adjuvant-like characteristics), these might generate an immune response. Tissue-derived danger signals such as caused by injury, infection, and inammation have also been proposed as possible causes for breaking of tolerance. Antidrug antibody formation in these cases is generally slow and often requires longterm administration. The relatively low incidence of
immunogenicity from human biotherapeutic proteins in patients with normal or suppressed (e.g., oncology patients), but not augmented (e.g., patients with lupus and other autoimmune diseases), immune systems5 suggests that the probability of the cascade of activation events (cf. the earlier text) is rather low. The mechanism for breakage of tolerance to autologous proteins is still unclear, but aggregates in the product are proposed as the key trigger, as discussed recently by Sauerborn et al.37 Antidrug Antibodies Antidrug antibodies are broadly classied as binding (BAbs) or neutralizing (NAbs). For biologics of human origin, BAbs and NAbs are of concern because of the possibility of impacting efcacy and pharmacokinetics. BAbs bind to the protein but do not neutralize it. They may mediate infusion reactions or alter the pharmacokinetic/pharmacodynamic prole of the therapeutic. BAbs can enhance clearance (clearing antibodies) or prolong systemic exposure (sustaining antibodies).14 They can, however, be precursor or triggers for the generation of NAbs through epitope spreading. NAbs bind to the therapeutic molecule and disrupt its ability to bind to the target, that is, neutralize its function. When present at low titers, the impact on efcacy may be minimal but efcacy and biological activity may be impacted at high titers. The most serious type of NAb response are those that cross-react and neutralize the function of the endogenous analog, especially one that serves a biologically unique function and has no redundancies.6,35,38 Note that the discussion in this paper does not cover the broader subject of immunotoxicity, which is closely associated with the therapy itself.39 Thus, events related to mechanisms, that is, immunosuppressive therapy (infectious complications and virusinduced neoplasias) and immunostimulatory therapy (acute cytokine release reactions, increased risk for autoimmune disease, and hypersensitivity reactions to unrelated allergens) are not considered. However, it must be kept in mind that by their very nature, immunomodulatory therapies will also skew the extent of unwanted immunological reactions, that is, ADAs observed.
FACTORS DETERMINING IMMUNOGENICITY

There are many factors that may be involved in the immunogenicity of a biotherapeutic to humans. These are broadly classied into two categories as given below, and listed in Table 1. (1) Patient (e.g., genetics, disease state, concomitant medication) and treatment regimen (e.g., dose, frequency, route) related factors
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Table 1.
Factors that May Impact Immunogenicity of Biotherapeutics Patient-Related Factors Disease state being treated General immune status of patient Genetic background (MHC genotype, HLA phenotypes) Concurrent illnesses and concomitant therapy Treatment-Related Factors Dose Route Frequency of dosing Length of treatment
Product-Related Factors Protein structure ( human/non-human, post-translational or chemical modications, T cell epitopes) Product quality parameters (isoforms, chemical and physical degradants) Contaminants and impurities Target (cellular or soluble)
(2) Product-related factors (including molecule design, manufacture, formulation, container, closure, stability, as well as excipients), also classied as CMC-related factors It is often difcult to deconvolute the impact of specic product attributes on immunogenicity from the numerous patient and treatment regimenrelated factors.2,13,40 Clinical trials are generally not conducted to specically assess the impact of product quality attributes. CMC data collected on clinical lots can be correlated to the information generated from these studies, including information from ADA assays, in an attempt to distinguish between productand patient-related factors, but this approach is far from satisfactory. Immunogenicity data is confounded by the multiple factors listed in Table 1, making correlation difcult. Clinical experience may also be limited to a few batches, reducing the range of product characteristics explored in the clinic. Animal models have been proposed to be used for this purpose (see, e.g., Ponce et al.14 ), and are discussed briey later in this review. Patient and Treatment Factors Genetic determinants that inuence susceptibility to antibody formation are outside the scope of this review. However, it has been shown that in at least two cases, variations in MHC Class II haplotype determine individual susceptibility to immune reactions against the same protein.41,42 Factors related to treatment route, patient disease status, and frequency of administration have a complex impact on the clinical immunological response. Among the common routes of administration, the probability of an immune response is generally highest after subcutaneous injection, followed by intramuscular, intranasal, and intravenous routes. Subcutaneous administration localizes and prolongs the exposure of the protein to a small area within close proximity of the lymph nodes where B and T cells are present.43 Lymphatic uptake can enhance exposure to APCs. Dendritic cells may potentially be activated if an adjuvant-like factor (e.g.,
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impurities, HCPs, endotoxin) or danger-sginal is present. Schellekens7 argues that the immune reaction is not predicated on choice of the route of administration: a substance or product is either immunogenic or not and a change in the route of administration cannot completely negate immunogenicity. Although the intrinsic nature of the molecule does not change, there are examples in which the route of administration did reduce or eliminate the frequency and titer of ADAs (see, e.g., Perini et al.44 ). Peng et al.45 also showed that antibody response to rFVIII in hemophilia A mice led to higher total titers via the subcutaneous route than via the intravenous route, but the titers for NAbs were similar for the two routes. However, there is at least one nonclinical counterexample from the pharmacology review section for adalimumab. A safety study conducted by the intravenous and subcutaneous routes in cynomolgus monkeys at two dose levels showed primate anti-human antibody (PAHA) titers higher in the intravenous group than in the subcutaneous group.46 Extensive clinical experience with pulmonary delivery of biotherapeutics is currently limited to insulin.47 Clinical studies with inhaled insulin found that switching patients from subcutaneous dosing resulted in larger ADA response in patients with both Type 1 and Type 2 diabetes.48,49 The antibodies were of the IgG class, were not neutralizing, and had no impact on clinical efcacy or safety. Presensitization with subcutaneous insulin was proposed as a possible explanation for the increased immunogenicity with inhaled insulin. This is supported by the observation that patients with Type 2 diabetes who were initially taking oral agents (noninsulin using) had lower, almost unchanged, antibody responses than insulin-treated patients who showed a slight increase.49 These results show that the pulmonary route has the potential to boost an existing immune response. However, its comparative capacity to induce a de novo response relative to the subcutaneous route is not clear from the clinical literature. Both the skin and the mucosal membranes make up the primary surface barriers to pathogens, and just beneath these lies the primary machinery (in the form of abundant professional APCs) to protect the body when these
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barriers are breached. These routes of administration thus may carry the greatest potential for generating an immunological response. The type of disease plays a role in the generation of treatment-related immune response, likely related to the immune status of the patient. Patients with weak or compromised immune systems or those on immunosuppression therapy are less likely to develop ADAs than those with intact immune systems. Interferon (IFN) seems more immunogenic in patients with multiple sclerosis (MS) than those with hairy cell leukemia, Kaposis sarcoma or condyloma acuminate, colorectal or breast cancer.50,51 Rituximab, a human mouse chimeric antibody directed against CD20 surface antigen, did not elicit an human anti-chimeric antibody (HACA) response in patients with B-cell lymphocytic leukemia, possibly because the antibody causes B-cell depletion, preventing the formation of HACA response. Furthermore, the patients were also on concomitant immunosuppressive drugs. In contrast, when administered to patients with autoimmune disease such as systemic lupus erythematosus and primary Sj grens syndrome, 65% and 27% o of patients developed HACA, respectively, despite immunosuppressive therapy.5 Patients with primed immune systems, such as in the case of acute bacterial infections, on the other hand, have a lower threshold for T-cell activation. Under these conditions, weakly immunogenic proteins, which would not have an impact on normal healthy individuals, might also trigger an immune response.52 Short-term therapy is less likely to be immunogenic than long-term therapy, although intermittent treatment is more likely to elicit a response than continuous therapy.53 Also, lower doses are generally more immunogenic than higher doses, a behavior that is probably akin to the phenomenon that the immune system is generally less tolerant of low-abundance proteins. In the primate study with adalimumab, all (16/16) monkeys receiving the low dose developed a PAHA response, with titers increasing with repeated administration. In contrast, only 2 of 16 monkeys in the high-dose group developed a PAHA response with low titers. Furthermore, within the low-dose group, the titers for animals receiving monthly doses was higher than those receiving weekly doses.46 High-dose regimens are therefore sometimes used as a mode of therapy to induce tolerance (e.g., for Factor VIII54 ). CMC Factors From the perspective of a product development scientist, the CMC factors that can play a role in the immunogenicity prole of the product begin with the molecule design and cell-line selection. On the basis of prior knowledge and experience, sequences can be mutated to avoid degradation and aggregation hotspots, as well as antigenic epitopes,5558 while
maintaining potency. The choice of host cell line determines the presence (or absence) of glycosylation and the glycosylation pattern. The upstream (bioreactor/fermentation, harvest) process impacts the distribution of glycoforms achieved and distribution of disulde isomers in (IgG2) antibodies and also determines the type of host cell impurities that may ultimately remain in the product. Although the overall objective of the post-harvest steps is to purify the protein by removing impurities (e.g., host cell proteins, DNA, endotoxins) and product variants (e.g., truncated, hydrolyzed, aggregated, deamidated, oxidized, charge variants, and improperly glycosylated forms), it is nevertheless impossible to completely eliminate these. The current state of purication processes is such that impurities are routinely reduced to levels well below what is considered a safety risk. Product variants are not as easily eliminated, however, and a certain minor fraction of some or all of these variants makes its way into the nal bulk solution, generally in the low percentage levels. The manufacturing process for a protein also subjects it to many stresses (see, e.g., Cromwell et al.59 ). The recombinant protein goes through highconcentration and high-temperature steps in the bioreactor or fermenter and a wide range of pHs, ionic strengths, shear stresses, airwater interfaces and material contact surfaces during capture, viral inactivation, purication, polishing, and formulation. Bulk protein solution stored in the liquid state is subject to interactions with the contact surface (metal or polymeric) and oxidative pressure (if stored in permeable containers). These contact surfaces can also be a risk factor for oxidation and/or aggregation, especially if incompatibilities exist between formulation (e.g., high chloride ion levels) and the surface.6062 Frozen storage of bulk creates a host of different stresses including the iceliquid interface, cryoconcentration, potential pH shifts, and ionic strength changes.63,64 Filtration and lling equipment leads to shear stresses and risk of shedding metal particles.65 Lyophilization also exposes the protein to low-temperature stresses, although the product after drying is generally quite stable. Exposure to light occurs in these operations, especially bright light during inspection, albeit brief, as well as light exposure during use. Siliconized container closures can shed silicone droplets that can interact with protein and lead to unfolding.66 Tungsten metal embedded in the neck of syringe barrels (during forming) has been shown to cause oxidation and aggregation.19,67 Product stored before use is subject to various protein degradation pathways.68 Shipping to the consumer subjects the product to a range of shear stresses, especially critical for liquids,69,70 and is potential for a range of temperature exposure, intentional or unintentional. Finally, the conditions at the use site could vary at the level of control,
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depending on the nature of the product. Clinic-based products are likely to have better control than homebased products from environmental (light, temperature, microbial) stresses as well as agitation/shear. In summary, the biotherapeutic product ultimately seen by the patient has undergone a complex series of processing steps. Although the manufacturing process steps are aimed at obtaining the puried and desired form of the protein, any or all of the steps can also subtly alter the protein structure through physical or chemical modications. These changes could be present immediately after the completion of manufacture or develop over time and can display novel epitopes in an otherwise nonimmunogenic protein. These structural changes could be present in only a fraction of the molecules, making them difcult to detect analytically, but still sufce to raise an immunological reaction. The CMC factors can therefore be broken into four general elements that cover a range of attributes with potential to impact immunogenicity, given in Table 2. (1) Molecule design and cell-line selection (2) Upstream processing (from thawing working cell bank to harvest) (3) Downstream processing (post-harvest to bulk formulation) (4) Drug product (ll nish to clinic/pharmacy) A number of the attributes are common across the various elements, as shown in Table 2. For example, the impact of molecular design or sequence (protein structure/variant) is directly relevant to its immunogenicity. However, the molecule design, and thus the sequence, also impacts its degradation characteristics as a drug product58 and therefore also indirectly the immunogenicity. It is recognized that the rst element (molecule design, cell-line selection) is generally a one-time activity in the development of a therapeutic biologic, whereas the other elements are repeated each time a new batch is made. A good understanding of the impact of all these elements on immunogenicity is required, but the last three elements also demand a level of control to ensure that the process can be run reproducibly over the lifetime of a product. We
Table 2.
therefore examine some of these attributes in the following text in more detail.
Choice of Protein Structure/Variant

