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GLOBAL BIOANALYTICAL SERVICES WHITE PAPER Implications of Immunogenicity in Drug Development Michele Gunsior, PhD, Covance Laboratories, Chantilly, VA efficacy. The latter is particularly prominent when immune responses abrogate efficacy of lifesaving therapies,” [Worobec, 2005]. The importance of immunogenicity to patient safety and drug efficacy has resulted in a number of guidance documents prepared by the regulatory community, the most recent of which is FDA’s Draft Guidance for Industry on Assay Development for Immunogenicity Testing of Therapeutic Proteins released December 2009. Introduction Biotherapeutics emerged in the 1970s as viable alternatives to small molecule drugs when the advent of recombinant DNA technology allowed for cost-efficient, large-scale manufacture of proteins in cultured cells under controlled conditions. In 1982, recombinant human insulin, developed by Genentech and Eli Lilly, was the first biotechnology therapy to be approved by the FDA. Since then the biotechnology industry has reached more than 50 billion dollars in annual revenue, and continued growth is expected as pharmaceutical companies shift more resources Immune Response into large molecule drug development, or make high-value The principal function of an immune system is to protect acquisitions of biologics companies. There are more than 200 the body against infection. For higher organisms this is biotechnology products currently on the market with 400-plus accomplished with the innate (immediate, non-specific) and additional therapies in clinical trials targeting diseases such as adaptive (specific, antibody-mediated) immune response. The cancer, Alzheimer’s, rheumatoid arthritis, multiple sclerosis, immune system must distinguish the organism’s own cells, tissues and HIV/AIDS. In 2014, it is predicted and proteins from non-self antigens, that 50% of the top 100 drug sales will although in autoimmune disorders such Biologics have the potential to induce an be biologics. This is a significant increase as rheumatoid arthritis and systemic antibody-mediated immune response that from 28% in 2008 and 11% in 2000 lupus erythematosus the immune system could produce severe consequences for [EvaluatePharma 2009]. humans…thus, immunogenicity testing is targets self tissues and proteins. Most an important part of developing non-self antigens will elicit an antibodylarge-molecule therapies. mediated immune response, termed In contrast to chemically synthesized immunogenicity, in animals and humans. drugs, biotherapeutics such as proteins, This adaptive response to a particular peptides, and antibodies generally possess antigen is heterogeneous; no two biological systems react in the large and complex structures often modified by glycosylation same manner, and the response of a single system will change or pegylation, and may be non-human in origin. These over time. The immune response is used advantageously in characteristics greatly increase the potential to induce an vaccinations, however, a primary concern in the development antibody-mediated immune response over their small-molecule of non-vaccine therapeutics is understanding, measuring, and counterparts, and in consequence may cause severe illness mitigating an unwanted, drug-induced antibody response. in humans. Real-world examples of therapeutics that have caused serious health consequences for healthy volunteers and patients include erythropoietin [Casadevall, 2002], factor VIII The myriad of factors that cause immunogenicity within a [Lavigne-Lissalde, 2005], and insulin therapy. Immunogenicity particular system can be categorized as drug-related or testing is thus an important part of developing a large-molecule patient-related. Some examples of drug-related factors drug for eventual FDA licensure and marketing. Worobec and are related to protein structure such as alterations due to Rosenberg state, “… the paramount concerns are safety and pegylation, glycosylation, and oxidation of the molecule. While Page 1 pegylation is often used to increase the bioavailability of population who lacks Factor VIII protein, and thus will label drug and reduce immunogenicity, anti-PEG-antibodies have the drug therapy “non-self ” and xenobiotic in origin. been detected and in some cases shown to be associated with changes to pharmacokinetics (PK) data and loss of efficacy Impact of Immune Response [Armstrong, 2007]. Structural characteristics of a protein Since a critical part of the drug development process is to such as repetitive amino acid sequences accurately assess drug exposure to (whether in secondary or tertiary