On 10 December 2020, BPI presented an “Ask the Expert” webinar with Jason Sterling, PhD (principal scientist and project director in analytical and formulation resources), and John Rockwell (group leader) of Catalent Pharma Solutions. Biophysical characterization is critical to understanding the makeup and behaviors of biologic therapies and vaccines both early in development and throughout scale-up for manufacturing. As biologics become more complex in structure and as scientists improve their understanding of the effects of structure on stability, efficacy, and safety, new and improved analytical methods for characterizing biologic products must be developed.

The experts discussed challenges in biophysical characterization and presented solutions for overcoming them. Sterling reported on three common techniques that are used to identify, characterize, and quantitate sialic acids; Rockwell highlighted a method for evaluating extractables and leachables (E&L) to measure residual-free toxins during antibody–drug conjugate (ADC) development.

Sterling’s Presentation
Sterling focused on techniques for glycopeptide, N-glycan, and sialic-acid analysis. Dozens of distinct monosaccharide structures are found in nature; sialic acids are among the most heterogeneous sugars known. They are found as terminal monosaccharides on N-glycans, O-glycans, and glycosphingolipids, and they cap the side chains of GPI anchors in vertebrates. Sialic acids are predominant sugars on all cell surfaces, forming complex sialomes that are integral to cell–cell interaction, recognition, and signaling; cytoprotection; immune response; protein stability; ion binding and transport; cancer metastasis; metabolism; and infection (influenza).

Glycopeptide Analysis: Sterling showed that glycopeptide mapping provides site-specific details on the N-glycans at each N-glycosylation site on a target protein. It even can be used to delve into a protein’s tertiary structure. Because of coelution tendencies, using this method to quantitate sialylated species can be difficult. The technique is based on reversed-phase ultraperformance liquid chromatography (RP-UPLC) with tandem mass spectrometry (MS/MS).

N-Glycan Analysis: Using amide-column–based UPLC-MS/MS enables accurate glycoprofiling. The method that Sterling described can incorporate additional steps, and results depend on the overall N-glycan abundance in a sample. This is the most commonly chosen technique of the three.

Sialic-Acid Analysis: This method is based on fluorescence high-performance liquid chromatography (HPLC). It doesn’t provide information about N-glycan structures, but it is the only technique available that provides quantitative results.

Rockwell’s Presentation
Rockwell described challenges in free-drug analysis during ADC development, focusing on E&L from labware used to store and test ADC drug substance and product. Free-drug/toxin levels must be kept below defined limits and measured for product release and stability testing.

A typical residual–free-drug assay uses RP chromatography for quantitation. Because toxins have similar properties to compounds used in labware manufacture, they can interfere in residual–free-drug assays based on hydrophobicity and spectral absorbance. The impact of E&L on free-drug assays necessitates exploration of compatible containers that are safe for biologics storage and do not contribute peaks to residual–free-drug assays.

Rockwell’s team found that typical long-term ADC storage in glass or polyethylene terephthalate glycol (PETG) copolyester vials does not present concerns. However, MS identification of E&L can be challenging because of their low abundance and poor ionization. It is critical to select cryovials that minimize leaching in free-drug assays. Glass is acceptable but may not be appropriate for cryostorage.

Questions and Answers
What are some other challenges besides extractables in ADC development?
Safety First: The free drug used in a conjugate is highly toxic. The powder form can become aerosolized and should be handled inside a glove box. Many solvents used to solubilize those toxins for analysis actually enhance their ability to pass through your skin.

Heterogeneous Mixtures: In addition to typical antibody heterogeneities, you have a varying number of free drugs bonded to those antibodies. That factor can change the molecule’s properties (e.g., for UV concentration measurements and size-based methods).

Given the many species of free drug that elute in a profile, can additional free-drug species remain unaccounted for outside the window? During method development and validation, you must evaluate whether you are accounting for all peaks. Spiking-and-recovery experiments can accomplish that. Linkers are highly labile, so whatever drug species fall off an antibody will react with something else.

Watch the webcast now.