A basic feature of the immune system is tolerance to self-proteins. An immune reaction to a foreign protein is more likely because of the higher probability of a foreign epitope being recognized by a T cell. A similar response can, however, be generated in people who do not have tolerance to a certain human protein. For example, patients with severe hemophilia A involving large deletions or nonsense mutations of the Factor VIII gene are more likely to have an antibody response to exogenous Factor VIII than patients with mild or moderate disease. The reason is that patients with the severe form of the disease do not express functional Factor VIII antigen and hence have no immune tolerance.71 Products used as enzyme replacement therapy for lysosomal storage disorders are also likely to create ADAs for the same reason.72 In these instances, the generation of ADAs may be considered as a reaction to a protein that is foreign to the patient. As in vaccines, the response is related to a number of factors such as number, frequency, and amount of dose administered, length of treatment, delivery route, and the presence of adjuvants.12 More surprising is the observation that self-proteins can induce an immune response even in individuals who are not decient in the protein but simply produce an insufcient amount for the desired biological effect.73 Foreign proteins can induce antibodies after a single injection, whereas human self-proteins may require longer exposure of up to 6 months.8 The fact that both types of proteins can induce antibodies implies that the molecular characteristic evoking antibody response is at least more complex than simply being self or nonself to the human system. Nature of the therapeutic (immunostimulatory vs. immunosuppressive; agonist vs. antagonist) proteins plays a role in the observed effect. Cell-surface binding therapeutic antibodies generally will have more potential to be immunogenic than those that interact with soluble targets. The impact of structural variants is illustrated by the experience from early insulin therapy that used
Biotherapeutic Product Elements and Attributes that Could Impact Immunogenicity Product Attributes that Could Impact Immunogenicity Protein structure/variant, degradation and aggregation hotspots, T cell epitopes, glycosylation, HCP, DNA, product-related impurities (charge variants, glycoform distribution, other post-translational modications) Glycosylation, HCP, DNA, endotoxins, Product-related impurities (glycoform distribution, charge variants, size variants, other post-translational modications, deamidation, oxidation) HCP, DNA, endotoxins, chromatography ligands (e.g., Protein A), product-related impurities (glycoform variants, charge variants, size variants, deamidation, isomerization, fragmentation, oxidation, aggregation) Formulation (excipients), endotoxins, degradation products (deamidation, isomerization, fragmentation, oxidation, aggregation), container, closure (leachables)
Product Element Molecule design Cell-line selection Upstream processing Downstream processing Drug product
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insulin from animal sources. Bovine insulin was found to be more immunogenic than porcine, which, in turn, was more immunogenic than human insulin. Porcine insulin differs from human insulin in the concealed Bchain and is therefore better tolerated than bovine insulin, which differs in the A-chain also. Other singlepoint mutants in the B-chain were also not found to be immunogenic in transgenic mice tolerant to human insulin but were all immunogenic in normal mice.74 Xenopeptides such as salmon calcitonin or exenatide are likely to have an increased probability for generating ADAs compared with human-derived proteins.75 Recombinant technologies used to produce human/ humanized versions of biopharmaceuticals, however, have not eliminated immunogenicity. Immune reaction to recombinant human insulin is still seen in patients with Type 1 and Type 2 diabetes. The generally higher observed frequency of antibody development and levels of antibody titers in patients with Type 1 diabetes than in patients with Type 2 diabetes is likely a consequence of the autoimmune nature of the disease in the former.76 In a study comparing recombinant methionylated human growth hormone (Met-rhGH) to bovine pituitary-derived hGH dosed intramuscularly to GHdecient children, Kaplan et al.77 found similar efcacy but higher ADAs in the Met-rhGH. The antibodies impacted efcacy in only 1 (of 26) child who was undergoing concomitant radiotherapy and chemotherapy. Moving this patient to the pituitary preparation restored growth. Similar ADAs were seen by Massa et al.78 in a study that followed children
over a 5-year treatment period, although no neutralizing effect were detected. Switching patients from Met-hGH to authentic rhGH eliminated the ADAs. Aston et al.79 reported on an monoclonal antibody that distinguishes between Met-hGH and pituitaryhGH, positing a possible antigenic difference between the two, although clearly not signicant enough to impact their bioactivity. The story of immunogenicity of growth hormone is, however, signicantly confounded by factors related to purity of the preparations used, be they are denatured monomers or aggregates caused by harsh extraction (from pituitaries) and purication conditions, or proteins from Escherichia coli expression systems (discussed later). Biochemical characteristics were generally not reported for these studies (see, e.g., Rougeot et al.80 and Valls et al.81 ), making it difcult to draw rm conclusions about CMC questions. Similarly, in the case of insulin, a number of studies related to immunogenicity to various CMC aspects were performed with bovine or porcine insulin. Apart from the differences in primary sequences from human insulin, the aforementioned factors arising as a consequence of the purication process probably account for some of the effects observed including antibodies against other contaminating pancreatic products (see, e.g., Robbins et al.82 and Ratner et al.83 ). The diversity of IFN products provides an interesting example of the impact of subtle variations in molecule design. Recombinant hIFN-"2 is produced in three different subtypes, as shown in Table 3, with a majority of human population expressing the allelic
Table 3. Product Roferon
Recombinant Human Interferon-" Product Characteristics and Their Immune Response Variant and Description rhIFN-"2a Lys,23 His34 Dose Various depending on disease Various depending on disease 5 MIU (18 :g)/m2 dailyb 1.53 MIU/m2 s.c. or i.m. three times per weekc Various depending on disease 5 MIU (20 :g)/m2 dailyb 3 MIU (15 :g)/m2 s.c. or i.m. three times per weekc 15 :g (3 MIU) s.c. three times per weeke Various depending on disease Various depending on disease 9 :g s.c. three times per weeke BAbs NAbs 25% (Package Insert) 4%44%a 24%b 20%c 0.8%13% (Package Insert)d 3%b 7%c <4%e 1%g 11% (Package Insert)h 11%e <4%e
31%b
IntronA
rhIFN-"2b Arg,23 His34
9%b
15%e
Beroforf Infergen
rhIFN-"2c Arg,23 Arg34 rhIFN-"con 88% homology to IFN-"2
Note: Because of differences in assay methodologies, comparison of immunogenicity measurements across studies should be carried out with caution. a Frequency of NAbs dependent on disease, dose, and length of treatment (Itri et al.87 ). b Antibodies detected between 60 and 750 days from start of treatment with the largest proportion detected after 150 days in patients with chronic myelogenous leukemia (Von Wussow et al.88 ). c Patients treated with lymphoblastoid IFN-"N1 (5 MIU/m2 , s.c. or i.m. three times per week) had 1% incidence of NAbs (Antonelli et al.89 ). d Frequency of NAbs dependent on disease, dose, and length of treatment. NAbs had low titers (Package Insert). e Antibody responses were transient and did not impact efcacy (Tong et al.90 ). f Not currently marketed. g From Steinmann et al.91 h BAbs developed at same frequency as with rhIfn-"2b (11% for 9 :g of Infergen; 15% for 3 MIU of IntronA; Package Insert).
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variant IFN-"2b.84 Some variations of human IFN" are O-glycosylated, but most are not.85 One IFN product (rhIFN-"con) has been designed by scanning sequences of several natural hIFN-" subtypes and assigning the most frequently observed amino acid in each corresponding position. Four additional amino acid modications were made and the recombinant molecule exhibits 88% homology with IFN-"2 (146/ 166 residues). Blatt et al.86 reported that the rhIFN"con was more biologically active than natural type 1 IFN but had similar effect on the induction of inammatory cytokines. Because of the higher potency, clinical doses could be smaller. Palleroni et al.92 showed that immunogenicity of IFN-"2a, b, and c in animal models is modulated by a number of treatment (frequency of injection, route of administration, aggregates) and host factors (concomitant immunostimulation, MHC genotype) but not by their minor amino acid variations. Using human transgenic IFN-"2b mice, they showed that IFN"-2a does not break tolerance and is not immunogenic to IFN-"2b transgenic mice. However, in comparative clinical trials, rhIFN-"2a was found to be more prone to antibody formation than rhIFN-"2b whereas the frequency with consensus rhIFN-" (rhIFN-"con) was found to be similar to rhIFN-"2b88, 90 (Table 3). Steinmann et al.91 showed much lower incidence of neutralizing antibodies for rhIFN-"2c (1%). Other experiments have shown that antibodies from patients treated with IFN-"2a do not specically recognize an epitope associated with IFN-"2a but are directed toward determinants common to both IFN-"2a and IFN-"2b.92 Thus, epitopes shared by several IFN-" subtypes may have different immunogenicity potential in each subtype.93 Porter3 concludes that the reason for the difference in the immunoreactivities of the 2a versus 2b forms is unresolved. Porter further suggests that the cause is unlikely to be the Lys to Arg substitution and proposes that formulation or contaminants may be playing a role. However, it is also known that point mutations can cause subtle but functionally signicant changes to folding and structure and thus to the biologic and antigenic properties of IFNs.94 Comparisons, as provided by von Wussow et al.88 and Tong et al.,90 however, again illustrate a gap in the reporting of such studies. There is no information about the quality aspects of the products used. As discussed later in the section on Aggregation, antibody response to rhIFN"-2a in various trials was dependent on the purity of the product as impacted by the storage conditions. From a CMC perspective, the results of these studies seem to exemplify the sensitivity of the immune system to small modications of the protein surface, but the conclusions about structure are confounded by potential impact of product-related impurities.
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IFN-$ is the other signicant human IFN used in therapy. It is available in two variants, as shown in Table 4. The lack of glycosylation, and the deletion and mutation in the sequence, has signicant consequences for rhIFN-$ structure.95 From a molecular design perspective, a lower specic activity was found for the nonglycosylated product. A single Tcell epitope that does not contain the Cys17 Ser region was shown to be key to the immune reaction in a large proportion of patients.41 Although the study was done with IFN-$1b only, it suggests that simply the presence or absence of glycosylation may be the critical design feature for the molecule instead of the glycoform prole. A comparative clinical study in MS patients showed that rhIFN-$1b lacking glycosylation is more immunogenic than rhIFN-$1a96, 97 (see data summary in Table 4). However, the incidence of immunogenicity also increases with frequency of injection and if given subcutaneously (Table 4 and Perini et al.44 ). Lack of glycosylation in rhIFN-$1b leads to signicantly higher aggregation levels in this product than in rhIFN-$1a, which could be a potential cause for its higher immunogenicity, as discussed later. Rudick et al.98 also showed that NAbs in IFN-$ therapy have a deleterious impact on clinical efcacy and are cross-reactive with other products, suggesting shared immunogenic epitopes between the variants. Incidence of immunogenicity is thus determined by the interplay between design- ( i.e. product) and treatment-related factors. Antibodies (Murine/Chimeric/Humanized/Human). As an increasingly important class of biotherapeutic products, monoclonal antibodies (MAbs) deserve special mention, as their design provides some interesting insights into the limitations of the ability to control immunogenicity. The rst murine antibody products had limited efcacy because of the generation of human anti-murine antibody (HAMA). Murine antibody, muromonab, leads to the formation of neutralizing antibodies within 1 or 2 weeks of treatment in all patients. Antibody engineering has been applied to generate chimeric (murine variable regions, human constant regions), humanized (murine CDRs, remainder human including the variable region framework), and fully human antibodies.15 The move from murine toward fully human structures has signicantly reduced but not eliminated immunogenicity.2,102,103 The two fully human products also show human anti-human antibody (HAHA) response: adalimumab dosed subcutaneously shows that 12% developed BAbs and 5% low-titre NAbs, and panitumumab dosed intravenously shows less than 1% high-afnity BAbs and 4.6% high- and lowafnity BAbs of which 2.4% were NAbs. In the case of adalimumab, the HAHA response is associated with loss of clinical response,104,105 although the loss of
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Table 4. Product Rebif
Recombinant Human Interferon-$ Products and Their Immune Response in Patients with Multiple Sclerosis Variant and Description rhIFN-$1a human, glycosylated Dose, Administration 6 MIU (22 :g), s.c., once weekly 6 MIU (22 :g), s.c., thrice weekly BAbs 58%a 89%a 27%b, ( ) NAbs +%a 31% (Package Insert) 7%b 15%c, ( ) 20%d 20%e 19%e 24% (Package Insert) 25%f 5% (Package Insert) 6%g 6%b 2%c 7%d 4%e 2%f 9%h 25% (Package Insert) ++%a 39%g 31%b 31%c, ( ) 38%d 32%e 39%h
6 MIU (22 :g), i.m., once/twice weekly 12 MIU (44 :g), s.c., thrice weekly Avonex rhIFN-$1a human, glycosylated 6 MIU (30 :g), i.m., once weekly 33%a 31.3%b, ( )
Betaferon/ rhIFN-$1b des Met1 , Betaseron Cys17 Ser, nonglycosylated
8 MIU (250 :g), s.c., every other day
97%a 80%b
Note: Because of differences in assay methodologies, comparison of immunogenicity measurements across studies should be carried out with caution. ( ), ( ) Numbers not signicantly different in pairwise comparison. a BAbs data at 12 months. NAbs at 6 months (80% Rebif; 25% Betaferon), but difference diminished after 24 months of therapy (Ross et al.96 ). b At 12 months (Scagnolari et al.97 ). c Persistent NAb-positive (2 consecutive positive samples) over 18 months of treatment (Bertolotto et al.99 ). d NAb-positive (1 positive samples) over 18 months of treatment (Bertolotto et al.99 ). e Persistent NAb-positive (2 consecutive positive samples) at 18 months of treatment (Bertolotto et al.99 ). f At 48 weeks (Panitch et al.100 ). g Duration of treatment 18 months or longer (Rudick et al.98 ). h Between 12 and 21 months of treatment (Cook et al.101 ).
response in HAHA-positive patients seems to be dose dependent.106, 107 As shown above, an expectation that immunogenicity will be completely eliminated is not realistic considering that the HAHA response can depend not only on the antibody construct but also on the target/function of the antibody, the patient (disease and immune status, MHC haplotype), and the dose/
Table 5. Factor Murine constant regions; V-region sequences; Human IgG allotypes Unusual glycosylation Specicity of antibody; Cell surface or soluble antigen; Formation of immune complexes with antigen Complement activation by antibody; Fc receptor binding by antibody; Inammation and cytokine release
frequency/route of administration. As an example, antibodies that deplete B cells (rituximab = chimeric, alemtuzumab = humanized), thereby attenuating the immune response, seem to be at the lower end of the immunogenicity scale than other MAbs.102 A more complete list of factors likely to impact immunogenicity of MAbs is given in Table 5. Only a few of these factors can be controlled by design, whereas the
Factors Related to Antibody Design and Function that May Impact Immunogenicity of Therapeutic Antibodies Comment Can be impacted by design (chimeric, humanized, human) Expression system Desired mode of action; Cell surface binding antibodies have higher risk for immunogenicity Desired mode(s) of action
Adapted from Clark.23
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rest are related to the function and desired mode of action. The examples in this section illustrate the importance of therapeutic protein structural variations (e.g., point mutations, antibody constructs) and features (e.g., glycosylation, discussed in more detail in the following text) on immunogenicity outcome. Carefully designed modications to reduce immunogenicity are possible, as exemplied by insulin variants on the market,108 but insulin is a relatively small and well understood molecule with a very wellcharacterized structure. Recognition by the immune system requires linear and/or conformational epitopes to be present. As discussed later, modeling and other tools are now becoming available to help remove such epitopes during the molecule design phase, even in the absence of detailed structural information. However, it is very likely that in all cases, the immunogenicity outcome will also be impacted by the clinical factors, as given in Table 1.
Post-Translational Modications: Glycosylation and PEGylation