determine bioavailability in the test structure) and the tendency to form The extent, duration, and severity of the species, it is important to understand aggregates can affect immunogenicity immune response can have a wide range how the presence of anti-drug rates as the immune system is primed of clinical consequences from little or no antibodies (ADA, also anti-therapeutic to respond to aggregates and other effect, to severe and life-threatening illness. antibodies, ATA) may impact this patterned, repetitive sequences. assessment. The antibodies produced Additionally, a particular drug meant as a result of drug challenge may be to mimic an endogenous counterpart may differ slightly sustaining, clearing, or neutralizing, all of which can affect the in sequence, causing the immune system to recognize the pharmacokinetic and pharmacodynamic properties of a the drug. therapeutic as “non-self.” A second drug-related factor is Sustaining antibodies prolong the drug half-life by protecting related to manufacturing process and drug formulation. it from normal clearance; in contrast, a clearing antibody more For example, drug production in eukaryotic or prokaryotic quickly decreases drug concentration below therapeutic levels systems may introduce immunogenic foreign proteins into the or removes drug from circulation. Binding antibodies may not product. Furthermore, the formulation itself may increase the clear a drug, but can interfere with pharmacokinetic analysis propensity towards aggregation as well as contain excipients or by preventing measurement of the therapeutic. Neutralizing impurities that induce an immune response. antibodies abrogate a drug’s therapeutic effect and potentially worsen the patient’s disease if not also inducing serious sideeffects. The extent, duration, and severity of the response can have a wide range of clinical consequences from little or no effect, to severe and life-threatening illness. The most wellknown example of severe illness is pure red cell aplasia in which neutralizing antibodies targeted against erythropoietin cross react with the endogenous protein. Finally, the presence of ADA can lead to other clinically important adverse events such as hypersensitivity reactions, anaphylaxis, serum sickness, and severe infusion reactions. Other drug-related factors include the route of administration, dose level, and frequency of dosing. It has been generally accepted that subcutaneous (SC) exposure is more immunogenic than either intramuscular (IM) or intravenous (IV) dosing. However, anecdotal evidence presented at a recent conference on immunogenicity (Boris Gorovits, Immunogenicity Summit, October 2009) indicates that this type of ranking is not necessarily supported by the data. A paper exercise evaluating data on 20 biotherapeutics of various classes indicated that in many cases, the rate of immunogenicity by IV exposure was equal to SC exposure. Given that many exceptions exist, specific testing of individual products is encouraged [Koren, 2008; Ponce, 2009]. Dose and frequency of drug administration effects are assessed more generally, as these are not truly independent of other factors. A single dose is usually not expected to produce a robust or well-developed immune response compared with multiple doses over time. However, frequent and high concentration dosing regimens is one mechanism used to induce drug tolerance in patients. The early immune response typically comprised of low affinity, low concentration IgM molecules, is of limited magnitude, and peaks within 7-14 days. As the immune response matures, often with repeated drug (antigen) exposure, isotype switching leads to a more robust response consisting largely of IgG antibodies. The presence of IgG4 indicates a well-entrenched immune response, with high affinity antibodies that are more likely to neutralize the effect of drug in higher concentrations. Understanding the antibody response to drug exposure is necessary to evaluating the significance of that response. Determining the magnitude of response is important in understanding and ultimately determining the safety and efficacy of a particular drug which may include: • Response duration - whether sustained or transient • Occurrence of adverse events such as allergic reactions • Evaluating the production of neutralizing antibodies • Correlating change in PK/TK profiles Patient or host-related factors, such as genetic disposition, immune status, disease state, and the presence of co-medications play a role in the immunogenicity rate. For example, a healthy individual will have a different immune response to a given drug than that of the targeted disease