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Discovery & Development Analytical Science, Bioprocessing - Upstream & Downstream, Quality & Compliance

Meet the Trendsetting Techniques of Biotherapeutic Molecule Characterization

Producing accurate analytical assessments of complex biological molecules places a heavy demand on lab workers and managers – and an even heavier demand on capabilities and operational resources. The demands are non-negotiable; developers absolutely need detailed functional and structural data to advance their programs. Assessing the therapeutic activity of today’s biotherapeutic molecules requires sensitive analytical technologies and systems that perform two key roles: i) characterizing the increasingly complicated protein structures, and ii) closely observe pharmacokinetic activity.

Analytical assessments of complex biological molecules rely on an array of functional assays to help determine whether the molecule under scrutiny elicits an appropriate cellular and immune response that correlates to potency in vivo. Fortunately, analytical technologies are evolving; breakthroughs in tech are rendering assessments more efficient and providing for as-yet unmet needs in biopharmaceutical analysis. Here, we examine the use and evolution of three key analytical technologies.

Hello, ELISA

One challenge biopharma developers face is the question of how to evaluate the potential functionality of the molecule in vivo. During early phase development, researchers often begin assessments using an enzyme-linked immunosorbent assay (ELISA) to determine the binding potency of their biological molecule. As binding potency assays typically require less time for development and qualification, this approach can accelerate the Investigational New Drug (IND) filing process.

ELISA is a plate-based assay technique designed for detecting and quantifying soluble substances, such as peptides, proteins, antibodies, and hormones. This popular early-development phase assay – also referred to as an enzyme immunoassay (EIA) – often serves as a jump-off point for biomolecular analysis (1).

There are several types of cell-based assays that can be used as a model to assess a molecule’s activity in-vivo.

In an ELISA, the target macromolecule is immobilized on a microplate and then complexed with an antibody linked to a “reporter” enzyme. By measuring the activity of the reporter enzyme via incubation with the appropriate substrate, a measurable detection is observed. In most cases, the most critical aspect of ELISA analysis is that it identifies a highly specific antibody–antigen interaction.

Because it can yield fundamental and critical data, ELISA is generally considered an early development tool. In some cases, a cell-based assay may be needed to assess proper function; for example, if the molecule has multiple functional domains that may interact with several molecules to produce the desired effect.

There are several types of cell-based assays that can be used as a model to assess a molecule’s activity in-vivo. Cell viability assays determine the ratio of live to dead cells. Cell proliferation assays assess the biological process of cells as they proliferate over time through cell division. Other varieties of assay include cytotoxicity, cell signaling, and cell apoptosis. Cell-based assays have a number of utilities – for example, certain assays can measure anticancer drug effects, and others can yield data to support CMC development and technical transfer (2).

A prize-winning approach: PCR DNA amplification

Polymerase chain reaction (PCR) is recognized as one of the most important scientific advances in the history of molecular biology. It revolutionized the study of DNA so completely that its creator, Kary B Mullis, was awarded the Nobel Prize for Chemistry in 1993. Described by the National Institutes of Health’s National Human Genome Research Institute (NHGRI) as molecular “photocopying,” PCR assays have proved to be an efficient means of “amplifying” or copying small segments of DNA (3).

According to the National Human Genome Research Institute, because significant amounts of a sample of DNA are necessary for molecular and genetic analyses, studies of isolated pieces of DNA are nearly impossible without PCR amplification. Once amplified, the DNA produced by a PCR assay can be used in many different laboratory procedures. Lastly, PCR also supports a number of laboratory and clinical techniques including DNA fingerprinting, detection of bacteria or viruses, and diagnosis of genetic disorders.

But PCR is also an essential analytical tool for biologics. For example, quantitative PCR is necessary for amplification of residual host cell DNA (HCD) – an upstream process-related impurity resulting from cell culture processes. HCD can elicit oncogenicity, infectivity, and possible immunomodulatory effects. Demonstration of viral clearance is also needed prior to clinical trials to ensure the purification process steps have reduced viruses that may have been introduced during production. During viral clearance studies, reverse-transcriptase PCR (RT-PCR) can be used to confirm the presence of viruses and determine their identity.

Glycan analysis: Not so sweet

Glycan analysis can be challenging because glycosylation occurs heterogeneously; multiple glycan structures are present on a single site but can also result from branching. Relative quantitative glycosylation analysis provides better information with more reproducible and robust results; however, such methods are time-consuming and laborious to accomplish in commercial settings. In contrast, high-throughput glycan analysis can save time but usually only offers qualitative results.