Glycosylation. Glycosylation represents the most common form of post-translational modication (PTM) in proteins, present in approximately 50% of human proteins and in about the same fraction (49/ 108) of approved biopharmaceuticals in the United States. Glycosylation can introduce heterogeneity in a product through the generation of different core oligosaccharides and variable addition of outer-arm sugars. The presence and nature of the glycoform may impact primary functional activity, folding, stability, trafcking, and immunogenicity. For this reason, the choice of the expression system is a critical activity in the development of a biotherapeutic. As mammalian expression systems produce mainly human glycans, these have become the dominant platform for the production of therapeutic glycoproteins. However, these platforms require good process control because they display an inherent glycan heterogeneity that is sensitive to culture conditions.109, 110 Glycosylation can have direct impact on immunogenicity, manifested as an IgE response instead of an ADA. CHO cells produce glycosylation patterns that are close to human, although these cells also express N-glycolylneuraminic acid (NGNA), a form of sialic acid not found in humans and reported as immunogenic.109,111113 Mouse cell lines (e.g., NS0, SP2/0) also produce NGNA in addition to or instead of the N-acetylneuraminic acid (NANA) present in human IgGs.114 High mannose glycan structures (e.g. expressed in yeast) are also considered a risk factor because they can be substrates for the mannose receptor commonly expressed on the surface of macrophages and antigen-presenting DCs.115 Specic cases of
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immunogenicity in the clinic due to these structures has, however, not been reported. Galactose (13) galactose linkages or terminal (13) galactose can also be expressed by murine cells (e.g., C127, NS0, SP2/0).112,116,117 Although a common expression platform, there is only one report of CHO cells also expressing this residue,118 suggesting that the risk with CHO cells is very small. This gal (13) gal epitope has been shown to be recognized by up to 1% of circulating IgG in humans.119 Reports of adverse events linked to these glycoforms in currently marketed antibody therapeutics produced in the murine cell lines are limited, with the exception of cetuximab, a chimeric antibody expressed in SP2/ 0 cells. This MAb has a second N-linked glycosylation site at Asn88 in framework 3 of variable region of the heavy chain (Fv or VH ). Of the 21 glycoforms found, 10 were capped by mono- or di-(13) galactose, 5 were capped by an NGNA, and 4 with both. These moieties were found only in the VH region.120 The gal (13) galactose moiety has been recently implicated in severe anaphylactic reaction from cetuximab because of pre-existing IgE antibodies.121 Interestingly, the frequency of incidence of the IgE antibodies to this epitope had a geographical variance. The adverse events as a consequence of glycoform variants are distinct from the ADA response that may be related to the chimeric nature of cetuximab. The same glycosylation epitope has also been shown to be the immunologic stimulus in bovine thrombin preparations; that is, it is not limited to recombinant products. It is implicated as a cause of generation of neutralizing antibodies to (bovine) Factor V present as an impurity in the bovine thrombin product.122 These anti-bovine Factor V antibodies are cross-reactive to endogenous human Factor V and lead to coagulation disorders.123 Under nonoptimal conditions, mammalian cells can produce a number of abnormally glycosylated products that may lack potency or be potentially immunogenic,112 although there are no examples of specic glycoform leading to ADAs. Much work has gone into engineering cells to reduce the levels of potentially immunogenic glycoforms, that is, forms that are not present in humans. CHO cells have been manipulated to reduce the expression of NGNA.124 Prokaryotic expression systems (e.g., E. coli) result in aglycosylated proteins, although attempts are being made to engineer glycosylation pathways into bacteria.125 Yeast expression systems add sugar side chains of high mannose content, but both yeast and fungi are being engineered to produce human glycosylation patterns.126,127 Insect cell lines patterns differ signicantly from characteristic mammalian patterns. Plants may add "(13) fucose and $(13) xylose that are reported to be immunogenic to humans.112 Excellent reviews on the topic of glycosylation and
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therapeutic relevance are available,128131 as well as on the choice of expression systems.114,125,132 Glycosylation can also have an indirect effect on immunogenicity through its impact on folding solubility and (structural) stability. Glycosylation can affect local secondary structure and thereby direct the generation of tertiary structure. Altered or absent glycosylation can therefore alter or eliminate epitopes or expose/generate new ones. Glycosylation can increase solubility by shielding hydrophobic patches and reducing tendency to aggregate and enhance stability by participating in intrachain stabilizing interactions.112,133 Thermal stability is also enhanced by glycosylation as shown for IFN-$, interleukin (IL)-5, rhEPO, and IgG1-Fc.95,130,134 In the case of the IgG1-Fc, the native glycosylated form was the most stable, followed by various truncated glycoforms, with the deglycosylated version being the least stable.95,130,134 Addition of Gal" to wasp venom peptides reduced their immunogenicity in mice in a dose (one or two Gal")-dependent manner.135 In contrast, complete deglycosylation of rhFVIII resulted in substantial loss of stability and increase in aggregation with loss of biological activity but did not show an increased immunogenic response in a hemophilia A mouse model.136 This is discussed later in the section on Aggregation. Some specic cases are discussed in the following text to further illustrate the indirect clinical relevance of glycosylation to immunogenicity. In a study with rhGM-CSF produced from yeast, E. coli and mammalian cells, 4 of 16 patients receiving the yeast-derived material rapidly developed ADAs to native GM-CSF. The antibodies were directed to sites on the protein backbone that are normally protected by O-glycosylation but were exposed in the yeast-derived material that was glycosylated at the N-linked sites only. (Mammalian and native GM-CSF have two O-linked and two N-linked glycosylation sites; yeast-derived material also has an Arg Leu substitution at position 23.) However, the ADAs were not neutralizing. (In this case, no antibodies were generated against the E. coliderived material but only three patients received nonglycosylated material of this origin.)137 Higher incidence of binding antibodies with the yeast-derived material (72%) than with E. coli-derived material (16%) was also seen in vaccination trials where GM-CSF as given as an adjuvant for immunostimulation.138,139 Similarly, as discussed earlier, unglycosylated rhIFN-$1b gives rise to significantly higher immunogenicity than the glycosylated rhIFN-$1a (Table 4). Natural human IL-2 exists in several forms, with two major forms being (heterogenous) glycosylated whereas a third minor species is unglycosylated.140 Recombinant IL-2 (Proleukin) is produced in E. coli and is not glycosylated and is further mutated at Cys130 to contain a Ser130 (r[desAla1 ][Ser125 ]IL-2).
The biophysical characteristics of these two molecules are very distinct, with rIL-2 exhibiting amphipathic behavior whereas the natural IL-2 is predominantly water soluble.141 Although Bergmann et al.141 conclude that this behavior is simply a result of the Cys to Ser switch, it would appear that glycosylation of the natural IL-2 plays a role in its solubility and the lack of this in rIL-2 plays a role in its immunogenicity, as discussed later. The reported frequency for BAbs during rIL-2 therapy ranges between 30% and 100%, whereas the limited trials with natural IL-2 reported 21% ( co-dosed with rIFN-"2b) and 0% (when co-dosed with rIFN-().142 The importance of control of glycosylation has been illustrated recently when the application for a scaled up manufacturing process for alglucosidase " (rhGAA) was disapproved by the Food and Drug Administration (FDA) because of concerns about the differences in glycosylation at the two scales, resulting in lack of comparability.143 In vitro studies showed differences in afnity for the mannose-6-phosphate receptor and uptake into Pompe broblasts between different sources (i.e. scales) of rhGAA, indicating possible differences in mannose glycans. Immunogenicity differences were also seen in the material produced at the two scales, although no direct evidence is available to link this to the differences in glycosylation. All patients treated with the 2000-L scale product developed ADAs with about 30% developing neutralizing antibodies. This was compared with about 89% antibody positives (10% inhibitory neutralizing) among those treated with the 160-L scale material. The earlier mentioned data were more disconcerting when examined in light of the fact that the patients in the 2000-L study were primarily late onset who would have had some prior exposure to endogenous enzyme, as opposed to the GAA-naive, infantile-onset patients treated with the 160-L material. The FDA was also concerned with possible potency differences between the two scales, although the impact on potency could not be denitely established. The FDA, therefore, required the two scales to be treated as two different products.144 PEGylation. One strategy to impact the immunogenicity of a biotherapeutic is by the covalent attachment of poly(ethylene glycol) (PEG) polymers to the molecule or PEGylation.145,146 The primary goal for PEGylation is often to increase the circulation time of therapeutics. Apart from the increase in serum halflife, PEGylation also tends to impact the potency (decrease) and solubility (increase). Reduced frequency of dosing is also expected to reduce immunogenicity. PEGylation can, however, also impact the immunogenicity of a molecule both by masking antigenic epitopes and by covering hydrophobic regions and making the molecule more soluble.147 PEGylated rIL-2
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had enhanced solubility and reduced immunogenicity (in a rabbit model) compared with rIL-2. The greater the degree of modication, the lower the IgG antibody titers detected.148 Mono-PEGylated-IFN-$1b was also found to produce a lower IgG response in rats (by subcutaneous or intramuscular routes) than unmodied IFN-$1b.149 PEGylation of solid TNF (tumor necrosis factor) receptor Type 1 reduced immunogenicity in chimpanzees.150 To create a wider choice in PEG attachment sites, Hersheld et al.151 used site-directed mutagenesis to introduce (Arg Lys) residues at three positions in E. coli porcine nucleoside phosphorylase (PNP). PEGylation at these sites could reduce immunogenicity in mice compared with the PEG-modied wild-type mice. More signicantly, the anti-PNP antibodies in mice treated with PEGwtPNP, while equally reactive to both unmodied enzymes, did not cross-react with PEG-mutantPNP, implying that some antigenic epitopes were masked. Success with the PEGylation approach to abrogate immunogenicity in the clinic is, however, mixed. Data in Table 6 compares the immunogenicity response for the products for which both non-PEGylated and PEGylated versions are available. The data do not
Table 6. Type Human (IFN-") Human (IFN-")
clearly delineate this benet for endogenous/humanproteins, with the advantage of PEGylation in reducing immune response more likely achievable for non human proteins. Improvement is also seen from rhIFN-"2a to PEG-rhIFN-"2a, but this variant of IFN-" is less prevalent in the population as discussed earlier.84 In the case of PEGylated asparaginase (PEGaspargase), the reduction in number of patients with high-titer antibodies in the rst delayed intensication phase of treatment (2% in the PEGaspargase group and 26% in the aspargase group; see Table 6) likely results in the faster remission in patients with acute lymphoblastic leukemia, although overall complete responder rates were similar.152,153 Apart from the products listed in Table 6, there are few other success stories of approved products from the immunogenicity perspective. PEG-modied (bovine) adenosine deaminase prevented the development of high afnity or high titers of clearing antibodies to bovine ADAs in severe combined immunodeciency disease in 90% of patients.154 This molecule went on to become an approved product. The PEGylated version of IL-2 was less successful. No reports on immunogenicity could be found, but in clinical trials,
Impact of PEGylation on Immunogenicity May Be Dependent on Whether the Molecule Is a Human or NonHuman Protein Name Roferon (IFN-"2a) Pegasys (PEG-IFN-"2a) IntronA (IFN-"2b) BAbs 43%a 9%b 29%a 15.2%c NAbs 25%b 37%a 3%b 13%a Low titer NAbs 0%13% depending on disease, dose, duration of treatmentb 2%d 0.8%1.9%c 2%b,d 0.8%1.9%c Noneb,e Noneb
Human (r-metHuG-CSF)
Non-human (bacterial)
PEG-Intron (PEG-IFN-"2b) Neupogen (Filgrastim) Neulasta (PEG-Filgrastim) Elspar (aspargase)
8%15%b 9.110.2%c 3%b 1%f <1%b 0%f 25%b ADAsg (6%15% in induction phase, 26%42% in delayed intensication phase 1, 8%22% in delayed intensication phase 2)
Oncaspar (PEG-aspargase)
High titer ADAsb (2% in induction phase, 10% in delayed intensication in phase 1, 11% in delayed intensication in phase 2) ADAsg (2%5% in induction phase, 2%12% in delayed intensication in phase 1, 11%18% in delayed intensication phase 2)
Note: All EPARs (European Public Assessment Report/Scientic Discussion) are accessible from http://www.ema.europa.eu/htms/human/epar/ eparintro.htm. Because of differences in assay methodologies, comparison of immunogenicity measurements across studies should be carried out with caution. a Data from Phase II exploratory dose and standard interferon study in patients with chronic hepatitis B. Antibody levels provided are overall incidence levels for all dose groups ( Pegasys EPAR 2005). b Information from Package Inserts for the commercial products (accessed December 2008). c Data from pivotal clinical trial comparing PEG-Intron and IntronA ( PEG-Intron EPAR 2005). d Approximately 2% of patients receiving PEG-Intron or IntronA with or without Rebetol developed low-titer neutralizing antibodies to PEG-Intron or IntronA (Package Insert PEG-Intron). e Data from trial comparing Neupogen and Neulasta (Package Insert Neupogen). f BAbs detected in Phase II comparative dose nding study; no NAbs detected ( Neulasta EPAR 2004). g Clearing antibodies (Avramis et al.157 ).
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signicant toxicity was seen along with elevated lymphocyte and eosinophil counts.155 In a clinical study with PEG-rhMGDF (dosed subcutaneously), three patients were found to have developed antibodies that cross-reacted with and neutralized endogenous thrombopoietin (TPO), resulting in thrombocytopenia. MGDF (megakaryocyte growth and development factor) is a nonglycosylated molecule that contains the rst 163 amino acids of the endogenous TPO (glycosylated molecule with total 332 amino acids). The anti-TPO IgG bound both rhTPO and PEG-rhMGDF equally well, indicating that the antigenic determinants were located in the rst 163 amino acids156 and that PEGylation in this case did not provide protection against immunogenicity. Ultimately, the success of a PEGylation strategy depends on a rational design approach to select site(s) and length of PEG to achieve the goal of reduced immunogenicity while maintaining activity.147,150 Antibodies against the PEG moiety itself are also becoming of concern.158 Low-titer IgM preceded the formation of IgG antibodies against the PEG moiety in patients treated with PEGuricase. The antibodies did not inhibit uricase activity but enhanced the clearance rate.159 Similarly, the presence of anti-PEG antibodies was strongly correlated with rapid clearance of PEG-asparaginase, while no relationship was found between these antibodies and serum asparaginase activity for patients treated with the unmodied enzyme.160 Accumulation of PEG in the kidneys due to glomerular ltration has been seen to lead to renal vacuolation in rats that is dependent on dose, frequency, length of dosing, as well as molecular weight of PEG.161 The effect is a consequence of the high water-binding capacity of PEG, which is also seen as a higher than expected ideal theory osmotic pressure. However, this is not identied as a possible adverse effect in the package inserts for any of the approved PEGylated products. Polysialylation has been proposed as an alternative to PEGylation, enabling the use of a biodegradable polymer of a natural glycan. In a report on polysialylation of asparaginase, Fernandes and Gregoriadis162 showed reduced immunogenicity with increasing polysialylation, although low modication levels prevented the complete abrogation of immunogenicity. Other experiments showed that polysialylation reduced the immunogenicity of asparaginase, while also allowing three to four times greater circulatory halflives than the native enzyme in preimmunized mice. Hyperglycosylation to enhance circulation time has been successfully applied to erythropoietin (EPO) to create darbepoetin alpha. Instead of the normal three N-linked glycosylation sites, darbepoetin alpha has ve. No differences in immunogenicity compared with rhEPO were reported with immunogenicity levels at
approximately 0%, although concerns were raised about the assay.163 In summary, PTMs such as glycosylation can have signicant impact on immunogenicity simply through their presence (or absence) as well as the nature of the glycans. Chemical modications such as PEGylation have been employed to reduce immunogenicity, but with greater success for nonhuman proteins.
Host Cell Proteins, DNA, and Chromatography Ligands