state population. For example, Factor VIII drug therapy is unlikely to induce the same response in healthy subjects whose immune system is more likely to see the drug as “self ” than the disease Page 2 The challenge then becomes developing sensitive and specific immunogenicity assays to determine the presence, specificity, magnitude, and neutralizing potential of ADA. levels of true positives. The screening cutpoint is determined by a statistical evaluation of a drug-naïve population representative of the study samples. Any false positives should then be distinguished from true positive (reactive) samples through a competitive assay, termed confirmation assay, with free drug. The specificity cut point is determined during evaluation of the screening cut point by incubating the same naïve samples (or, alternately, naïve samples spiked with low positive control) with an excess of drug and evaluating the reduction in signal of spiked versus unspiked samples. A detailed explanation for determining both the screening and confirmatory cutpoints can be found in Shankar, et al. Methodology for Immunogenicity Testing and Analysis A typical immunogenicity analysis follows a tiered approach that includes three types of analysis (Figure 1): an initial screen, in which study samples are assessed for the presence of ADA, a confirmation assay, which determines whether putatively positive samples react specifically with free drug (versus nonspecific reactivity), and a titer evaluation that provides a relative level of ADA. When assessment of neutralizing antibody potential is required, a fourth type of assay, most often cell-based, is implemented to determine whether ADAs in confirmed positive samples specifically prevent drug from binding to its target. Screening Assay Negative Positive Confirmatory Assay Positive Immunogenicity assessments can be made using a number of analytical platforms such as: • Enzyme-linked Immunosorbent Assay (ELISA) • Radioimmunoassay (RIA) • Electrochemiluminescence (ECL) • Surface Plasmon Resonance (SPR) Characterization: Neutralizing Titer Domain Specificity Ab Class Each of the platforms has advantages and disadvantages; a well-thought out development plan will take into consideration these practicalities in designing the immunoassay. For example, ECL assays are often the platform of choice as a homogenous bridging assay. ECL eliminates multiple wash steps and increases the ability to detect low affinity antibodies as well as various isotypes. For further characterization of the immune response, SPR, such as the Biacore system, may be used to determine the specific antibody isotype or binding characteristics. Negative Figure 1. Tiered approach for immunogenicity sample analysis. The screen assay to determine whether samples are positive or negative for the presence of ADA is performed first, typically followed by a confirmatory analysis, then further characterization such as ADA titer and drugneutralizing potential. An inadequately designed ADA assay can hinder the ADA assays are considered qualitative (or sometimes quasiappropriate assessment of clinical findings and delay quantitative) because the positive control does not accurately product development or marketing. The ability to correlate reflect the heterogeneity of immune system response, or, immunogenicity and clinical events therefore relies upon a usually, originate from the species of properly developed and validated assay. interest. The positive control is often a The approach to immunogenicity testing An inadequately designed ADA assay monoclonal antibody or polyclonal sera has undergone significant changes in can hinder the appropriate assessment derived from hyper-immunization of recent years. For example, Shankar et al., of clinical findings and delay product a particular species, and will vary from (2008) published a more concrete plan for development or marketing. the study sample in terms of affinity, developing and validating reliable testing avidity, specificity, and isotype. Given methods than was previously considered. the inherent dissimilarity between positive control and study Specifically they expanded upon previous industry opinions sample, the use of mass units in defining the level of response [Mire-Sluis, 2004] by recommending a more statistical and is of limited value, and not recommended by the FDA [U.S. objective approach in evaluating certain method parameters Dept. Health and Human Services, 2009]. The purpose of such as cutpoint and confirmatory cut-off limits. a positive control is therefore to demonstrate the method The screening cut point is defined as the level at and above functionality