Tuesday, December 23, 2014

Potency Testing of Biopharmaceutical Products


Potency determination refers to the quantitative measurement of the biological activity of a given product. Biological activity is a critical quality attribute; therefore, potency testing is an essential component of quality control. Various procedures, including animal-based assays, ligand and receptor binding assays, cell culture-based assays, or other biochemical assays (such as enzymatic assays), may be used for potency testing based on the mechanism of action of the product. This article provides a review of the more commonly adopted assays—specifically ligand and receptor binding and cell-based potency assays, as well as recent advancements in statistical analysis for potency determination and strategies for phase appropriate method development and validation.

Ligand and Receptor Binding Assays

Many biological products, such as monoclonal antibodies, exert their function via binding to a cellular or soluble target, which subsequently triggers appropriate downstream cellular events. For these products, a binding assay offers direct measurement of the product’s affinity to its intended target and may be suitable for potency testing. The most common type of binding assays is the Enzyme Linked Immuno-Sorbent Assay (ELISA), which can be developed relatively quickly and typically offers robust performance. With the advancement of technology, various “homogeneous” immunoassays have been developed and successfully utilized for potency measurement in QC settings. Examples are Time Resolved Homogeneous Fluorescence Resonance Energy Transfer assays, Amplified Luminescence Proximity Homogeneous assays (such as AlphaLISA) and Proximity Based Electrochemiluminescence Immunoassays. These homogeneous immunoassays eliminate the need for wash steps, and the simple “mix and go” procedures result in decreased assay time and potential analyst error. In some cases, superior signal-to-noise ratio and better overall assay performance, as compared to traditional ELISA, may be achieved. However, custom protein conjugation may be required, and assay performance is much dependent on the quality of these critical reagents (tagged proteins, donor and acceptor beads, etc). In addition to immunoassays, Surface Plasmon Resonance (SPR) assays have also been utilized to measure product binding to its intended target. In an SPR assay, protein-protein interaction is detected in real time through changes in mass due to adsorption at the chip surface. Data generated can be used to calculate the binding constant; therefore, SPR assays can be particularly useful during product development. To date, SPR assays have not been used as widely as QC methods for potency measurement but have been adopted sometimes for product characterization, in particular in the field of Biosimilars as part of the comparability study to the innovator products.

Cell-Based Potency Assays

Cell-based potency assays are often the preferred format for potency determination, since they measure the physiological response elicited by the product, which may or may not be extrapolated solely based on demonstration of protein interactions between the product and its intended target. Cell-based potency assays should be developed based on the mechanism of action (MOA) of the product, and therefore, they come in many different formats. The most common types of cell-based assays used to characterize recombinant protein/monoclonal antibodies include proliferation and cytotoxicity assays, apoptosis assays, and assays that measure induction/inhibition of functionally essential signal molecules (such as phosphorylated proteins, enzymes, cytokines and cAMP). Cell proliferation and cytotoxicity assays are essentially cell viability assays. They are most often utilized for products that act through promoting or inhibiting cell growth/killing, such as recombinant growth factors, and Antibody-Drug Conjugate products, which are a common class of cancer therapeutics. Proliferation and cytotoxicity assays typically require prolonged cell culture incubation time and measure viable cell number via quantification of metabolic activity or metabolic substrate (such as ATP). For products that induce cell death via apoptosis pathways, an apoptosis assay measuring the caspase activity offers an alternative, faster method. Activation of the caspase activity is one of the early cellular events that take place in cells undergoing apoptosis. As a result, caspase-based apoptosis assays can often be accomplished within hours, compared to the 2 to 5 days required for traditional cell viability assays. Assays that measure induction/ inhibition of signal molecules tend to be more complex as the quantitation of signal molecules are often accomplished through an ELISA or enzymatic assay. Consequently, both the cell culture treatment as well as the follow up ELISA/enzymatic assay need to be optimized. When a “native” assay poses significant technical challenges that are difficult to overcome, a surrogate assay may be used with sound scientific rationale. For example, reporter gene assays have been frequently used when the intended biological effect has been shown to be mediated through relevant transcriptional regulation events. Reporter gene assays in general offer the advantages of easy set-up, short assay time (1 to 2 days), and reliable assay performance. In addition, once a reporter gene cell line is established, it may be used for the testing of multiple products that have a similar MOA and become a “platform” potency assay. It is of note that recently, Antibody Dependent Cell Cytotoxicity (ADCC) reporter gene assays have been developed and demonstrated with significantly more robust performance than the traditional Peripheral Blood Mononuclear Cells based ADCC assays. The effector reporter gene cell line can be coupled with an appropriate target cell line to assess the ADCC function of any given product. More specialized cell-based potency assays such as phagocytosis assays, cell transduction assays, cell differentiation assays, and viral plaque assays are also employed, whenever appropriate, based on product mechanism of action.