Host cell proteins and DNA as well as chromatography ligands are potential process-related impurities that carry the risk of functioning as adjuvants and thus triggering an immunologic reaction to the therapeutic, given the appropriate antigenic determinants. For example, early recombinant preparations of hGH led to high antibody levels (greater than pituitary preparations) but were related to E. coli proteins (HCPs) remaining in the preparations and not to the N-terminal Met in the rhGH.164 This was shown when an improved preparation of recombinant Met-hGH containing less than 30 ppm HCPs (compared with 200 ppm in an earlier version) reduced the incidence of antibody formation in the clinical study conducted by Kaplan et al.77 Wadhwa et al.165 reported antibodies against two E. coli proteins in 80% of patients (100% of high-dose group and 64% of low-dose group) when treated with GM-CSF preparations containing these proteins as trace contaminants. However, the presence of these HCPs did not enhance the immunogenicity against GM-CSF itself, with a purer product producing more neutralizing antibodies of greater specicity than the less pure product. Bacterial DNA contains unmethylated CpG motifs that are known to activate Toll-like receptors and are themselves being studied as adjuvants for vaccines.166 However, for biotherapeutics, current purication processes reduce these host cell and processrelated impurities to very low levels, making them unlikely to be able to function as adjuvants. As an example of levels achieved in commercial products, rFactor VIIa RT167 species a maximum of 1.2 ng/mg of mouse IgG, 30 ng/mg of bovine IgG, and 19 ng/mg of HCP (from BHK cells and media). The World Health Organization limit for residual cellular DNA contamination is 10 ng per dose. Protein A capture step is a common rst chromatographic operation in the purication of monoclonal antibodies. This is a cell-wall protein derived from Staphylococcus aureus and binds a range of mammalian IgGs, primarily at the Fc domain. It is immobilized on a resin when used in column chromatography and thus carries the potential risk of leaching into the process stream when the captured antibody is eluted. As a bacterial protein, it is potentially immunogenic, even though an extensive safety database exists with its use in mAb purication processes as well as in
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plasma apheresis.168 Therefore, demonstrated clearance of Protein A is a part of regulatory requirement for approval. Despite improvements in purication and analytical technology, such process-related impurities remain a critical determinant of product quality. The scientic summary available for the registration of a biosimilar for rhGH (rhSomatropin, Omintrope R ) provides an interesting perspective to the earlier mentioned observations. The rst version of growth hormone used in the development program led to almost 60% of patients developing anti-GH antibodies. This was traced to high levels of HCPs from E. coli, leading to enhanced antibody reaction against GH and development of anti-HCP antibodies.169 These anti-GH antibodies were not neutralizing (in agreement with literature discussed earlier; e.g., Massa et al.78 ) and the patients showed efcacy similar to those who did not have these antibodies. Improved purication process lowered the incidence rate compared with that of other products. Similarly, in their recent review of the regulatory ling for alglucosidase " (rhGAA, Myozyme), the European Medicines Agency cited concerns with HCP and DNA impurities in the 160-L product and only approved the 2000-L scale product.170 In summary, process-related impurities have the potential to function as adjuvants, providing the danger signal to trigger costimulation and an immunologic reaction. These impurities must therefore be carefully monitored and controlled.
immunogenicity are few. However, in vivo PTMs of endogenous proteins (acetylation, cysteinylation, deamidation, deimination, dimerization, isoaspartylation, phosphorylation, as well as glycosylation) are implicated in several autoimmune diseases (see, e.g., Anderton171 and Doyle and Mamula 172 ). Deamidation. Direct clinical examples of the immunogenicity of deamidated forms of therapeutic proteins could not be found. Porcine desamido-insulin was shown not to be immunogenic in rats compared with unmodied version.173 However, Chen et al.174 have raised cytotoxic T lymphocytes (CTLs) that recognized succinimide derivatives of peptides but were only weakly cross-reactive at high concentrations with the parent peptide. Analogously, CTLs raised against the parent peptide did not recognize the succinimide derivative (formed as intermediate during asparagine bond rearrangement in deamidation or isomerization). The implication is that deamidation or isomerization can lead to autoimmune recognition or present novel epitopes to the immune system. In fact, deamidation of the "-gliadin component of gluten by tissue transglutaminases generates a highly stimulatory antigen for CD4+ T cells and is the reason for intestinal inammation in the autoimmune celiac disease.175 Deamidation, however, must be considered as a likely fate of biotherapeutics injected in vivo because it is one of the most common PTMs in cells.176 For example, Huang et al.177 showed that an IgG1 monoclonal antibody continuously deamidated in blood, with all forms having similar clearance rates. Distinguishing the clinical immunogenicity of in vitro deamidated protein from the impact of its in vivo modication would be very difcult. Oxidation. Oxidation of proteins is also associated with immunogenicity, although again no clinical examples with therapeutic proteins could be found. Oxygen free radicals have been shown to upregulate several DC surface markers that lead to an enhanced ability to activate T cells. Since oxygen free radicals are a common by-product of inammation, this suggests that they could serve as the danger signal required for initiating an immune response.178,179 More directly, oxidation of protein antigens (hen egg lysozyme, bovine "-lactalbumin, and ovalbumin) by performic acid or HOCl improved the processing by macrophages and enhanced recognition by specic T cells.180 Oxidation was shown to allow unfolding and thereby enhance exposure of antigenic epitopes, even though in the case of hen egg lysozyme and ovalbumin, none of the residues in the epitopes themselves were susceptible to oxidation (Trp, Met, Cys). Oxidatively [by reactive oxygen species (ROS)] modied human serum albumin (HSA) was also found to be a potent immunogen in rabbits, inducing high-titer
Product-Related Impurities and Degradation Products