and monitor assay performance. The ideal positive which a sample is considered positive (or reactive) for ADA control is derived from the species of interest, which is usually and below which a sample is considered negative. A risk-based not possible for human subjects. If the positive control is from approach requires a cutpoint selection that leads to a 5% false a different species, it should be diluted into the matrix of positive rate as it is more appropriate for a certain number of study samples. In method design and execution, low, high, and samples be considered false positive than false negative. The negative controls are used to monitor assay performance. A high false positive samples can then be proven non-reactive in the positive control with signal at the upper end of linear response confirmatory assay ensuring that the method can detect low is useful in monitoring method performance over time, reagent Page 3 qualifications, and troubleshooting, or when a method has a demonstrable hook effect. In contrast, it is the low positive control that ensures reliable assay performance and provides confidence that the assay can detect low levels of ADA. clinical immunogenicity rate was only 12% [Ponce, 2009]. As many large molecule drugs are human in origin, it is expected that the animal species will recognize these as non-self proteins and mount an immune response. The development of ADA and magnitude of the response can have clear impacts on the ability to interpret study findings. As discussed earlier, antibodies can enhance the clearance of drug (reducing drug exposure and limiting efficacy, as well as altering PK and PD profiles), sustain bioavailability which may result in increased toxicity or increased efficacy, or neutralize the drug’s pharmacological effect which can decrease efficacy and toxicity. Thus the main purpose of evaluating immunogenicity in animals is to help interpret the PK and PD properties of the drug, as well as any toxicity findings, or more simply, ADA + PK + PD + toxicity = immunogenicity assessment. As a result, careful consideration should be made when selecting appropriate animal models. Both drug quantitation and ADA determination assays are subject to interference from matrix components. For drug quantitation assays this includes the presence of ADA; ADA assays are similarly prone to interference from free drug. Antibody drugs in particular have long half-lives, and ADA may stabilize drugs thereby prolonging the half-life. Effort is made to minimize these interferences as much as possible: time points for ADA analysis are typically chosen when the drug is in the elimination phase, which may be months following drug challenge, or the assay format may include an acid-dissociation step to remove or decrease antibody:drug complexes. The affect of drug on antibody detection, e.g. tolerance, in an analytical Accurate assessment of ADA in study samples is impacted by method is evaluated by incubating increasing levels of drug several factors, including the concentration of drug in matrix with the low positive control. The lowest concentration of drug and the choice of positive controls. The effect of high drug that prevents detection of ADA is termed the drug tolerance; concentration can sometimes be minimized by the assay format, drug tolerance depends, like sensitivity, upon the particular or by the use of techniques such as acid dissociation to decrease positive control chosen. It is therefore suggested that ADA drug:ADA complexes. The other limitations of an ADA method validations are designed to appropriately assess the method are those imposed by the choice of positive control, drug tolerance over the range of possible drug concentrations which will not adequately represent the heterogeneity of the that may be seen in test samples. Caution is required, however, study samples. A biological system mounts a polyclonal immune when interpreting results of study samples with measurable response in which antibodies produced are of differing affinity quantities of drug; since drug tolerance is dependent upon the and avidity. Excellent method chosen positive control – which drug tolerance and sensitivity itself is not representative of all The main purpose of evaluating immunogenicity in animals may be illustrated with the use possible immune responses – the is to help interpret the PK and PD properties of the drug, of a high affinity monoclonal assessment of ADA (positive or as well as any toxicity findings: antibody control, but this is negative) and the corresponding ADA + PK + PD + toxicity = immunogenicity assessment. not reflective of the biological level (titer) may