Valerie Quarmby

Recommendations for the validation of immunoassays used for detection of host antibodies against biotechnology products

Most biological drug products elicit some level of anti-drug antibody (ADA) response. This antibody response can, in some cases, lead to potentially serious side effects and/or loss of efficacy. In humans, ADA often causes no detectable clinical effects, but in the instances of some therapeutic proteins these antibodies have been shown to cause a variety of clinical consequences ranging from relatively mild to serious adverse events. In nonclinical (preclinical) studies, ADA can affect drug exposure, complicating the interpretation of the toxicity, pharmacokinetic (PK) and pharmacodynamic (PD) data. Therefore, the immunogenicity of therapeutic proteins is a concern for clinicians, manufacturers and regulatory agencies. In order to assess the immunogenic potential of biological drug molecules, and be able to correlate laboratory results with clinical events, it is important to develop reliable laboratory test methods that provide valid assessments of antibody responses in both nonclinical and clinical studies. For this, method validation is considered important, and is a necessary bioanalytical component of drug marketing authorization applications. Existing regulatory guidance documents dealing with the validation of methods address immunoassays in a limited manner, and in particular lack information on the validation of immunogenicity methods. Hence this article provides scientific recommendations for the validation of ADA immunoassays. Unique validation performance characteristics are addressed in addition to those provided in existing regulatory documents pertaining to bioanalyses. The authors recommend experimental and statistical approaches for the validation of immunoassay performance characteristics; these recommendations should be considered as examples of best practice and are intended to foster a more unified approach to antibody testing across the biopharmaceutical industry.

Recommendations on risk-based strategies for detection and characterization of antibodies against biotechnology products

The appropriate evaluation of the immunogenicity of biopharmaceuticals is of major importance for their successful development and licensure. Antibodies elicited by these products in many cases cause no detectable clinical effects in humans. However, antibodies to some therapeutic proteins have been shown to cause a variety of clinical consequences ranging from relatively mild to serious adverse events. In addition, antibodies can affect drug efficacy. In non-clinical studies, anti-drug antibodies (ADA) can complicate interpretation of the toxicity, pharmacokinetic (PK) and pharmacodynamic (PD) data. Therefore, it is important to develop testing strategies that provide valid assessments of antibody responses in both non-clinical and clinical studies. This document provides recommendations for antibody testing strategies stemming from the experience of contributing authors. The recommendations are intended to foster a more unified approach to antibody testing across the biopharmaceutical industry. The strategies proposed are also expected to contribute to better understanding of antibody responses and to further advance immunogenicity evaluation.

Method Validation and Measurement of Biomarkers in Nonclinical and Clinical Samples in Drug Development: A Conference Report

No HeadingBiomarkers are increasingly used in drug development to aid scientific and clinical decisions regarding the progress of candidate and marketed therapeutics. Biomarkers can improve the understanding of diseases as well as therapeutic and off-target effects of drugs. Early implementation of biomarker strategies thus promises to reduce costs and time-to-market as drugs proceed through increasingly costly and complex clinical development programs. The 2003 American Association of Pharmaceutical Sciences/Clinical Ligand Assay Society Biomarkers Workshop (Salt Lake City, UT, USA, October 24–25, 2003) addressed key issues in biomarker research, with an emphasis on the validation and implementation of biochemical biomarker assays, covering from preclinical discovery of efficacy and toxicity biomarkers through clinical and postmarketing implementation. This summary report of the workshop focuses on the major issues discussed during presentations and open forums and noted consensus achieved among the participants on topics from nomenclature to best practices. For example, it was agreed that because reliable and accurate data provide the basis for sound decision making, biomarker assays must be validated in a manner that enables the creation of such data. The nature of biomarker measurements often precludes direct application of regulatory guidelines established for clinical diagnostics or drug bioanalysis, and future guidance on biomarker assay validation should therefore be adaptable enough that validation criteria do not stifle creative biomarker solutions.

Bioanalytical method validation for macromolecules in support of pharmacokinetic studies.

The development and validation of ligand binding assays used in the support of pharmacokinetic studies has been the focus of various workshops and publications in recent years, all in an effort to establish a guidance document for standardization of these bioanalytical methods. This summary report of the workshop from 2003 focuses on the issues discussed in presentations and notes points of discussion and areas of consensus among the participants.