Mammalian proteins in the body can undergo PTMs, either enzymatically or otherwise, which can enable immune recognition of neo-self-epitopes resulting in inammatory and autoimmune disorders.171 Among the number of in vivo mechanisms (e.g., deimination, methylation, isomerization, deamidation, hydroxylation, phosphorylation, glycosylation), only deamidation and oxidation are commonly encountered in vitro. These fall into the category of impurities and degradation products that must also be controlled from a CMC perspective. Product-related impurities and degradation products often overlap and are not readily distinguishable. For example, charge variants encoded as a consequence of the cell-line (e.g., sialylation) and/or generated in the upstream/downstream processes will often overlap with deamidation/isomerization products. Oxidation of susceptible residues can occur at any stage in the production process or subsequent storage and use, as can fragmentation/ hydrolysis. Finally, size variants such as truncated, misfolded, and aggregated species can also arise at all stages. Reported instances of these forms of PTMs of therapeutic proteins and their impact on
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antibodies, whereas titer against the native HSA was low. The IgG antibodies were ROS-HSA specic, indicating generation of neoepitopes by oxidation.181 Indeed, signicant structural modication was observed as a consequence of modest levels of oxidation (<0.2 mol of carbonyl/mol of HSA). Hydrophobic regions are exposed and ligand-binding properties of (one of the major drug-binding region on HSA identied as) site II are decreased.181,182 Lack of crossreactivity to the native protein would imply that oxidative modications could be tolerated barring impact on functionality, but the risk of epitope spreading to the native molecule cannot be discounted. Oxidation of a single Met residue of IFN-"2b was shown to have no impact on bioactivity. The functionspecic immunological activity of this modication could not be assessed but suggests that the oxidation altered the conformation of the monoclonal-specic epitope.183 Hochuli184 also showed that oxidized (Met to Met sulfoxide) monomeric forms of IFN-"2a present in vials stored at 25 C were signicantly more immunogenic than the unmodied molecule in a mouse model. However, the oxidized forms of IFN-"2a also had a strong tendency to aggregate, implying a possible indirect effect of the oxidative modication. Aggregation. Aggregation, involving association of multiple protein molecules in partially/wholly unfolded forms and even in their native state is one factor that raises a great degree of concern around immunogenicity of a biologic product from a CMC perspective.18,19 Aggregates can result due to a variety of interactions between the protein molecules including hydrophobic interactions as well as due to structural changes caused by chemical modications such as oxidation or deamidation (see, e.g., Nilsson et al.185 ). Other factors that can impact the level of aggregation in a protein solution include conditions such as pH, temperature, concentration, ionic strength, and stresses such as freezethaw, shear stress, airliquid and liquidsolid interfacial stresses, or other mechanical stress. A full discussion of the mechanisms of aggregation is outside the scope of this review here but a number of good reviews are available. Aggregates arising as a result of different stresses are different in their characteristics (e.g., driving forces, size, structure; see, e.g., Chi et al.,186 Wang,187 and Mahler et al.188 ). Thus, when reviewing the literature on the impact of aggregation on immunogenicity, it must be kept in mind that the term aggregates covers a wide variety of proteinbased species. However, there are very few (preclinical) studies in the literature that have examined the connection between aggregation and immunogenicity and also characterized their aggregates well. This kind of detail about the aggregates in clinical studies is, on the other hand, nonexistent.
Mechanisms for Immunogenicity of Protein Aggregate.

Aggregates are posited to cause immunogenicity via the T-cellindependent pathway discussed earlier. Because they comprise a large number of individual protein molecules, there is the potential for the surface to comprise a repetitive array of (novel) epitopes that may be seen by the immune system as resembling the external surfaces of pathogens, triggering the pattern recognition receptors (Tolllike receptors) on APCs.189 The immunon model has been used to dene the characteristics of the aggregates that could potentially induce immunogenicity by this mechanism.190 The model states that in order for polymeric antigens to activate B cells by the Tcellindependent pathway, the antigens must display a minimum of 10 to 20 epitopes at a characteristic repetitive spacing of approximately 100 , referred to as an immunon. Immunons greater than 100 kDa in molecular weight have been shown to successfully activate B cells in studies using virus-like particles.191 By extension, it has been proposed that aggregates of therapeutic proteins could potentially contain such structures and thus induce an immune response.18 As described earlier, this T-cellindependent pathway leads to an IgM response. However, it is proposed that unlike activation by small molecule haptens in Dintzis model or by polysaccharide antigens, B cells activated by protein aggregates could function as APCs to recruit T cell help and thereby switch to an IgG response. Although B cells are relatively poor APCs, their presentation potential is enhanced following specic recognition of antigen, thus contributing to initiation of T cell responses. An alternate mechanism is proposed for the generation of antibody response by particulate or insoluble aggregates analogous to that for particulate vaccines.192 Such particulate aggregates in the size range of a virus (20200 nm) could be endocytosed, preferentially ingested by DCs, cleaved, and presented by the MHC Class II molecule. The displayed epitopes could potentially activate B cells against the monomeric molecule by the Tcelldependent pathway described earlier. Larger sized particles (0.55 :m) are, on the other hand, primarily ingested by macrophages via phagocytosis or macropinocytosis.192 Receptors for such aggregates have, however, not been found yet, and nonspecic uptake by phagocytosis is much less efcient than receptor-mediated endocytosis. Current understanding suggests that particulate aggregates are more immunogenic than soluble aggregates, but small particulates are more immunogenic than large ones. Native aggregates in which the protein retains a large part of its structure are considered of greater concern because antibodies could be generated against epitopes that are present on the native
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monomeric version. Antibodies generated against nonnative aggregates (generated by misfolded species or chemical modications) could still result in increased clearance as well as raise potential safety concerns. However, to the best of the authors knowledge, there are no published reports examining the structure of naturally occurring protein aggregates in therapeutic protein products, which either prove or discount the presence of the repetitive motifs required by the immunon model. Furthermore, it is not clear where the costimulatory or danger signal, required for the activation of B or T cells, respectively, would arise from.32,36 One view is that the aggregates themselves provide the danger signal, functioning as adjuvants when taken up by DCs.193,194 However, the threshold for activation is not dened. The eld of vaccines has validated the immunon concept. The antigens in Gardasil R comprise large multimeric protein constructs akin to virus-like particles, although an adjuvant is added to generate the required immune response. White et al.195 found that insoluble micron-sized particles of ovalbumin were more immunogenic in mice than submicron-sized particles at the same total mass dose, as measured by an in vivo CTL activity assay. These aggregated particles were also found to generate a better anti-ovalbumin IgG response in the mice than solubilized aggregates, suggesting that size is more important than number. Whether this size versus number translates to a human clinical situation with a protein that is not inherently immunogenic (unlike ovalbumin to mice) is, however, not known.
Evidence for Immunogenicity of Protein Aggregates

Although the earlier mentioned arguments for the immunogenic impact of altered conformation or aggregates of therapeutic proteins have long been made, the actual clinical evidence, as reviewed in the following text, is limited and not very clear-cut. Animal models have also often been used to elucidate the impact of aggregates. Both types of analyses are reviewed here. The most direct evidence for the impact of aggregation on clinical immunogenicity comes from Moore and Leppert,196 who looked at the aggregate levels in pituitary-derived hGH formulations and correlated it with antibody response. Patients treated with different hGH preparations by the subcutaneous route were monitored over 6 years and classied on the basis of the pattern of antibody formation measured. Differences were seen over time in antibody generation and binding capacity of the antibody. Group 1 consisted of patients who rapidly (within 3 months) developed antibodies that were persistent, had low binding afnity, and high capacity. These patients received preparations containing 50% to 70% aggregated protein and the antibodies persisted even after
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they were switched to purer preparations containing about 10% aggregates. Group 2 consisted of patients who developed antibodies in 6 to 9 months that had high afnity and lower capacity. These antibodies were transient and decreased or disappeared on switching to a different preparation with lower aggregate levels. These patients had received preparations containing less than 5% aggregates. Finally, a third group comprised patients who did not develop antibodies despite prolonged therapy with multiple preparations (varying aggregate levels). No signicant difference in growth rate was seen between any of the groups (except for one patient in group 1 who had the highest titer). There was no decline in growth rate that correlated with the development of antibodies. The authors concluded that development of antibodies to hGH is dependent on the presence of aggregated species as well as individual susceptibility. They suggested that the percentage of patients developing antibodies may be similar when treated with or without aggregated hGH, but the use of low-aggregated material alters the immune response so that the antibodies are transient. Prolonged stimulation with aggregated hGH resulted in persistent antibodies in susceptible individuals. In this example, as in other such clinical reports, the immunological response to aggregated hGH is confounded by individual susceptibility. Furthermore, the levels of aggregates is extreme, raising questions about the impact of purication process on the state of the remaining hGH and the high likelihood of non-hGH proteinaceous impurities present in the preparations as discussed earlier in this paper. In a more recent analysis, Ahangari et al.197 found that about 8.5% patients developed antibodies to a modern rhGH preparation with presumably low aggregate levels. Patient-related factors (Table 1) likely determine individual susceptibility to the various aggregate levels, whereas patients with mutations in endogenous hGH (hGH-null) will develop antibodies regardless of the quality of preparation. It is worth noting that the hGH circulating in vivo consists of at least three monomeric variants (including an acidic species) as well as oligomeric aggregates up to pentamers,198,199 suggesting that the body would not recognize hGH aggregates up to a certain size as antigens. Although this size of aggregates is smaller than that estimated to generate an immunologic response,190 it is also likely that the presence of relatively high levels of hGH in the circulation makes it a well-tolerated protein for patients who are not hGH-null. A recent publication194 on the use of mice to study the impact of aggregates of rhGH on immunogenicity is interesting in this respect. Aggregates were generated by agitation and freezethaw stress in two commercial formulations of rhGH. The aggregates were well characterized by various biophysical techniques, and in the situation where insoluble
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particulate aggregates were formed, the structure was shown to be nearly native-like. Solution-state rhGH was also shown to have minimal perturbation of secondary and tertiary structures despite increased levels of soluble aggregates. These aggregates generated ADAs in naive adult mice and neonatally primed mice but not in hGH transgenic adult mice. Presence of insoluble aggregates led to the greatest titers, whereas reducing the size of aggregates by pressureinduced disaggregation reduced the titers.194 Lack of response to any size or level of aggregates in the transgenic mice agrees with the clinical observation that hGH products are relatively nonimmunogenic. In the extensive discussion provided around the development history of biosimilar rhGH (rhSomatropin, Omnitrope R , discussed earlier), no mention is made of aggregation as a characteristic of concern with respect to the antibody reactions observed.169 Thus, a part of the explanation for observations of the type reported by Moore and Leppert196 possibly lies in the detrimental impact of solvent-based purication processes used for the extraction of hGH from pituitary preparations on the structure of the hGH, as well as non-hGH proteinaceous impurities in the product dosed. Similar to hGH, insulin has been used as a therapeutic over a long period of time, starting from porcine-derived material. Anti-insulin antibodies were common in patients receiving animal-derived insulin, but, in general, these were not neutralizing. Recombinant human insulin has lower immunogenicity but can still elicit antibodies in patients who have not received previous insulin therapy.3 Reports have shown distinct antibodies directed against a covalent dimer aggregate of insulin, as opposed to the monomer in patients with Type 1 diabetes using beef/ pork-derived material.82 Dimeric forms of (beef/pork/ human) derived insulin have been implicated in cutaneous allergy, with the human-derived material showing lower and dose-dependent allergic response.83 Rapid-acting insulin preparations have been found to be less allergenic than intermediate- or long-acting versions, presumably because of the natural tendency of insulin to aggregate into dimers and hexamers that have been designed out in the rapid-acting varieties.76 Long-acting preparations such as zinc suspensions are basically crystalline aggregates of insulin. Higher insulin IgG antibody levels have also been observed in patients receiving insulin via implantable pump devices intraperitoneally than via regular subcutaneous therapy. Clinical studies also conrm that insulin from the implantable intraperitoneal pump system give rise to higher ADAs (in patients with Type 1 diabetes) than an externally worn pump system delivering the insulin by subcutaneous route. The main difference in the two systems is the temperature that the insulin is subject to, with the implanted insulin kept
at body temperature for 6 weeks whereas the subcutaneous device stores insulin at ambient temperature for 5 days.200 Jeandidier et al.201 found higher aggregate levels (20.6 and 41.2 U/mL) in samples removed from the pump reservoir after 60 and 90 days, respectively, than the corresponding solution held in a vial (0 U/mL). When injected intraperitoneally into rats, the pump reservoir material induced signicantly higher ADAs. The route of administration (intraperitoneal vs. subcutaneous) was ruled out as the cause of the difference in immune response. Fibrillar aggregates had also been observed in the intraperitoneal pump reservoir and ejection system.202 In rabbit immunization experiments, partially brillated insulin samples exhibited a tendency toward increased formation of IgG antibodies compared with unbrillated control samples. In addition, a clear correlation was seen with IgE response. Brange et al.203 proposed that it was the size and not the quantity of brils that was important for the allergic response. In the case of inhaled insulin, the presence of aggregates in the product ExuberaTM was proposed as a possible reason for higher ADAs from pulmonary delivery than subcutaneous delivery (discussed earlier). However, no alterations in structure could be found in the ExuberaTM powder, and a study with inhaled liquid droplets of insulin led to similar levels of ADAs. The site of delivery and individual susceptibility were the factors governing ADA response, as discussed earlier.49 Because the above-cited reports suggest immunological response to insulin (dimeric and hexameric) aggregates, which are much smaller than implicated by the immunon model, part of the explanation may also lie in the likely presence of small amounts of larger aggregates in these preparations. Insulin autoantibodies are detectable in insulin-naive patients who have a high likelihood of developing Type 1 diabetes. Development of antibodies to exogenous insulin therapy is thus largely unavoidable because of patient susceptibility.76 The history of development of interferons provides some interesting lessons on the impact of formulation and process, although the above-mentioned lack of clarity in specifying the aggregates is pervasive. Ryff204 analyzed antibody titers in serum samples from ve different clinical trials on IFN-"2a, using preparations with varying degrees of purity and with different formulations. The analysis showed that highly puried refrigerated lyophilized product or similarly puried HSA-free products were less immunogenic (as percentage developing NAbs and titer levels) than less pure material stored either as lyophile or as liquid containing HSA. Unfortunately, the purity of the material was not dened but was likely related to presence of IFNIFN or IFNHSA aggregates and oxidized variants in the less pure material.184 Hochuli,184 as discussed earlier, reported
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that the oxidized monomeric form of IFN-"2a was more immunogenic (in a normal mouse model) than correctly folded IFN, or other variants. However, the oxidized molecule also had a high tendency to aggregate. Storage of product in vials at 25 C was found to lead to oxidation and aggregate formation (IFNIFN and IFNHSA). IFN-"2a product from vials stored at ambient temperature was signicantly more immunogenic (in the normal mouse model) than that from vials stored at 20 C or fresh vials. Immunogenicity was, therefore, related to storage temperaturedependent degradation including aggregation, and the recommendation was made to move to 2 to 8 C storage while removing HSA and adding PS80 to the formulation. This study illustrates the impact formulation and storage can have on the product immunogenicity but unfortunately does not clearly discriminate the impact of oxidation from oxidationinduced aggregation and of homotypic (IFNIFN) or heterotypic (IFNHSA) aggregates. Palleroni et al.92 showed that injection with IFN-"2a aggregates produced by glutaraldehyde cross-linking or improper storage caused an immunogenicity reaction in human transgenic IFN-"2 mice. Subcutaneous and intraperitoneal injections produced high-antibody titers, whereas intramuscular and intravenous administrations did not produce any antibodies. Frequent injections (three per week instead of one per week) caused higher Ab titers. Similarly, Braun et al.205 showed that IFN-" aggregates (IFNIFN, IFNHSA, and IFNMouseSA) were signicantly more immunogenic than the monomers in transgenic mice. IFNIFN aggregates were from an old sample of IFN and contained approximately 50% dimers and very small amounts of larger oligomers. Glutaraldehyde was also used to crosslink IFNIFN and the albuminIFN aggregates. The IFNIFN aggregates (natural as well as glutaraldehyde-based) induced antibodies in normal and transgenic mice. In transgenic mice, breaking of tolerance resulted in immunogenicity to the aggregates as well as the monomers. Although these studies demonstrated the impact of aggregates on immunogenicity, the lack of proper characterization of the aggregates (size, amount) tested makes it difcult to draw a rm conclusion whether it is the dimers or the oligomers that were the main causative factor. Furthermore, the use of glutaraldehyde to create aggregates raises questions about how representative these of a normal situation were. The Braun study, however, also illustrates the role of dosing frequency and route of administration in the generation of immunogenicity, though only with the nonaggregated material. Use of IFNs to elucidate the relationship between structure and immunogenicity has been ably continued by Jiskoot and colleagues206,207 Hermeling et al.206 studied the impact of structure on immunoDOI 10.1002/jps
genicity by degrading IFN-"2b through various routes and testing on wild-type and immune-tolerant mice. The ADA levels in wild-type mice varied with the type of degradation (metal-catalyzed oxidation > peroxide treated nontreated boiled), with glutaraldehydetreated material not inducing antibodies. In transgenic mice, only the metal-oxidized material could break immune tolerance. Structurally, the lowest level of monomer was contained in the boiled solution (0%), followed by glutaraldehyde-treated (27%), metal-catalyzed (43%), and peroxide-catalyzed (99%) materials. The ndings of this study afrm the role of structure of aggregate on immunogenicitynot all aggregates are equal in this respect. The highly aggregated material (glutaraldehyde-treated or boiled) was not immunogenic, but the metal-oxidized material was immunogenic. Because the peroxide-treated material was not immunogenic, it is likely that the aggregates formed by the metal catalysis were the cause of immunogenicity and not the oxidation per se. In a continuation of their work, Maas et al.207 also showed that misfolded proteins, bearing the hallmarks of amyloid-like structures, can break tolerance in these transgenic mice. The complication with using aggregates as a catch-all term in assessing risk for immunogenicity is also illustrated by the work of Purohit et al.208 . They prepared aggregates of rhFVIII by thermal treatment in Tris buffer at 80 C for 2 min. This aggregate preparation was mixed with untreated rhFVIII to obtain solutions containing 5% and 20% aggregated material. On the basis of SEC and SDS-PAGE analyses, the aggregated material was expected to consist of at least six or more individual rhFVIII molecules, bound by both noncovalent and covalent interactions. The antigenic epitopes (detected using MAbs to known epitopes) and structural features of the aggregates and normal material were different. However, when tested in hemophilia A mice, the antibody titers were higher toward the untreated rhFVIII than the aggregated, 5% aggregated, or 20% aggregated material. In vitro Th-cell proliferation studies and cytokine analysis studies suggested that the uptake and intracellular processing of aggregated material by APCs might vary from those of the untreated rhFVIII. This was also seen in their study with deglycosylated rhFVIII where despite higher aggregate levels, no impact was seen on immunogenicity in mice.136 The aggregates in this case were non-native in their conformational structure. Apparently, aggregated material, irrespective of how it is generated, does not enhance the immunogenicity toward rhFVIII but rather acts as a distinct antigen.208 Although not examined in their studies, it is feasible that antibodies may have been generated toward the aggregates themselves, and one may speculate whether they would have been cross-reactive with the monomer or not.
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These results from studies on the interferons and rhFVIII reviewed earlier show that the statement aggregates cause immunogenicity is too simplistic. Immunogenicity due to aggregates is likely to be determined by the number, size, and orientation of the epitope, as well as the structure of protein in the aggregates. Thus, aggregates of the same therapeutic formed by different stresses may have different immunogenicity potential. Complete loss of native conformation may make the aggregate unrecognizable as an antigen, thereby producing no reaction (to the original molecule), whereas native-like aggregates may be more likely to break tolerance.
Functionality of the Protein Therapeutic and Immunogenicity of Its Aggregates