not be accurate. response, which likely contains Similarly, since ADA may many high affinity antibodies and a host of low affinity/avidity interfere with quantitation of drug, the concentration of drug antibodies. The drug tolerance and sensitivity obtained from obtained from a particular sample may not be a true reflection method validation should not be applied to study samples, of the actual level. or used cautiously with careful consideration of the type of positive control and its similarity to the biological response; very Preclinical Implications of different sensitivities and drug tolerance results will be obtained Immunogenicity with a polyclonal preparation versus a single high-affinity Until recently, the focus of regulatory agencies and opinion monoclonal antibody control. papers has largely been the design and application of immunogenicity studies on human subjects, but an important Immunogenicity assessment in animals is best used to interpret part of any drug development program are preclinical study findings and assess the validity of toxicology studies. toxicology studies in which the effects of drug are evaluated During protocol development, blood samples should be in animal models. The particular choice of animal will depend collected for any study in which animals are exposed to drug on the drug characteristics and physiological function so as to for greater than seven days. In an effort to manage sample better understand the mechanism of action and effect in vivo as analysis, Ponce, et al. (2009) provided a decision tree that guides well as to assess toxicity. However, animal models are generally the investigator on whether ADA analyses will meaningfully poor predictors of immunogenicity in humans. For example, the contribute to data interpretation. For example, if suitable PK recombinant human monoclonal antibody Humira produced assays and PD biomarkers are available, and no change in a 100% induction of ADA in cynomolgus monkeys yet the profile is seen, then an ADA screen may not be warranted. If Page 4 however, changes are observed, then at minimum the screening assay should be performed, followed by titer if shown positive for ADA. Though a tiered approach (screen, confirmation, titer) is recommended for clinical sample analysis, confirmation is generally not needed in animal studies, nor is the fourth tier of neutralizing antibody assessment. Risk Level Drug Characteristics Examples Consequences Low Not structurally identical to endogenous protein Enzymes Infusion site reactions Antibody drugs Mild allergic reactions Non agonistic Clinical Implications of Immunogenicity Medium Partially or completely identical to endogenous protein It is widely understood that “the induction of immunogenicity in animal models is not predictive of a potential for antibody formation in humans” [ICH S6 Guidance] and thus the immunogenicity rate in a particular animal model does not necessarily preclude the initiation of clinical studies. An approach that takes into account a number of factors including the causes and consequences of immunogenicity is applied to any new biotherapeutic under consideration for marketing and licensure; proper risk assessment considers the probability of generating an immune response and the consequences of such a response [Rosenberg; Koren, 2008]. The risk is then categorized as low, medium, or high and the bioanalytical testing strategy is defined accordingly [Shankar, 2007]. Loss of efficacy Replacement therapy as Factor VIII Antibody drugs Endogenous counterpart is either missing or redundant Same as low risk Overstimulation of endogenous mechanism Immune complex formation or Not structurally identical to endogenous protein/agonistic High Partially or completely identical to endogenous protein Erythropoeitin GM-CSF Same as medium risk Neutralization of endogenous counterpart To appraise the probability of an immune response, the causes Endogenous Autoimmune of immunogenicity discussed under the Immune Response counterpart is deficiency section above are considered along with other factors such as: not redundant • Presence of drug conjugates (as to a toxin) • Exposure of neoantigenic epitopes upon drug denaturation The type of risk inherent to a particular drug dictates the or fragmentation testing and sampling strategy. Low and medium risk drugs • Natural tolerance or use of immunosuppressive drugs should have ADA samples taken frequently in Phase I • Length of drug treatment and Phase II trials to help understand the human immune • Homology of drug product to endogenous counterpart response to the drug; testing may be less frequent in Phase • Redundancy of