POTENCY TESTING


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


10 American Pharmaceutical Review | Biopharmaceutical Supplement 2014


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


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 “parallelism testing.”


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.


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