Robust Bioassays Needed for Increasingly Complex Analytical Workflows

Bioassays play a crucial role in biologic drug development as a means to determine potency, toxicity effects, pharmacological activity, stability, purity, and other product qualities. The complexity of biological molecules, however, requires a wide range of analytical procedures to adequately characterize the product. In particular, the concern over immunogenicity in the use of biologics is driving the need to develop more highly sensitive assays. Because of this concern, there is a continual need to refine biological assays to increase accuracy and reproducibility. There is a specific need to replace in-vivo bioassays with appropriate in-vitro assays (1).

In vitro and in vivo in biologics development

Both in-vitro and in-vivo bioassays are important tools employed during drug discovery/drug development, according to Weihong Wang, PhD, director of Technology Development, Eurofins BioPharma Product Testing. In-vitro bioassays use cell culture and offer the benefit of low cost and high throughput, both of which are essential during early development. An in-vitro bioassay also has the necessary robustness needed for use as a lot-release assay for product quality control later in the drug development life cycle, Wang explains.

In addition, an in-vitro bioassay is a more efficient method that allows researchers to clearly characterize product mechanism of action in a controlled environment, says Catherine Liloia, director, Cell & Microbiology, PPD clinical research, Thermo Fisher Scientific. “This approach is scalable and reproducible across production and can assess product stability,” she states.

In comparison, in-vivo bioassays (non-clinical) use animal models and are considered more relevant to potential clinical outcome, says Wang. In-vivo bioassays are often costly, however, and are less robust and subjected to strict regulations and compliance standards. “Regulatory agencies across the world have been encouraging sponsors to replace, reduce, and refine (the three Rs) dependence on animal studies by advancing development of, and evaluating new, fit-for-purpose methodologies,” Wang explains.

Liloia adds that in-vivo bioassays are more representative of the full biological complexity and pharmacokinetics of the human body; they can, therefore, more broadly assess potential patient safety impacts stemming from metabolic changes and other side effects. The downside, however, is that in-vivo bioassays require high investment, strict animal care protocols, and are not as conducive to high-level analytics, according to Liloia.

Generating needed data

Bioassays, both in vivo and in vitro, generate valuable data needed for regulatory filings, such as investigational new drug (IND) applications. In-vitro bioassays support determination of product potency, for instance, says Liloia. “When included in analytical panels during release and stability testing, potency data can demonstrate lot-to-lot consistency and long-term stability of the product,” she says.

In addition, bioassays provide data to better understand the impact of manufacturing process changes through process development. “Early in-vitro methods may be needed to help discern true product mechanism of action in biological pathways that are complex or not well characterized. These methods can sometimes be consolidated or replaced on a product release panel as data [are] collected to determine a single method’s capability to reliably determine product potency across production samples,” Liloia explains.

Meanwhile, in-vivo methods are necessary when an in-vitro method cannot by itself model the biological response in a reliable manner, as is the case with immunogenicity and other safety testing, Liloia adds.

IND-enabling studies, including pharmacology/pharmacodynamic, pharmacokinetics, and toxicology studies, are typically done through both in-vitro and in-vivo assays, states Wang. She adds that additional in-vivo/in-vitro assays to address product safety and quality, such as viral testing, and potency testing, are also necessary later on for product licensing.

The cell-based bioassay alternative

Cell-based bioassays have also become an important tool in the analytics arsenal for biologics drug development. According to Wang, cell-based bioassays measure relevant physiological responses in cell cultures, but unlike in-vivo bioassays, cell-based bioassays do not involve the use of live animals and often have different types of assay readouts/endpoints. “Nevertheless, the physiological responses are reflective of mechanism of action, and, therefore, cell-based assays are considered necessary components of product characterization, in addition to other biophysical/physiochemical analytical methods,” she states.

Cell-based bioassays utilize biologically relevant cell lines to measure product potency, says Benjamin Ziehr, senior group leader, PPD clinical research, Thermo Fisher Scientific. He notes that there are several types of cell-based bioassays that vary depending on what is being measured. “The classic cell-based bioassay compares the response of cells treated with drug sample or reference standard. Typically, sample and standard are diluted in a manner suitable to produce a four-parameter response curve in the target cell line. Well-defined response curves are critical for maintaining and demonstrating control of the assay and drug product in these assays,” he explains.

Ziehr further explains that the relative activity of sample drug material to reference standard drug material is reported as the relative potency of the drug product. In comparing cell-based bioassays to other types of bioassays, the key distinction lays in the fact that cell-based bioassays allow mechanism-of-action-dependent measurements, whereas other bioassays measure physical characteristics of the drug product, such as epitope binding or receptor occupancy. “Therefore, cell-based bioassays allow measurement of the cellular processes responsible for a drug product’s effects on the patient,” says Ziehr.