an endogenous III. By contrast, a high risk drug, one counterpart with potential to cross react with and • Mechanism of action Proper risk assessment considers the neutralize an endogenous counterpart, probability of generating an immune might require frequent sampling and response and the consequences of Following assessment of immunogenic real-time ADA analysis (including such a response. potential, consideration of the possible neutralization potential) at all stages of clinical sequelae, as well as whether the clinical development, or sequential patient target indication is a life-threatening dosing (rather than a cohort design) to better mitigate risks. disease is needed to develop a risk-management plan. In mildAt a minimum, all immunogenicity assessments during clinical to-moderate responses to ADA, the patient may experience trials should include screen, confirmatory, and titer analysis. loss of drug efficacy, or allergic hypersensitivity reactions such The timing for implementation of a neutralizing antibody as an infusion-site reaction. More serious consequences include assay, and type of format required, will vary with drug product overstimulation of endogeneous mechanisms such as that and associated risk to the patient. A cell-based assay for a following treatment with TGN1412 [Medicines and Healthcare monoclonal antibody designated as low risk may be of limited Products Regulatory Agency, 2006], anaphylactic shock, or value (while taking considerable time and effort to develop and cross reactivity with, and neutralization of endogenous protein validate). leading to an autoimmune deficient syndrome (Table 1). Approaches to attenuate immunogenicity and minimize patient risk include drug redesign (to remove epitopes of Table 1. Risk assessment for clinical immunogenicity. Page 5 high immunogenic potential), change in dose and/or dosing procedure (IV administration versus inhalation), and exclusion of sensitive subjects (a patient with known allergies to a similar product could be prescreened with a skin test, or be administered drug in a clinical setting where severe allergic reactions can be readily treated). Once immunogenicity is observed, the clinician must decide the best path forward for a patient. If ADA inhibits the function of a life-saving therapy, it may be necessary to institute a tolerizing regimen to inure the immune system against the therapeutic. These decisions are made on a case-bycase basis with consideration of all available information. production of a sound risk-assessment strategy and a fit-forpurpose approach in testing of preclinical and clinical samples. Whatever the application, properly designed and validated immunoanalytical methods provide confidence in the data used for these purposes. References Armstrong, J.K., Hempel, G., Koling, S., Chan, L.S., Fisher, T., Meiselman, H.J., and Garratty, G. “Antibody Against Poly(Ethylene Glycol) Adversely Affects PEG-Asparaginase Therapy in Acute Lymphoblastic Leukemia Patients.” Cancer. 2007, 110(1) 103-111. Follow-on Biologics Casadevall, N., Nataf, J., Viron, B., Kolta, A., Kiladjian, J.J., Martin-Dupont, P., Michaud, P., Papo, T., Ugo, V., Teyssandier, I., Varet, B., and Mayeux, P. “Pure Red-Cell Aplasia with Antierythropoietin Antibodies in Patients Treated with Recombinant Erythropoietin.” New Engl. J. Med. 2002, 346(7), 469-475. Follow-on biologics, or biosimilars, are large-molecule therapeutics similar to a licensed drug, but manufactured and marketed by a third-party following patent expiration of the innovator molecule. While there is currently no mechanism for approval and licensure of biosimilars in the US, the EU and Japan both have a legal, regulated process that includes assessment of immunogenicity between the innovator drug and follow-on biologic at all stages of the development process to ensure comparable quality, safety, and efficacy. EvaluatePharma, “Biotech set to dominate drug industry growth.” 2009. (http://www.evaluatepharma.com/Universal/ View.aspx?type=Story&id=188700§ionID=&isEPVantage =yes) Paramount in assessment of immunogenicity is that the methods show comparable sensitivity and specificity. Key questions to consider when developing a single method that shows equal sensitivity and specificity to both the innovator and biosimilar drug products [U.S. Dept. Health and Human Services, 2009] include the following: • What is best the assay format and positive control to use? • If a bridging assay is employed, should the capture and detection reagents be innovator molecule or biosimilar? • Should the positive control be one generated from the reference product or follow-on biologic, or perhaps