It must be noted that the high immunogenicity of IFNs is partly related to their function as immunostimulatory cytokines.209 Interferons have, therefore, been a good vehicle to study the impact of productrelated factors on immunogenicity but perhaps at the risk of leading to conclusions that may be exaggerated because of this intrinsic activity. Complications in separating the impact of aggregation from the impact of intrinsic functionality or characteristics of the molecule is also illustrated by a critical examination of the information on recombinant interleukin 2 (rIL-2) (r[desAla1 ][Cys125 Ser]IL-2) (Proleukin). The rIL-2 molecule is very hydrophobic and tends to form aggregates in solution with a Kd of approximately 600 nM (at room temperature).210 The size of the aggregates is reported to be 27-mer, but the anionic surfactant sodium dodecyl sulfate (SDS) is added to the product for solubilization and the concentration of protein is only about 70 nM,211,212 implying that at least between 2 and 8 C, aggregates should theoretically not form in this solution. The ionic surfactant can also impact the tertiary structure of the protein.213, 214 In solution, rIL-2 also forms covalent dimers and trimers spontaneously, although these have been observed in natural IL-2 too.141,215 In addition, as discussed earlier, the two major forms of natural IL-2 are glycosylated whereas rIL-2 (Proleukin) is not. Bergmann et al.141 found signicant differences in phase-partitioning behavior of natural IL-2 compared with the rIL-2, with the rIL-2 being substantially more amphipathic. The substitution [Cys125 Ser] also increased the hydrophobicity, as judged by chromatographic retention time.216 The biophysical data thus show that the rIL-2 product differs signicantly from endogenous IL-2 both in its functionality and in its structural characteristics.141, 217,218 This is reected in the clinical data also. Systemic rIL-2 therapy is associated with severe toxicity and with the generation of ADAs as well as NAbs, especially in longer treatments. On the other hand, therapy with natural IL-2 also induces ADAs but to a lesser extent,
although there are limited trials with this material142 (data shown earlier). The conclusion in the literature is that the hydrophobicity and the resultant aggregation of rIL-2 are the cause of the ADAs.18,142 However, there is another aspect to the immunogenicity of rIL-2 that must be considered. IL-2 is a potent immunostimulant. Local therapy shows reduced toxicity related to the fact that endogenous IL-2 does not naturally circulate systemically but is produced physiologically for local effect215,219 and therefore has limited spatial distribution and very low plasma concentration. It is therefore quite likely that in the case of rIL-2, the systemic treatment modality, patient disease state, and its immunostimulatory function are just as signicant contributors to the observed immunogenicity as is its aggregation state. Aggregates of molecules with immunostimulatory function will have a greater probability of generating an immunological reaction than molecules of immunosuppressive or other functionality.
Process Changes, Aggregation, and Immunogenicity

With degradation products in general and aggregates in particular, implicated as contributing factors for immunogenicity, signicant resources are expended to improve process and formulation to minimize them in therapeutic protein products. This was shown earlier by the example for IFN-"2a (Hochuli184 ). Similarly, a new formulation for IFN-$1a was developed to improve tolerability and immunogenicity prole for the product while eliminating HSA from the product.220223 Only 2.5% showed NAbs with the new formulation compared with 14.6%, whereas 11% had high titers compared with 20% historically.224 These product performance improvements were suggested to be due to the HSA-free formulation(s) not containing IFNHSA and IFNIFN aggregates. Unfortunately, no data were provided to enable a direct comparison of the aggregate levels (or types) as well as any other formulation characteristics in the solutions studied. Inadvertent impact of process changes on conformation and aggregation is a risk. For example, increased incidence of antibodies to Factor VIII was temporally correlated to the introduction of a new FVIII concentrate in Netherlands.225 The new FVIII concentrate was produced from plasma and pasteurized for virus inactivation. Another incident was reported by Peerlinck et al.226 from Belgium, when a concentrate was pasteurized at 63 C for 10 h. Characterization of the double-virusinactivated (pasteurized) preparation provided by Robinson et al.227 seems to indicate that the pasteurized material was no different from nonpasteurized in terms of electrophoretic mobility, SDS-PAGE (with and without immunoblotting), and in its association with von Willebrand factor. It was concluded that no obvious
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denaturation had occurred and that the aggregation levels had not changed because of the heating step. The discrepancy between the characterization results and the patient experience can likely be explained as follows: (1) the analytical techniques were not of sufcient resolution to pick up differences between preparations, and/or (2) differences developed over time post-manufacture and release. Wang and Kelner228 have shown that high-molecular-weight species increase with thermal treatment of rhFVIII over time. Recombinant FVIII without added stabilizers shows an onset of melting at around 56 C, whereas the addition of stabilizers increases the temperature to some extent.229 However, the pasteurization temperature of 63 C is close enough to this denaturation temperature and that the formation of aggregates or other misfolded species is not unexpected. In another example, Hochuli184 reported that an improved process for IFN-"2a (specically an inactivation step involving acidication of fermentation broth) did not lead to an incorrect tertiary structure (determined as ID, purity, and folding) and therefore did not impact the immunogenicity, with the molecule retaining its bioactivity. However, Kudela et al.,230 using mAbs to various epitopes, showed that subtle changes could occur to an acid-labile epitope in IFN-"2 when the acid treatment at pH 2 lasted for at least 1 h. As emphasized by Rosenberg and Worobec,231 changes to highrisk manufacturing steps could require more detailed evaluation, including animal or clinical studies.
the molecule design stage itself, as briey discussed later.
Excipients
A review of the formulation composition for biologics shows that the vast majority of biologics comprise a buffer, a tonicity modier, cryo- or lyoprotectant, and a surfactant. Other additives such as chelator, antioxidant, and preservative are occasionally found.235238 It is beyond the scope of this article to review the potential immunogenic or allergenic aspects of each excipient but a good survey is available.239 A few notable cases are covered to exemplify how adverse events can arise from seemingly innocuous sources, both directly and indirectly. Excipients are not necessarily biologically inactive. The direct impact of excipients is generally anaphylactic or anaphylactoid in nature and is not connected to the immunogenicity of the therapeutic; this aspect is briey covered in this section for completeness. Indirect impact is implied through their potential to generate neoepitopes or to act as adjuvants and thereby elicit an immunological reaction to the therapeutic. Among the common ingredients is the surfactant polysorbate 20 or 80 (PS20 or PS80), comprising partial fatty acid esters of sorbitan and its anhydrides copolymerized with ethylene oxide. Polysorbates (or other nonionic surfactants) are added to protein formulations to stabilize against interface (airliquid or solidliquid)-induced aggregation and also prevent surface adsorptive losses. These are present in a large number of therapeutic products and are generally regarded as safe. Polysorbate 80 has been known to cause anaphylactic reactions in dogs, especially at high doses (5 mg/kg).240 In humans, symptoms of hepatomegaly, ascites, and thrombocytopenia have been reported in neonates at high doses [72 mg/ (kg day) i.v.]. Transitory decrease in blood pressure has also been observed in humans at relatively low intravenous doses (6 mg/kg) and may have been associated with histamine release.241 Polysorbate 80 has been implicated in many nonimmunogenic anaphylactoid reactions.242 Among biotherapeutics, Price and Hamilton243 have recently reported on an anaphylactoid reaction after successful long-term therapy in two patients receiving omalizumab by subcutaneous administration and point to PS80 as the most likely cause. Oleic acid, present in a residual form from incomplete esterication as well as a hydrolytic degradation product, has been proposed as a cause of hypersensitivity reactions.244,245 Mueller et al.246 recently reported that degradation products of oleic acid could upregulate the expression of CD86 costimulatory cytokine by DCs, a key event in the induction of an immunological response. However, oleic acid itself had no effect.
Aggregation and Immunogenicity