Statistical Analysis in Potency Assays

Many statistical considerations are necessary to support the development of potency assays and to establish suitability for use. In this article, we focus on the concept and implementation of “parallelism testing.” Typically in a potency assay, dose response curves of the test sample and the reference standard are generated, and test sample results are reported as “relative potency” compared to the reference standard. The sample and reference standard dose response curves are compared to determine similarity, or “parallelism.” Only when the dose response curves are parallel, can a meaningful relative potency result be calculated. Historically, classical hypothesis testing (Difference Testing) has been adopted for measuring parallelism. In recent years, there has been a move in the potency testing field towards the “Equivalence Testing” approach. In the new USP bioassay chapters (<1032>, <1033>, and <1034>), theoretical advantages, practical challenges as well as several recommended approaches for implementing the Equivalent Testing are well described. Ideally, the equivalence limit should be set taking into consideration both assay capability and knowledge of product characteristics. Sufficient assay data, generated from the reference standard comparing to itself, to multiple manufacturing lots, and to known “non similar samples” (for example, degraded samples), whenever possible, should be evaluated to determine appropriate acceptance criteria.

Phase Appropriate Potency Assay

Development and Validation Development of a biopharmaceutical product requires significant time and resources and carries a high level of uncertainty. Therefore, it is pragmatic to adopt a phase appropriate strategy for potency method development and validation. During the early stage of development, a binding assay (if appropriate based on MOA) is often preferred over a cell-based potency assay since a binding assay is much easier to develop and implement in a QC environment. However, as the project advances, especially when moving into pivotal clinical trials, a cell-based potency assay is often necessary and is typically preferred by regulatory authorities since it is more physiologically relevant and can sometimes reveal differences in product quality that are not detected in binding assays. It is important to note that some products may have multiple MOAs. In such a case, multiple assays may need to be established to sufficiently demonstrate product efficacy as well as lot-to-lot comparability. As an example, for monoclonal antibodies that are expected to function through direct inhibition of receptor-induced proliferation, as well as Fc function (such as ADCC and Complement Dependent Cytotoxicity [CDC]), a toolbox containing a cell proliferation assay, an ADCC assay, a CDC assay, and an array of chemio-physical assays may be necessary to support both product development and quality control. Once sufficient knowledge has been obtained on product consistency and correlations between results from these different potency assays have been established, it is possible that only one of the assays is selected as a lot release assay to support routine manufacturing campaigns.
Once a potency assay is developed, the sponsor needs to perform a method qualification or validation to demonstrate suitability for intended use. Method qualification/validation also often follows a “phase appropriate” approach. During the early phase of clinical trials, the potency method should at minimum be qualified to demonstrate sufficient accuracy, precision, linearity, and range. The focus on accuracy and precision ensures meaningful interpretation of dose escalation studies. Comprehensive method validation should be implemented as the product moves into Phase III clinical trials and in anticipation of commercialization. A late phase validation study is typically more extensive than that of an early phase qualification and performed under a written protocol that clearly defines the scope of the validation, the target acceptance criteria, and data analysis plan. Multiple analysts and instruments are often employed, and the number of necessary assay runs is justified based on assay variability and intrinsic bias (if known). Method accuracy can be established by testing a sample with known relative potency prepared from the reference standard. In addition, representative routine sample types (drug substance, drug product, etc) should also be tested to confirm suitability of sample handling procedures and method precision. Representative degraded samples—obtained through long term or forced degradation studies—are also frequently included for testing during method validation to confirm the method’s stability-indicating property. Although method robustness may have been established using results generated during method development, a Design of Experiment can often be included within the validation to demonstrate tolerance to varying critical assay conditions.
After successful completion of the method validation, proper assay maintenance should be performed to prevent assay drift. Critical reagents need to be qualified prior to use, and new lots of reference standard need to be calibrated and bridged to the old lot of reference standard. Trending and periodic review of method parameters, such as EC50, signal-to-noise ratio, assay failure rate, as well as relative potency results are also essential components of assay maintenance, especially in a QC environment.

Final Comments

Potency determination is a critical part of product quality control. Potency assays may present in many different formats based on the MOA of the product. Phase appropriate method development and validation strategies help to reduce patient and business risk and are an integral part of product development.

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