Currently, appropriate glycan analysis method(s) may be chosen based on selection criteria, information needed, and suitability relative to the process stage at which the data is needed. However, an understanding of how glycan structure affects the protein activity of the molecule will help determine which method is appropriate.

Extractables and leachable analysis and stability

The vast majority of finished biopharmaceuticals are liquid and parenterally delivered. Throughout manufacturing, the actives and compounds come into contact with a variety of materials used in handling and bioprocessing. Similarly, to reach patients doses, liquid biotherapeutics must be filled into a variety of primary packaging. Regardless, every surface the formulation comes into contact with – from the factory to the point of patient infusion – must be analyzed for all possible interactions.

Some extractables and leachables (E&Ls) can be toxic, carcinogenic, or immunogenic. And some E&L compounds can affect the quality of the therapeutic biological protein by altering its physico-chemical properties. For instance, metal ions can significantly affect glycosylation profiles and can, in some cases, cause aggregation. As biological molecules have multiple potential sites for interaction, leachables can bind to the molecule, leading to unfolding, truncation, or aggregation.

Developers and biopharmaceutical manufacturers need to comprehensively understand the potential of E&L compounds to interact with the molecule – and do so early enough to mitigate potential stability issues before they disrupt a program entirely.

Evolving analysis: capillary electrophoresis

At the turn of the 21st century, release and stability analytical analysis for size variants was typically performed using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), a discontinuous electrophoretic system commonly used to separate proteins with molecular masses between 5 and 250 kDa. Similarly, isoelectric focusing (IEF) gels were used to assess and correlate charge variants.

Increasingly complex molecules such as bi/multi-specific antibodies, fusion proteins, or recombinant proteins are posing new challenges because they require novel techniques and don’t quite fit into the traditional analytical “box.”

Both of these techniques are time-consuming and expensive because processing the gel slabs and processing/imaging the gels can be labor and material intensive. The result is a higher than necessary cost of goods (CoG) profile that is not economically sustainable in the long term, especially for labs purchasing pre-cast SDS-PAGE and IEF gels.

Since then, capillary electrophoresis (CE) has become more ubiquitous and central to CE-SDS and (i)cIEF analysis. Capillary electrophoresis allows for separation of the molecule based on size or charge within a capillary, which enables more robust and reproducible results.

Increasingly complex molecules such as bi/multi-specific antibodies, fusion proteins, or recombinant proteins are posing new challenges because they require novel techniques and don’t quite fit into the traditional analytical “box.”

Evaluating and assigning the critical quality attributes (CQAs) for complex molecules can be difficult. For example, consider the characterization of multi-specific antibodies; traditional size exclusion high-performance liquid chromatography (SE-HPLC) and CE methods may not have the sensitivity to resolve minor differences in protein product variants, such as chain mis-pairings. For optimal results, the above-mentioned platform methods should be optimized to ensure that the intricacies of more complex molecules (product-related variants, impurities, and post translational modifications) can be resolved in a reproducible and robust manner.

Advanced analytical methods lead the way ahead

Industry experience tells us that when manufacturers combine fit-for-purpose cell line development platforms with advanced structural and functional analytical methods, it can optimize development and accelerate project timelines.

Incorporating phase-appropriate analytical platform methods along with high-throughput techniques to accurately characterize molecules in development will play an increasingly crucial role, helping biologics developers bring safe and efficacious drugs to patients faster.

Image Credit : Markus Winkler / and Wikimedia Commons

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  1. Thermo Fisher Scientific, “What is an ELISA (enzyme-linked immunosorbent assay)?”, Thermo Fisher Scientific (2015). Available at:
  2. Ruti Goldberg, “Analytical Methods for Cell Therapies: Method Development and Validation Challenges”, BioProcess International (2021). Available at:
  3. National Human Genome Research Institute, “Polymerase Chain Reaction (PCR) Fact Sheet”, National Human Genome Research Institute (2020). Available at:
About the Author
Eliza Lee

Eliza Lee is a Lead Scientist at Samsung Biologics, a contract development and manufacturing organization (CDMO). With over 15 years of extensive experience in the fields of R&D and biopharma, Eliza plays a significant role in ensuring that the analytical program management at SBL is fit for purpose and is suitable throughout the product lifecycle. She acquired her Bachelor of Bioengineering from University of California, Berkeley

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