Among the various product-related impurities and degradation products, aggregation is considered a strong risk factor for immunogenicity. It is also the attribute that is the most difcult to control because there are multiple factors that can lead to the formation of aggregates in a product. Aggregates are also difcult to exactly measure and characterize because the term encompasses heterogenous species ranging from soluble dimers to visible particles comprising millions of monomers.232 Aggregates created by different stress are not equal in their potential to cause immunogenicity, and this potential is also likely to be related to the function of the biotherapeutic. The mechanisms relating aggregation to immunogenicity are still debated,36,37 and the clinical data reviewed here are not unequivocal. Fundamental research in this area is therefore needed, although it is possible that a clear link may never be established and the actual impact of aggregates may have to be assessed on a product-by-product basis.233,234 From a safety perspective, the level of aggregation in a product is generally regarded as a critical quality attribute that must be controlled. Well-designed process and formulation are the key for this. However, efforts are now also being made to enable control of this attribute from
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Apart from the earlier mentioned direct effects, polysorbates can, however, also induce protein degradation through oxidation247 and even aggregation.70,248 Association of EPO with PS80 micelles, with the formation of multimeric structures or the loss of structure, has also been hypothesized as a possible cause for immunogenicity of EPO and the increased incidence of pure red cell aplasia (PRCA), although this has been debated in the literature249251 (and discussed in more detail later). Clear evidence for immunogenicity mediated by polysorbates through this indirect mechanism could not be found, but the possibility is mentioned in the EMEA (draft) Guideline on Immunogenicity Assessment of Biotechnology-Derived Therapeutic Proteins.252 Intravenous immunoglobulin (IVIG) therapy has been associated with numerous episodes of acute renal toxicity and osmotic nephropathy because of a very high sucrose load in some formulations.253,254 IVIG is fractionated from plasma. The sucrose is added to the product to reduce the formation of aggregates as a consequence of the pathogen-removal steps in the process.253257 Sucrose and sorbitol, as well as maltitol and fructose, can also be contraindicated in patients with hereditary fructose intolerance, or in the rare cases of glucose-galactose malabsorption syndrome or congenital sucrose-isomaltase deciency.258 Sucrose has also the potential to hydrolyze in solution (generally at acidic pHs) to give the reducing sugars fructose and glucose. These can react with the protein through the Maillard reaction leading to glycation at the lysines and eventually even aggregation. The rate of reaction is generally very slow at the normal refrigerated storage conditions of proteins.259,260 However, considering the fact that, in general, the molar ratio of sucrose to protein in formulation is very high, it would not require a large number of sucrose molecules to hydrolyze to potentially have an effect. Trehalose has greater stability against hydrolysis into reducing sugars than sucrose261, 262 (also inhouse unpublished data) and is a good alternative in liquid formulations. Recently, the liquid formulation of sargramostim, rhGM-CSF (Leukine R ), was recalled by Bayer because of a temporal connection between a change in the formulation to include disodium edetate and an upward trend in spontaneous reports of adverse reactions including syncope (fainting). The dose of disodium EDTA for a patient through leukine is about 1.9 mg/day i.v. or s.c for a standard patient (2 m2 ). The liquid formulation has been replaced by a lyophilized formulation not containing EDTA.263 Interestingly, in the same time frame, a new formulation of fosaprepitant dimeglumine (Emend R ) containing disodium EDTA was approved, with a dose (of EDTA) up to 14.4 mg per injection.
The FDA has adopted a guidance for industry, Nonclinical Studies for Safety Evaluation of Pharmaceutical Excipients, which focuses on issues associated with development of safety databases that will support clinical use of excipients in drug products.264,265 Although immunogenicity/allergenicity is not specically mentioned, qualication of (new) excipients with respect to proposed levels, duration of exposure, or route of administration is expected.
Container Closure
The FDA guidance document on container closure considers inhalation aerosols and solutions, injections, and injectable suspensions as products with the greatest level of concern when accounting for the route of administration and risk for packaging componentdosage form interaction.266 The concern for packaging componentdosage form interaction for biologics again arises because of the potential for alteration of the structure of the protein through surface adsorption or chemical degradation pathways such as oxidation. Some components that come into contact with the product include the container (glass or plastic and vial or syringe), closure (stopper), administration and infusion components (syringes, bags, infusion catheters). Some common leachables from these include metals, antioxidants, plasticizers, lubricants, as well as their degradation products. A full discussion about leachables is outside the scope of this review, but a few illustrative examples are considered in the following text. No direct examples of surface adsorptioninduced loss of structure and subsequent impact on safety or efcacy could be found. However, the discussion in the following text clearly illustrates the potential risk that leachables can pose. Probably, the most serious case of immune reaction in which the reaction was temporally related to a formulation change and its interaction with container closure system is the incidence of antibody-mediated PRCA in Europe in the late 1990s and early 2000s. The event, and its associated investigational history, has been well documented.267272 The dramatic increase in the incidence of PRCA occurred at the same time as the change in formulation of the implicated brand of EPO. The stabilizer HSA was replaced by PS80. It has been shown that the PS80 caused an increase in the amount of organic leachates from the uncoated stopper used in the prelled syringe. Leachates were not detected in syringes containing a uoropolymer-coated stopper. The leachates were proposed to act as adjuvants upon subcutaneous injection and provided the trigger for generation of anti-EPO antibodies leading to PRCA.273, 274 Other investigations into the manufacturing, storage, and handling processes, as well as more detailed analytical investigation, did not reveal any changes to the molecule
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and the structure. Leachates from uncoated stoppers were shown to serve as adjuvants when treating mice with a surrogate antigen, ovalbumin.274 Risk mitigation strategies put into place after the identication of the incidence (contraindication for the use of subcutaneous route with the implicated product, especially in patients with chronic renal failure, improved cold chain and handling and replacement of uncoated stoppers with coated stoppers) signicantly reduced the incidence. Although agreeing with the temporal link between formulation changes and PRCA incidence (HSA vs. PS80 and uncoated vs. coated stoppers), the mechanistic explanation has been considered inadequate by Schellekens and Jiskoot,275 who raise the question that with the leachables functioning as adjuvants, there had to be some other cause for the induction of the actual immunological reaction. They suggested that PS80 as a poorer stabilizer than HSA may have led to greater levels of aggregate formation over time and these aggregates could be the root cause for the immune response (also see Haselbeck276 and the discussion earlier about polysorbates in formulations). Interestingly, De Groot et al.55 report that in silico tools for identifying T cell epitopes accurately predict the potential immunogenicity of erythropoietin. The incidence shows that immunogenicity potential of an otherwise nonimmunogenic protein is realized when inammatory conditions (danger signals) are generated55 through a combination of factors that promote adjuvancy in an immunologically provocative (subcutaneous) treatment modality. In studying the immunogenicity potential of stopper extractables, Mueller et al.246 found that extractables (after incubation at 100 C for 4 days) or leachables (after incubation at 40 C after 1 year) in aqueous formulations from both coated or uncoated stoppers could not activate human DCs as measured by upregulation of CD86. Formulations containing PS80 did lead to an upregulation, but this was related to the degradation products of PS80, specically of the oleic acid moiety as discussed earlier. On the subject of stoppers, the use of natural rubber closures has been associated with dermal allergic reactions with leachates, and it is strongly recommended that these should not be used for parenteral products because suitable synthetic substitutes are readily available.277 Poland et al.,278 however, could not detect latex allergens in reconstituted smallpox vaccine vials or stoppers. A number of product labels have carried or currently carry a warning note about the presence of natural rubber in the needle cover of prelled syringes (e.g., Humira R , Kineret R , Neulasta R ) or vial stopper (Somavert R ). Phthalates are commonly used as plasticizers in plastic components and can be found in infusion bags and infusion sets. Diethyl hexyl phthalate (DEHP) has been shown to be an oxidant
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in vivo279 and its extraction is aided by surfactants in formulation.280 DEHP has also been shown to function as an adjuvant when administered by the subcutaneous route.281 Other phthalate species have varying adjuvant potency.282 Testing of compatibility of the biologic product with infusion components is a useful exercise to ensure suitability for use in terms of its impact (physical and chemical) on the biologic. If an impact is seen, a risk assessment based on knowledge about molecule is needed to address the ability to use such components. The impact of any possible adjuvancy by DEHP is not simple to elucidate, but the possibility should be recognized. Although DEHP-free infusion components (bags and lines) are now available, the use of DEHP-containing components [e.g., polyvinyl chloride (PVC)] remains widespread. It is interesting to note that the package insert for the recently approved rhFVIII product, XynthaTM , carries a bolded warning about the possibility of extraction of DEHP from PVC by the PS80 in the formulation.283 Silicone oil coating is commonly used on stoppers and on the inside of syringes or cartridges as a lubricant to enable movement of the plunger. Silicone oil contamination by the syringes used for injecting insulin has been well documented (e.g., Chantelau et al.284 and Bernstein285 ). Current processes for siliconization of prelled syringes or cartridges apply well-controlled amounts and involve baking of the silicone emulsion. This tends to reduce the levels of silicone oil extracted into the formulation but does not eliminate it.286 The impact of silicone oil emulsion on immunogenicity is not clearly documented. Naim et al.287 showed variability in adjuvancy effect of silicone gel used in breast implants by measuring anti-OVA antibodies in mice. Different formulation/ products differed in the amount of anti-OVA titer generated. However, silicone gels used in breast implants differ very signicantly in their molecular weight and cross-linking compared with the silicone oil emulsions used in parenteral products, which have been shown to be well tolerated.286 Silicone oil droplets in a protein solution can lead to the formation of aggregates by serving as nuclei for hydrophobicity-driven interaction and proteinprotein association. Fibrous aggregates have been shown to form in an mAb formulation when drawn into syringes containing silicone oil288 and for a number of model proteins incubated with silicone oil.66 Silicone oil mixed in a 1:1 volume ratio with insulin solution (containing 400 U/mL with 10 :g/ mL of polyethylenepolypropylene glycol) was shown to lead to high ADA levels in rats compared with just the insulin formulation. However, this level of silicone oil (500 :g/mL) was signicantly higher than that found in clinical studies (0.65 :g/mL) and adjuvancy in the clinical setting was considered improbable.201 In summary, utilizing siliconized,
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single-use, disposable plastic syringes to prepare or administer protein products represents a risk (see, e.g., Abatacept289 ) and should be avoided. Evidence for immunogenicity caused by product impacted by silicone oil on stoppers, prelled syringes, or cartridges is limited or not available. However, this factor should be part of the design consideration when choosing and evaluating the primary package or dosing instructions.
all potential scenarios have been observed in dossier evaluation, ranging from an almost absent immune response in animals and a high immunogenicity in humans to a high immunogenicity in animals and low immunogenicity in humans (which is the usual case).
ANIMAL MODELS FOR ASSESSMENT OF IMMUNOGENICITY

