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European Biopharmaceutical Review

Quality Assurance

Biological activity is the specific capacity of a product to achieve a defined organic effect, as well as a critical quality attribute. Quantitative measure of biological activity, or potency determination of a product, can be accomplished using various procedures, including animal-based, cell culture-based, ligand and receptor binding, or other biochemical and enzymatic assays. Among these, cell culture-based potency assays are often the preferred format for the purpose of product release and quality control (QC). This is because they have the advantage of measuring a direct physiological response elicited by the product, when compared to ligand and receptor binding assays, and are generally more robust and cost-effective than animalbased assays.

Assay Formats

To support product licensing and commercial release, it is critical that appropriate cell-based potency assay formats are selected based on the mechanism of action of the product. Some common types of cell-based assays used to characterise recombinant protein/ monoclonal antibodies (mAbs) include proliferation, cytotoxicity, and apoptosis assays, and those that measure induction/ inhibition of functionally essential signal molecules – such as phosphorylated proteins, enzymes, cytokines and cyclic adenosine monophosphate.

Surrogate assays may also 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. More specialised assays – such as phagocytosis, cell transduction, cell differentiation and viral plaque assays – are also employed, whenever appropriate.

It is important to note that some products may have multiple mechanisms of action. In such a case, multiple assays may need to be established to sufficiently demonstrate product efficacy, as well as lot-to-lot comparability. A case in point is the characterisation of mAb products. A recent paper (Nature Reviews Drug Discovery 10, February 2011) proposed the classification of mAb products based on their intended target and the cellular response they trigger.

With scientific rationale, the paper described recommended phaseappropriate potency assays to characterise each class of the mAb products. In particular, Class 1 mAb products that bind to target cells and elicit Fc-mediated effector functions often require several cell-based potency assays, in addition to an array of chemiophysical assays, to support both product development and QC. Another example where multiple assays may be necessary is bispecific/dual specific or multi-targeting antibodies, which have received renewed attention in recent years.

When developing a cell-based potency assay intended for product release and QC, careful considerations need to be taken to ensure both scientific soundness, as well as successful implementation in a regulated environment.

Cell Line Selection

Selection of the assay format should be based on the product’s mechanism of action, as the choice of indicator cell line is one of the most critical aspects of assay development. Ideally, the cell line should be from a lineage close to the cell/tissue type targeted by the drug. Furthermore, due to the often quantitative nature of the cell-based potency assays, the cell line should be sensitive enough to respond to varying concentrations of the product in a dose dependent and consistent manner, while remaining relatively insensitive to small, intrinsic variations that occur during cell handling.

Often as part of the cell line selection, multiple assay formats are evaluated. For example, for a product that induces cell death in target cells, assays measuring an early readout, such as induction of caspase activity, or a late readout, such as reduced viable cell number, both reflect the mechanism of action. In such a case, both assay performance (signal to noise ratio, accuracy, precision, range, linearity, specifi city and robustness), as well as practicality (ease of operation, assay turnaround time and implementation cost) may need to be carefully considered to decide on the assay format best suited for QC purposes. Once the indicator cell line and assay format are selected, a master and/or working cell bank should be prepared and tested to confirm identity, purity, growth characteristics and functional stability.

Relative Potency

Another critical aspect of developing cell-based potency assays intended for QC testing is the data analysis. Results of cell-based potency assays are typically expressed as the ‘relative potency’ when compared to a reference standard. The use of relative potency allows direct comparison between the test sample and the reference standard within the same assay, therefore reducing the impact of run-to-run variability on final reportable results. However, relative potency is only meaningful when the reference standard and sample dose response curves are similar and parallel.

To this point, parallel line analysis (PLA) is now considered the industry’s standard methodology for calculating relative potency. PLA calculates relative potency results by comparing the response generated by the test sample versus that generated by the reference standard over the entire dose titration curve, rather than relying on response comparison of a single dose pair (historically EC50). PLA also generates statistical values that measure the parallelism between reference standard and sample dose response curve.