some combination of both? • If a single method can not be developed for immunogenicity comparison, then do the separate methods show equivalent sensitivity and specificity? de Galan, B.E., Simsek, S., Tack, C.J., and Heine, R.J. “Efficacy and safety of inhaled insulin in the treatment of diabetes mellitus.” Neth. J. Med. 2006, 64(9), 319-325. Koren, E., Smith, H.W., Shores, E., Shankar, G., Finco-Kent, D., Rup, B., Barrett, Y.C., Devanarayan, V., Gorovits, B., Gupta, S., Parish, T., Quarmby, V., Moxness, M., Swanson, S., Taniguchi, G., Zuckerman, L.A., Stebbins, C.C., and Mire-Sluis, A. “Recommendations on risk-based strategies for detection and characterization of antibodies against biotechnology products.” J. Immunol. Methods. 2008, 333, 1-9. Lavigne-Lissalde, G., Schved, J. Granier, C., and Villard, S. “Anti-factor VIII antibodies: a 2005 update.” Thromb. Haemost. 2005, 94(4), 760-769. Answers to these and other questions are important to distinguish between the effects of different manufacturing processes, purity of biosimilar versus originator therapeutic, or biosimilar itself from differences in assay performance. Medicines and Healthcare Products Regulatory Agency, “Investigations into Adverse Incidents during Clinical Trials of TGN1412.” 2006. (http://www.circare.org/foia5/ clinicaltrialsuspension_interimreport.pdf ) Conclusion In contrast to new chemical entities, biotherapeutic drugs are potentially immunogenic, and, in rare cases, treatment with such products can lead to severe and devastating illnesses in humans. Therefore, the importance in understanding how immunogenicity affects drug exposure, efficacy, and toxicity at all stages of the drug development process can not be overstated. The complexity of the immune response necessitates Mire-Sluis, A.R., Barrett, Y.C., Devanarayan, V., Koren, E., Liu, H., Maia, M., Parish, T., Scott, G., Shankar, G., Shores, E., Swanson, S.J., Taniguchi, G., Wierda, D., and Zuckerman, L.A. “Recommendations for the Design and Optimization of Immunoassays used in the Detection of Host Antibodies Against Biotechnology Products,” J. Immunol. Methods. 2004, 289(1-2), 1-16. Page 6 Ponce, R., Abad, L., Amaravadi, L., Gelzleichter, T., Gore, E., Green, J., Gupta, S., Herzyk, D., Hurst, C., Ivens, I.A., Kawabata, T., Maier, C., Mounho, B., Rup, B., Shankar, G., Smith, H., Thomas, P., and Wierda, D. “Immunogenicity of biologically-derived therapeutics: Assessment and interpretation of nonclinical safety studies.” Reg. Tox. Pharm. 2009, 52(2), 164-182. U.S. Dept. of Health and Human Services, Food and Drug Administration, “Guidance for Industry: Assay Development for Immunogenicity Testing of Therapeutic Proteins, Draft Guidance.” FDA-2009-D-0539, 2009. Worobec, A., and Rosenberg, A.S. “A Risk-Based Approach to Immunogenicity Concerns of Therapeutic Protein Products, Part 1: Considering Consequences of the Immune Response to a Protein.” BioPharm International. 2005, 17, 22-26. Proceedings of the International Conference on Harmonization (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. “Tripartite guideline S6 Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals. 1997. (http://www.ich.org/LOB/media/ MEDIA503.pdf ) Rosenberg, A.S., and Worobec, A. “A Risk-Based Approach to Immunogenicity Concerns of Therapeutic Protein Products, Part 2: Considering Host-Specific and Product-Specific Factors Impacting Immunogenicity.” BioPharm International. 2004, 17, 34-42. Rosenberg, A.S., and Worobec, A. “A Risk-Based Approach to Immunogenicity Concerns of Therapeutic Protein Products, Part 3: Effects of Manufacturing Changes in Immunogenicity and the Utility of Animal Immunogenicity Studies.” BioPharm International. 2005, 17, 32-36. Shankar, G., Pendley, C., and Stein, K.E. “A risk-based bioanalytical strategy for the assessment of antibody immune responses against biological drugs.” Nat. Biotech. 2007, 25(5), 555-561. Shankar, G., Devanarayan, V., Amaravadi, L., Barrent, Y.C., Bowsher, R., Finco-Kent, D., Fiscella, M., Gorovits, B., Kirschner, S., Moxness, M., Parish, T., Quarmby, V., Smith, H., Smith, W., Zuckerman, L.A., and Koren, E. “Recommendations for the validation of immunoassays used for detection of host antibodies against biotechnology products.” J. Pharm. Biomed. Anal. 2008, 48(5), 1267-1281. Covance is an independent, publicly held company with headquarters in Princeton, New Jersey, USA. Covance is the marketing name for Covance Inc. and its subsidiaries around the world. The Americas +1.888.COVANCE (+1.888.268.2623) +1.609.419.2240 Europe/Africa +800.2682.2682 +44.1423.500888 Asia Pacific +800.6568.3000 +65.6.5677333 www.covance.com CSBIO007-1210 Page 7