The objective of this review was to relate productrelated factors to immunogenicity data generated primarily through clinical studies of biotherapeutics. However, it is clear that product quality parameters are seldom an explicit part of the design of clinical studies, and animal data are often the only data available. Preclinical studies therefore play an important role in the study of impact of these CMC or productrelated factors.14, 290,291 Animal models have traditionally been used to evaluate the safety of (bio)pharmaceuticals, but their utility in evaluation or prediction of clinical immunogenicity is controversial. The selection of the species itself is often a challenge.292 Data generated from the animal models must be placed in context of the type of molecule. Bugelski and Treacy293 grouped recombinant proteins into classes on the basis of preclinical immunogenicity. For some classes (e.g., bacterial proteins), immunogenicity in animals is often predictive for humans. For others, such as fully human proteins, even data from nonhuman primates can have little predictive value. Nonhuman primates with a high level of sequence homology to the human target are often seen as most relevant. However, the evidence for success is limited and mainly governed by the degree of conservation across species.13,292,293 Functional domains beyond the target may have reduced (structural and functional) homology, thus negating the predictive value of nonhuman primates.290 Limited homology means that the animal models will generally be overpredictive of human immunogenicity. If used for comparability studies to support process or formulation changes, a large number of animals may be required to obtain statistical signicance. The EMEA draft guidance on immunogenicity assessment also supports the use of animal models as part of comparability exercise.252 Wierda et al.294 advocated animal models as useful for predicting relative immunogenicity of human proteins, using a hyperimmunization and rechallenge protocol. However, given the lack of correlation to human response, the relevance of such studies is questioned.14 Jahn and Schneider53 Summarized their regulatory experience by stating that
In certain cases, transgenic animals (generally mice) that express the appropriate human transgene have been developed to allow the protein to be tested without generating a xenogenic response, that is, these are tolerant to the human protein. Such nonHLA transgenic models have limited utility because murine MHC differs from human leukocyte antigen proteins at the amino acid level. Peptide from the therapeutic protein that might be displayed by the human T cell may not do so in murine MHC, and vice versa. Other models have been developed that are transgenic to the human HLA. A more appropriate model would aim to cover both: the HLA most representative of the target population and also be tolerant to the human protein.231, 295298 Limitations on the use of animal models are magnied when trying to decipher the relative impact on immunogenicity of a few percentages of product degradants. To be able to detect such changes, the animal models must have a low baseline immune response or a slow development trajectory for immunogenicity whereas the studies have to be carefully controlled. Models with immune tolerance are likely important for recombinant human proteins given to patients who express the endogenous protein. Composite transgenics as described earlier could be useful for this purpose. However, immune tolerance may not be important for patients receiving foreign proteins (whether human or not). In summary, the utility of animal models would primarily lie in assessing the relative immunogenicity risk of CMC-related factors. However, the translation of an observation of increased or decreased immunogenicity in the animal model to the human clinical response would still be difcult. Appropriate animal models could also be used to identify the product-related factors most important for determining the immunological response. Due to a lack of other important determinants of human immune response such as disease state and concomitant therapy, animal models should not be used to predict immunogenicity in patients.
MINIMIZATION OF IMMUNOGENICITY
Given the signicance of clinical immunogenicity to the safety and efcacy of biotherapeutics, signicant efforts are made for its minimization. Strategies for minimizing immunogenicity build upon impacting as many of the factors listed in Table 1 as possible.11
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For example, expression systems are being improved to eliminate immunogenic glycoforms.125, 299 Cell engineering is being utilized to produce selected homogenous glycoforms, for example, by eliminating mannose or producing fully galactosylated forms.115 Progress in processing technology has also reduced levels of process and product impurities.300,301 Welldesigned and robust formulations can lead to products with improved storage stability.302 Improvements in packaging and in the supply chain can ensure that the product reaching the patient is of consistent quality. Patten and Schellekens9 state that the best method to prevent immunogenicity is optimizing production, purication and formulation of the biopharmaceutical protein to generate soluble, nonaggregated, native protein free of contaminating adjuvants. Although true to a certain degree, the fact that clinical immunogenicity is a function of several coincident factors means that simply preparing a pure drug product will not eliminate immunogenicity. Once a molecule has been selected, the ability to impact immunogenicity through CMC factors is limited. The greatest impact on immunogenicity will be seen from the proper selection of the molecule itself. Molecules should be designed or selected to have improved solutionstate properties, thus increasing solubility and chemical stability and reducing aggregation propensity, resulting in improved expressability, processability, and shelf-life. Computational analysis to guide sitedirected mutagenesis has been shown to have the ability to impact aggregation propensity, while preserving activity/potency.57,58,303,304 In effect, the druggability of the biologic must also be a selection criteron.305 Apart from improvements in product quality, an important aspect of the design of the molecule that is becoming computationally tractable is the removal of epitopes that lead to recognition by T cells. In silico prediction tools have been developed to identify T-cell epitope content of a protein. Because the T-cell epitopes presented by MHC Class II molecules are linear, sequence-based screening to evaluate the binding potential of overlapping peptides to the binding pockets of common HLA Class II alleles is performed. Immunogenicity potential of the whole sequence can be scored, allowing the possibility to identify, modify, or completely remove epitopes to prevent their display by MHC.30,55,306 Such guidance can be useful in early development of new biologics, although the models currently lack adequate validation and do not capture the complex interplay between in tolerance and immunogenicity that actually determines clinical outcomes.10 The ability to predict structural B-cell epitopes is much poorer but not as critical. Along the same lines, patients can also be screened for the presence of MHC genotypes that have the potential to raise antibodies against the protein therapeutic and treatment decisions made accordingly.
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In the case of therapeutic mAbs, their design has evolved from murine to chimeric to humanized and even to fully human for the express purpose of addressing immunogenicity15,23 and has succeeded in reducing but not completely eliminating immunogenicity. Harding et al.307 recently reported that the residual immunogenicity (assessed by detecting T-cell epitopes via T-cell proliferation assay) of humanized and fully human antibodies resides in the CDRs (i.e., the afnity-determining regions). In parallel, Wang et al.308 report, from a computational analysis, that the CDRs also contain potential aggregation-prone regions. These coincidences provide opportunities for rational design approaches to address multiple determinants of immunogenic potential while maintaining the activity of the molecule.
CONCLUSIONS AND RECOMMENDATIONS

Virtually all therapeutic proteins induce some level of antibody response. The immune reaction can vary from low-titer, low-afnity, transient IgM antibodies to a high-titer, high-afnity IgG response, with consequences ranging from none to severe or life threatening. Analysis of the causes of safety-related regulatory actions with approved biotherapeutics (US, EU 19952008) shows that the main factors were (1) their parenteral mode of administration manifested as injection site reactions, and (2) their immunomodulatory function manifested as infections and infestations, immune system disorders, and neoplasms (tumors).309 Immunogenicity as a consequence of CMC-related factors did not make the list. There are a multitude of factors that determine the appearance of immunogenicity, some of which can potentially be impacted by CMC aspects of the product. These, together with patient- and treatment-related factors, determine the type, level, and consequence of the immunogenicity seen clinically. Individual susceptibility plays a key role, whether determined by genetics or disease or a combination of both. Control of CMC factors alone will therefore never be able to completely eliminate immunogenicity. However, a number of steps can be taken to address the impact of these factors. Attempts to minimize immunogenicity need to begin at the molecule design stage itself, encompassing selection of sequence (elimination of immunogenic epitopes and favorable physical and chemical stability and solubility) and PTMs. Subsequent development of process and formulation has to be directed toward minimizing process- and product-related impurities and degradants that could act as adjuvants or danger signal. Awareness of the possible impact of these adjuvants allows the process engineer and formulator to design the appropriate process based on a knowledge of the strengths and weaknesses of
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the molecule to minimize the risk factors. A risk assessment strategy from a CMC perspective for a specic molecule/class would involve the evaluation of the following, with respect to their possible impact on the molecule and thereby on its immunogenic potential. (1) Expression system: Glycosylation and other PTMs (2) Impurities (3) Product: Processing steps, formulation, container closure (4) Degradation products The risk assessment would be in the context of the relevant therapeutic function, and patient and treatment factors, and could thus vary between molecules. Prior knowledge and experience can be used to assess the above factors. Evaluation of degradation products could also be performed in a preclinical setting in animal models as part of forced degradation studies utilizing well-characterized degradants. Among the CMC factors reviewed here, aggregation carries the greatest concern as a risk factor for immunogenicity, as it often is the least well-understood characteristic of the product. Aggregates resulting from different stresses are not similar. The connection between (the nature of the) aggregate and immunogenicity is also far from clear. Further research is required on this subject, but it is possible that a general rule connecting the two does not exist and that products or classes of product may have to be evaluated on a case-by-case basis. From a risk management perspective, aggregation is generally considered to be critical quality attribute, and the process and product have to be designed to minimize their formation. Preclinical studies with properly designed models may be useful to rank the factors as to their potential for impacting immunogenicity. In must, however, be kept in mind that translation of animal data to humans is not often predictive. The safety and efcacy prole of biotech products is established during research and development by appropriately designed nonclinical and clinical studies. Immunogenicity is monitored and evaluated during clinical studies, using the clinical study material, and thus any impurities/degradants/aggregates that could be potentially relevant for safety and/or efcacy are qualied. This qualication requires that material tested both in preclinical models and in the clinic settings be well characterized and that there is good communication between the clinicians and the product development scientists to ensure that information regarding any observed adverse events or differences in clinical outcomes is evaluated in the context of immunogenicity and can be collated
and examined for possible correlations to quality parameters. Preclinical and clinical experience with the product (or products from a class) can help to build a safety design space for product quality attributes. This design space can be enhanced by incorporating knowledge and understanding of the mechanism of action, and of the connection between safety and efcacy of the molecule to its quality attributes, where possible
ACKNOWLEDGMENTS
The author is grateful to Deborah Finco and Ellen McCormick, as well as the reviewers, for critical reading of the manuscript and numerous insightful suggestions.
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REVIEWS Impact of Product-Related Factors on Immunogenicity of Biotherapeutics

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 2, FEBRUARY 2011

IMPACT OF PRODUCT-RELATED FACTORS ON IMMUNOGENICITY OF BIOTHERAPEUTICS

FACTORS DETERMINING IMMUNOGENICITY

IMPACT OF PRODUCT-RELATED FACTORS ON IMMUNOGENICITY OF BIOTHERAPEUTICS

IMPACT OF PRODUCT-RELATED FACTORS ON IMMUNOGENICITY OF BIOTHERAPEUTICS

Choice of Protein Structure/Variant

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 2, FEBRUARY 2011

Table 3. Product Roferon

rhIFN-"2b Arg,23 His34

rhIFN-"2c Arg,23 Arg34 rhIFN-"con 88% homology to IFN-"2

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 2, FEBRUARY 2011

IMPACT OF PRODUCT-RELATED FACTORS ON IMMUNOGENICITY OF BIOTHERAPEUTICS

Table 4. Product Rebif

Betaferon/ rhIFN-$1b des Met1 , Betaseron Cys17 Ser, nonglycosylated

8 MIU (250 :g), s.c., every other day

Adapted from Clark.23

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 2, FEBRUARY 2011

IMPACT OF PRODUCT-RELATED FACTORS ON IMMUNOGENICITY OF BIOTHERAPEUTICS

Post-Translational Modications: Glycosylation and PEGylation

IMPACT OF PRODUCT-RELATED FACTORS ON IMMUNOGENICITY OF BIOTHERAPEUTICS

PEG-Intron (PEG-IFN-"2b) Neupogen (Filgrastim) Neulasta (PEG-Filgrastim) Elspar (aspargase)

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 2, FEBRUARY 2011

Host Cell Proteins, DNA, and Chromatography Ligands

IMPACT OF PRODUCT-RELATED FACTORS ON IMMUNOGENICITY OF BIOTHERAPEUTICS

Product-Related Impurities and Degradation Products

Mechanisms for Immunogenicity of Protein Aggregate.

IMPACT OF PRODUCT-RELATED FACTORS ON IMMUNOGENICITY OF BIOTHERAPEUTICS

Evidence for Immunogenicity of Protein Aggregates

IMPACT OF PRODUCT-RELATED FACTORS ON IMMUNOGENICITY OF BIOTHERAPEUTICS

Functionality of the Protein Therapeutic and Immunogenicity of Its Aggregates

Process Changes, Aggregation, and Immunogenicity

IMPACT OF PRODUCT-RELATED FACTORS ON IMMUNOGENICITY OF BIOTHERAPEUTICS

the molecule design stage itself, as briey discussed later.

Aggregation and Immunogenicity

IMPACT OF PRODUCT-RELATED FACTORS ON IMMUNOGENICITY OF BIOTHERAPEUTICS

ANIMAL MODELS FOR ASSESSMENT OF IMMUNOGENICITY

IMPACT OF PRODUCT-RELATED FACTORS ON IMMUNOGENICITY OF BIOTHERAPEUTICS

CONCLUSIONS AND RECOMMENDATIONS

IMPACT OF PRODUCT-RELATED FACTORS ON IMMUNOGENICITY OF BIOTHERAPEUTICS

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 2, FEBRUARY 2011

IMPACT OF PRODUCT-RELATED FACTORS ON IMMUNOGENICITY OF BIOTHERAPEUTICS

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 2, FEBRUARY 2011

IMPACT OF PRODUCT-RELATED FACTORS ON IMMUNOGENICITY OF BIOTHERAPEUTICS

IMPACT OF PRODUCT-RELATED FACTORS ON IMMUNOGENICITY OF BIOTHERAPEUTICS

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 2, FEBRUARY 2011

IMPACT OF PRODUCT-RELATED FACTORS ON IMMUNOGENICITY OF BIOTHERAPEUTICS

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 2, FEBRUARY 2011

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