Assessment and Reporting of the Clinical Immunogenicity of Therapeutic Proteins and Peptides—Harmonized Terminology and Tactical Recommendations

Immunogenicity is a significant concern for biologic drugs as it can affect both safety and efficacy. To date, the descriptions of product immunogenicity have varied not only due to different degrees of understanding of product immunogenicity at the time of licensing but also due to an evolving lexicon that has generated some confusion in the field. In recent years, there has been growing consensus regarding the data needed to assess product immunogenicity. Harmonization of the strategy for the elucidation of product immunogenicity by drug developers, as well as the use of defined common terminology, can benefit medical practitioners, health regulatory agencies, and ultimately the patients. Clearly, understanding the incidence, kinetics and magnitude of anti-drug antibody (ADA), its neutralizing ability, cross-reactivity with endogenous molecules or other marketed biologic drugs, and related clinical impact may enhance clinical management of patients treated with biologic drugs. To that end, the authors present terms and definitions for describing and analyzing clinical immunogenicity data and suggest approaches to data presentation, emphasizing associations of ADA development with pharmacokinetics, efficacy, and safety that are necessary to assess the clinical relevance of immunogenicity.

Recombinant human growth hormone poly(lactic-co-glycolic acid) (PLGA) microspheres provide a long lasting effect

The treatment of growth hormone deficiency requires the daily administration of recombinant human growth hormone (rhGH). Long lasting formulations of rhGH have the potential to increase patient compliance, improve quality of life, and increase the efficacy of rhGH (lower total dose). One approach to these formulations is the use of biodegradable, injectable microspheres consisting of poly(lactic-co-glycolic acid) (PLGA). rhGH PLGA microspheres (12% w/w rhGH) were produced using a conventional double emulsion process. Initial in vitro studies of these microspheres indicated an initial release of 35% (1 day) and a continuous release for 21 days. A single administration of these microspheres (200 mg) in rhesus monkeys resulted in an initial elevation of serum hGH levels followed by a lag phase (return to baseline) until day 12 and then a sustained level of hGH greater than 5 ng/ml for 30 days. Development of improved in vitro release methods yielded release profiles comparable to those observed in vivo as determined from a detailed pharmacokinetic analysis of the hGH serum data. Serum hGH concentrations at or above 5 ng/ml provided a maximal response in two primary indicators of hGH biological activity, insulin-like growth factor-I (IGF-I) and IGF-binding protein 3. Further assessment of serum samples in an in vitro cell-based assay indicated that the rhGH released in vivo from the microspheres was bioactive. The rhGH PLGA formulations were well tolerated with mild to moderate inflammation and fibrosis. One of four animals developed a low titer anti-hGH antibody response, but transgenic mice expressing hGH did not develop an immune response to rhGH released from the PLGA microspheres. Therefore, this formulation had a low immunogenicity similar to the commercial rhGH formulation (Nutropin®). Overall, these studies demonstrated that a single administration of rhGH PLGA microspheres provide a prolonged release of bioactive rhGH that is well tolerated. With the improved in vitro release methods, future rhGH PLGA formulations may be designed without a lag phase to yield a one month continuous release of bioactive rhGH.

Association of Endogenous Anti–Interferon-α Autoantibodies With Decreased Interferon-Pathway and Disease Activity in Patients With Systemic Lupus Erythematosus

OBJECTIVE Numerous observations implicate interferon-α (IFNα) in the pathophysiology of systemic lupus erythematosus (SLE); however, the potential impact of endogenous anti-IFNα autoantibodies (AIAAs) on IFN-pathway and disease activity is unclear. The aim of this study was to characterize IFN-pathway activity and the serologic and clinical profiles of AIAA-positive patients with SLE. METHODS Sera obtained from patients with SLE (n = 49), patients with rheumatoid arthritis (n = 25), and healthy control subjects (n = 25) were examined for the presence of AIAAs, using a biosensor immunoassay. Serum type I IFN bioactivity and the ability of AIAA-positive sera to neutralize IFNα activity were determined using U937 cells. Levels of IFN-regulated gene expression in peripheral blood were determined by microarray, and serum levels of BAFF, IFN-inducible chemokines, and other autoantibodies were measured using immunoassays. RESULTS AIAAs were detected in 27% of the serum samples from patients with SLE, using a biosensor immunoassay. Unsupervised hierarchical clustering analysis identified 2 subgroups of patients, IFN(low) and IFN(high) , that differed in the levels of serum type I IFN bioactivity, IFN-regulated gene expression, BAFF, anti-ribosomal P, and anti-chromatin autoantibodies, and in AIAA status. The majority of AIAA-positive patients had significantly lower levels of serum type I IFN bioactivity, reduced downstream IFN-pathway activity, and lower disease activity compared with the IFN(high) patients. AIAA-positive sera were able to effectively neutralize type I IFN activity in vitro. CONCLUSION Patients with SLE commonly harbor AIAAs. AIAA-positive patients have lower levels of serum type I IFN bioactivity and evidence for reduced downstream IFN-pathway and disease activity. AIAAs may influence the clinical course in SLE by blunting the effects produced by IFNα.