Meanwhile, there are several other modes of cell-based bioassays that provide information about other attributes of the drug product. Ziehr points out two common examples of these other modes from cell and gene therapy product testing: infectivity and replication competency assays. “These assays measure the infectious potential of the drug product and the presence of replication-competent viral particles, respectively,” he states.

In other words, rather than measuring a cellular response, these infectivity and replication competency assays measure the accumulation of viral genetic material following infection of permissive cell lines. “While these assays don’t measure product potency, they do provide critical process and safety data and detect lot-to-lot variability of products,” says Ziehr.

Cell-based bioassays become a clear front runner in this movement, where applicable, remarks Wang, who points out that, in other circumstances, sponsors may be able to eliminate certain in-vivo assays based on risk analysis. For example, per the International Council for Harmonisation Q5A(R2) (2), in-vivo testing is no longer necessary for well-characterized cell lines such as Chinese hamster ovary, non-secreting murine myeloma, and SP2/0 (a standard mouse myeloma cell line), based on cell-line history, prior knowledge, and other risk-based considerations, notes Wang.

Despite their advantages, however, cell-based bioassays are limited in their ability to represent more complex, distal biological responses in a patient, cautions Liloia.

Challenges with cell-based bioassays

Meanwhile, the challenges in working with cell-based bioassays include not only the fickleness of working with certain cell lines, but also the fact that the day-to-day variability is much higher in terms of assay performance, observes Ziehr. The state in which cells enter the bioassay affects their response in the bioassay.

“Cellular state is dictated by many factors, notably metabolic stimuli, cellular density—both in culture leading up to a bioassay and in the bioassay itself—and cellular age. Stable transfections, optimized banking, and robust maintenance controls are needed to minimize cell variability,” Ziehr says.

Ziehr explains that, typically, robust maintenance controls involve the use of well-defined and controlled culture protocols, qualification of critical reagents, and substantial system suitability criteria in the bioassay. “Consistency of technique is challenging, even with clear procedure, so robust training with clear connection between biological processes and cell handling technique remains important,” he remarks.

Because cell-based assays utilize live cells, they are subjected to intrinsic assay variability that are often larger than that of physiochemical methods, says Wang. Thus, the selection of an appropriate cell line that is not only relevant to product mechanism of action, but also able to support a robust response in a dose dependent manner, is the single most critical factor for successful method development, she emphasizes.

“Many details need to be considered and optimized to produce a robust assay suitable for quality control. In addition, for products that may have multiple mechanisms of action, a carefully selected matrix containing multiple cell-based assays may be necessary to fully characterize the product,” Wang says.

Wang also points out, however, that it may yet be difficult to develop a cell-based bioassay for some products that either have not known defined mechanisms of action or have complex mechanisms of action. Furthermore, some cell-based assays still require extended testing time, which can be a cause for concern regarding products that require rapid factory-to-patient delivery. In such cases, Wang suggests that development of surrogate assays and/or orthogonal assays may be beneficial, though subject to discussions with the relevant regulatory agency.

In terms of advantages conferred, compared to in-vivo bioassays, cell-based bioassays often offer the advantages of a more streamlined testing procedure, higher assay throughput, quicker turnaround time, and less assay variability, according to Wang. “It is generally easier to develop a cell-based assay with an accurate quantitative endpoint than an in-vivo assay, which is particularly essential when the method is intended to be used for quality control,” she states.

Liloia notes that cell-based methods are quite challenging in many areas. Solutions such as liquid handling automation helps in providing accurate and precise sample dilution pipetting and transfers to the low concentrations and small volumes often needed in a cell-based assay. She also notes, however, that analytical workflows have become more complex because of the diversity of analytical readouts needed to support expanding product areas.

The growing complexity of analytical workflows has resulted in a greater need for coordination across functional areas, broadened cross-training, and design changes to laboratory spaces to maintain efficiency while also maintaining process flows that consider biosafety and contamination prevention, Liloia says. “Data analysis expectations continue to evolve as laboratories and agencies gain experience with newer products, requiring strong statistical support and the ability to apply industry best practices in a new way,” she states.

Overall, cell-based bioassays are considered a critical tool to predict quality and safety of drug products in the patient. “When established early in the development process, cell-based bioassays can provide early indicators of lot-to-lot variability in manufacturing, drug potency stability, and impact from process changes,” states Ziehr.

“Bioassays have an essential role throughout the life cycle of product development of biologics.Many aspects of product discovery, development, characterization, and quality control require careful construction and implementation of bioassays in order to thoroughly assess product safety, efficacy, and stability,” Wang adds.

References

1. Mire-Sluis, A.R. Progress in the Use of Biological Assays During the Development of Biotechnology Products. Pharm Res. 2001, 18, 1239–1246. DOI: 10.1023/A:1013067424248.
2. ICH, Q5A(R2) Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin, Draft version (2022).

About the author

Feliza Mirasol is the science editor for BioPharm International.