Protein characterization broadly covers assays related to a molecule’s chemical composition (including stereochemistry), structural determinations (e.g. primary, secondary, tertiary, and quaternary), purity/homogeneity, and activity (including suitability to purpose or, for protein drugs, efficacy). Different levels of rigor apply to proteins used in foods, as reagents, or as drugs.
Commercial biotechnology and its leading revenue source, biopharmaceuticals, are responsible for elevating the art and science of protein characterization to the highest levels of sophistication. Today the market for biologic drugs is estimated at around $125 billion in the U.S. alone. Every protein molecule in drug development undergoes extensive characterization for composition and effectiveness—not just at the point of sale or use, but, in the form of stability testing, throughout its commercial life. With 23 biosimilars already approved in the U.S. as of this writing, the need for protein characterization has become even more acute.
Characterizing proteins has become an expert-driven activity reliant on advanced instrumentation, compendial methods, and deep, broad scientific knowledge. Where the characterization of protein drugs used to occur almost exclusively within the drug sponsor’s own laboratories, the field has become so specialized—yet at the same time so dependent on diverse analytical skills—that today many biopharmaceutical companies outsource the activity to third-party entities.
What drug sponsors need
Primary structure (amino acid sequence) is arguably the most fundamental characteristic of proteins. Thanks to advances in mass spectrometry, drug sponsors can unravel not just sequences, but post-translational modifications experienced by specific amino acid residues.
Higher-order secondary and tertiary protein structures require another set of analytical methods, including circular dichroism, differential microcalorimetry, Fourier-transform infrared spectroscopy, and other biophysical methods. Aggregation status—of significance in determining actual titer and one aspect of a drug formulation’s immunogenicity—relies on separation or purification methods like size-exclusion chromatography.
The third major group of characterization exercises related to safety, efficacy, and stability requires ongoing, lifecycle-long vigilance and technical expertise, from discovery to beyond licensure. Since stability so often depends on formulation, analytical labs charged with quantifying drug substance activity over time must possess deep familiarity with buffers, excipients, stabilizers, and their interactions in a finished product. Once a biopharmaceutical’s activity has been established, developers must come up with robust biochemical and cell-based assays to assure that the product, whose identity has already been confirmed, behaves as it should in vivo. Activity or mechanism of action demands expertise in a wide range of biochemical assays plus cell biology and the emerging organoid/spheroid disciplines.
Clearly, the depth and breadth of characterization studies required to establish the identity, activity, and safety of protein drugs rarely exists at any one discovery company or analysis lab.
“A protein characterization service provider should understand a wide array of projects to support research during all development stages,” says Ulrike Herbrand, Ph.D., scientific director, global in vitro bioassays at Charles River Laboratories. “Service companies must possess the latest instrumentation and technology to ensure rapid and sensitive testing for analyzing complex protein structures. Throughout the process, all reporting and analysis must conform with safety and quality guidelines such as Good Manufacturing Practices and Good Laboratory Practices, while complying with and adhering to best-practice data integrity policies. This combination of expertise and credibility is essential toward generating trustworthy data for regulatory submission.”
Focus on safety
Many of the quality attributes assumed under the heading of “protein characterization” serve multiple ends. For example, homogeneity assays address chemical purity, efficacy, and safety which, according to Mark Whittaker, manager, analytical core facility at Bio-Techne, raises “significant concerns” surrounding this characteristic on the part of drug developers.
One technique for measuring homogeneity, analytical ultracentrifugation (AUC), works by separating proteins by molecular weight. AUC covers aggregates over a molecular weight range of kDa to MDa. Size-exclusion chromatography (SEC) is the method of choice for analyzing submicron aggregates and for stratifying them according to molecular weight.
Regulatory issues are a significant factor in determining how to approach and use an outside service provider, Whittaker says. “One of the challenges a customer may face is that, since applications are custom, a test may not be validated for the specific molecule of interest.”
At that point, customers operating in regulated environments face the challenge of either accepting the data as-is or paying a quite significant premium for molecule-specific validation.
“We have generally recommended operating within the realm of Good Laboratory Practices on validated instrumentation with appropriate process controls, rather than moving to a fully validated, molecule-specific process,” Whittaker adds. “We have found that this very often serves the customer’s needs without the large upfront costs. Making this work demands maintaining a dialog with the customer to accurately determine their needs, to define a statement of work, and to be willing to adapt as needed to changing market and scientific demands.”
Depth and breadth of knowledge, as well as access to a comprehensive portfolio of instrumentation and methods are critical in selecting a protein characterization services provider, Whittaker says. “Also helpful is finding a provider capable of out-of-the-box thinking. Some analyses appear simple on the surface, but turn out to be quite involved once the layers of complexity have been peeled away. The ability to switch gears and move in a different direction if needed is a clear plus.
Whittaker also advises discovery companies to examine a contract lab’s operations. “A lab may have $20 million in equipment, but if it is deployed inefficiently or overly restrictively, that lab may be unable to provide sufficient support. From my perspective as a service provider, I like to create an environment where the customer feels we are partners, and that communication can be open and productive.”