Evaluation of total IGF-I assay methods using samples from Type I and Type II diabetic patients.

Measurements of circulating insulin-like growth factor-I (IGF-I) levels are an important part of many studies on growth and development. Circulating IGF-I levels are growth hormone (GH) dependent and are also impacted by age, gender, nutritional status and disease. Moreover, IGF-I is the main pharmacodynamic marker of GH activity. The majority of circulating IGF-I is associated with high affinity insulin-like growth factor-binding proteins (IGFBPs), making accurate and precise measurements of total IGF-I concentrations in biological matrices technically challenging. Many total IGF-I assay methods combine an immunoassay with a sample preparation method aimed at removing IGFBPs. However, not all sample preparation methods efficiently remove all IGFBPs or BP fragments (BPFs), and there is currently no reference method for IGF-I measurement against which these IGF-I assays can be calibrated. We have evaluated a number of IGF-I immunoassays and sample preparation methods using plasma samples from normal donors and from donors with Type I and Type II diabetes mellitus. In order to eliminate the variability between assays due to differences in assay standardization, we used the same preparation of highly pure, fully characterized IGF-I as the standard for all assays. We found that the data produced by many of the IGF-I assay methods showed good agreement when IGF-I levels in samples from normal individuals were measured. However, we found that these agreements were quite poor when IGF-I levels in samples from diabetics were measured. This was true of methods that claimed to physically separate IGFBPs from IGF-I either by acid/ethanol extraction or by acid chromatography. Several methods have recently been developed that physically separate IGF-I from IGFBPs followed by a chemical displacer to displace any residual BPs or BPFs from IGF-I. We found that the data generated by these displacement methods showed good agreement when assaying samples from diabetic as well as normal donors. There is considerable discussion in the literature as to whether individuals with diabetes have normal circulating levels of IGF-I. Many of the published studies are based on assays that may not accurately measure IGF-I levels due to problems with assay standardization and/or with assay methodology. Displacement methods may enable us to more accurately measure IGF-I levels in diabetes.

Quantitative evaluation of fucose reducing effects in a humanized antibody on Fcγ receptor binding and antibody-dependent cell-mediated cytotoxicity activities

The presence or absence of core fucose in the Fc region N-linked glycans of antibodies affects their binding affinity toward FcγRIIIa as well as their antibody-dependent cell-mediated cytotoxicity (ADCC) activity. However, the quantitative nature of this structure-function relationship remains unclear. In this study, the in vitro biological activity of an afucosylated anti-CD20 antibody was fully characterized. Further, the effect of fucose reduction on Fc effector functions was quantitatively evaluated using the afucosylated antibody, its “regular” fucosylated counterpart and a series of mixtures containing varying proportions of “regular” and afucosylated materials. Compared with the “regular” fucosylated antibody, the afucosylated antibody demonstrated similar binding interactions with the target antigen (CD20), C1q and FcγRIa, moderate increases in binding to FcγRIIa and IIb, and substantially increased binding to FcγRIIIa. The afucosylated antibodies also showed comparable complement-dependent cytotoxicity activity but markedly increased ADCC activity. Based on EC50 values derived from dose-response curves, our results indicate that the amount of afucosylated glycan in antibody samples correlate with both FcγRIIIa binding activity and ADCC activity in a linear fashion. Furthermore, the extent of ADCC enhancement due to fucose depletion was not affected by the FcγRIIIa genotype of the effector cells.

Determination of critical quality attributes for monoclonal antibodies using quality by design principles

Quality by design (QbD) is a global regulatory initiative with the goal of enhancing pharmaceutical development through the proactive design of pharmaceutical manufacturing process and controls to consistently deliver the intended performance of the product. The principles of pharmaceutical development relevant to QbD are described in the ICH guidance documents (ICHQ8-11). An integrated set of risk assessments and their related elements developed at Roche/Genentech were designed to provide an overview of product and process knowledge for the production of a recombinant monoclonal antibody. This chapter describes the identification of critical quality attributes (CQAs) as an important first step for QbD development of biopharmaceuticals. A systematic scientific based risk ranking and filtering approach allows a thorough understanding of quality attributes and an assignment of criticality for their impact on drug safety and efficacy. To illustrate the application of the approach and tools, a few examples from monoclonal antibodies are shown. The identification of CQAs is a continuous process and will further drive the structure and function characterization of therapeutic proteins.