Several statistical theories – such as F-statistics, chi-square statistics, both a difference test, as well as various equivalence tests – have been proposed and implemented for measuring parallelism in bioassays. Despite the many different opinions on what might be the best methodology, the statistical model, along with the appropriate system suitability criteria, should be thoroughly evaluated using a sufficient number of representative data sets generated from a given assay, before implementation in a QC environment.

Validation Design

Adequate evaluation of product potency via a properly designed and qualifi ed method is expected by the regulatory agencies in support of clinical trials. In general, a ‘phase appropriate’ method qualifi cation strategy is adopted within the industry. During the early phase of clinical trials, the potency method should, at minimum, be qualified to demonstrate sufficient accuracy, precision, linearity and range. A properly qualified method capable of detecting lot-to-lot variability ensures meaningful interpretation of dose escalation studies. Results generated from a qualified method with clearly defined test procedures provide valuable information on assay performance over time, as well as an understanding of the product manufacturing processes as they are being optimised.

As the product moves into a Phase 3 clinical trial, and in anticipation of commercialisation, comprehensive method validation should be planned and implemented. Validation study design should consider all aspects of the testing procedures and be supported by sound statistical rationale. Method accuracy and range is typically assessed using reference standard prepared at known potency. However, representative routine sample types (drug substance, drug product) should also be tested to confirm lack of matrix effect, as well as suitability of sample handling procedures if these are different from those of reference standard.

Degraded samples that have significantly altered bioactivity, obtained through long-term or forced degradation studies, are also frequently used to demonstrate the stability indicating properties of the assay. Although method robustness may be established using results generated during process development, it is advisable to include robustness studies on certain key factors – such as cell number, cell passage number and incubation time – within the validation.

Method Transfer

Compared to method validation, fewer regulatory guidelines are available for the transfer of a cell-based assay. Due to their nature, cell-based assays tend to have higher inter-laboratory variability and may require a more extensive transfer scope when compared to other analytical methods. A phase appropriate approach may also be considered for method transfer.

Multiple assay runs involving multiple analysts are desired. During the early clinical phase of the product, a comparability study to demonstrate establishment of the assay in the receiving lab is typically performed. Results from actual manufacturing samples obtained at the receiving lab are compared to those results obtained at the originating one. Alternatively, or in addition to the routine samples, the originating lab may prepare mock samples with known potency to assess method accuracy at the receiving end.

For late-phase products, more comprehensive testing is likely to be required. Multiple lots of product are typically included in the comparability study, and method transfer almost always involves multiple analysts and assay runs at the receiving lab to provide better assessment of system precision. Often, a receiving lab may be required to perform a partial validation to confirm performance of key process parameters. In almost all cases, statistical analysis is conducted to ensure comparability of method performance between labs, and to prevent potential changes in data trend.

Furthermore, despite the intrinsic variability due to the use of a biological system, consistent cell-based assay performance can be accomplished with proper assay maintenance. Qualification of critical reagents, and proper calibration and/or bridging between new and old lots of reference standard, are both critical components of assay maintenance. Trending of key method parameters, such as EC50, signal-to-noise ratio, assay failure rate, as well as relative potency results, are also desirable to help reveal and correct assay drift over time.

Safety and Efficacy

Cell-based potency assays are an important part of establishing the safety and efficacy profi le of biopharma products. Therefore, development of these assays should be, first and foremost, based on science. For a cellbased potency assay intended for QC, a successful outcome requires additional considerations, including statistical analysis, phase appropriate validation and transfer strategy, and proper long-term assay maintenance.

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Dr Weihong Wang is Technology Development Manager for Eurofins Lancaster Laboratories’ Molecular and Cell Biology group. She oversees assay development/ validation projects and serves as a subject matter expert to assist clients with testing needs. Weihong has 12 years of experience developing and characterising biopharma products using ELISA and cell-based assays. She earned a PhD in Cell and Molecular Biology from Brandeis University, and completed postdoctoral research at Harvard Medical School.
Weihong Wang
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