Characterization of Biologic Drugs: An Overview

Intact Mass and Subunit Fragment Analyses

LC-MS analyses of intact and subunit biologic molecules are essential tools for characterizing the detailed modifications of complex proteins and peptides. These information-rich analyses are utilized throughout the product development lifecycle, including GMP release testing, comparability studies, and routine product characterization. The consistency of intact mass results provides strong evidence of reliable drug production and manufacturing processes. Additionally, intact LC-MS can serve as an identity (ID) test in commercial release settings. These analyses are broadly applicable to protein- and peptide-based biopharmaceuticals, such as monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs).

Intact mass analysis is performed via LC-MS with minimal sample preparation. The drug substance or drug product is typically diluted and injected directly into the LC-MS system. The resulting data confirm the protein’s expected mass, and can detect major glycosylation patterns, along with C-terminal or N-terminal modifications. Subunit mass analysis requires additional preparation, such as the reduction of inter-chain disulfide bonds, enzymatic cleavage of the hinge region in mAbs, or enzymatic release of N-glycosylation. In some cases, a combination of these approaches is used. Fragment analysis enables detection of additional modifications like oxidations and glycations, and allows for the assignment of these modifications to specific subunits. Analysis of deglycosylated proteins provides further insight into the core structure of the biopharmaceutical, often required by internal quality protocols.

At Emery Pharma, intact and fragment mass analyses are conducted using the Thermo Scientific™ Exploris 240 UPLC-MS system, which includes a Biopharma option and offers a high mass range (m/z up to 8000), suitable for intact mass analysis of large biomolecules.

Peptide Mapping

Peptide mapping involves enzymatically digesting a protein—commonly with trypsin—into smaller peptides. These peptides are separated by liquid chromatography and analyzed by either LC-MS or LC-UV. To improve sequence coverage, additional enzymes like Lys-C, Glu-C, or chymotrypsin may be employed. LC-MS-based peptide mapping provides detailed information about the primary sequence of the protein, confirming identity, mapping disulfide bonds, and identifying post-translational modifications (PTMs) such as glycosylation. It can also detect sequence misincorporations and amino acid changes.

Although less specific, LC-UV-based peptide mapping is more cost-effective and accessible for routine use in many laboratories. It is commonly performed following the establishment of an LC-MS-based peptide map during drug development and is often employed as an identity test during GMP release and stability studies.

Emery Pharma has extensive experience with both LC-MS and LC-UV-based peptide mapping, using high-resolution Orbitrap mass spectrometry to deliver precise, in-depth analyses.

Glycan Profiling

N-glycosylation is a co-translational modification that typically occurs during protein expression in mammalian and other eukaryotic cells. The resulting glycan profile—comprising various oligosaccharides attached to the biologic molecule—is a critical quality attribute. Glycan profiles impact drug safety, efficacy, and immunogenicity. Maintaining a consistent glycan profile during development and commercialization reflects process stability and product integrity. Unrecognized glycans can elicit immune responses, and changes in the glycan profile can also indicate cell health during protein expression.

Emery Pharma employs several analytical methods to characterize glycan profiles. While intact and fragment mass analyses can identify the most abundant glycans, the most sensitive and definitive method involves enzymatic glycan release, chemical labeling, and analysis via UPLC-MS. Glycans are identified by their mass spectra and retention time comparisons to authentic standards.

Host Cell Proteins (HCPs)

Host cell proteins are residual impurities originating from the expression system used in biologic production. These impurities, often present in small amounts, must be carefully monitored and minimized, as they can compromise biologic potency, stability, and immunogenicity. HCP analysis is a regulatory requirement and is validated prior to commercialization.

At Emery Pharma, HCPs are characterized using modern UPLC-MS techniques with high-field Orbitrap mass spectrometry. In later stages, a combination of analytical tools—such as LC-MS/MS, ligand binding assays, and ELISA—is used to monitor and quantify HCPs across the production pipeline.

Size Exclusion Chromatography (SEC), Ion Exchange Chromatography (IEX), and other HPLC Analysis of Biologics

Size Exclusion Chromatography (SEC) and Ion Exchange Chromatography (IEX) are specialized HPLC (High-Performance Liquid Chromatography) techniques that serve as essential analytical tools in the analysis of biologic compounds. These methods are particularly valuable for release testing and stability testing of biologics. Emery Pharma has the expertise and advanced instrumentation to perform comprehensive HPLC analysis of biologics, offering detailed interpretations and robust data.

SEC methods are highly effective for assessing size variation, aggregation, and molecular weight distribution in biologic samples. In contrast, IEX methods are utilized to analyze charge variants, isoforms, and purity. These methods are frequently employed for identification (ID) testing by comparison to authentic standards. Additionally, both SEC and IEX techniques can be scaled up for semi-preparative isolation, aiding in the characterization of detected components and verification of the chromatographic purity of the primary molecule.

Furthermore, reversed-phase HPLC and reversed-phase UPLC (Ultra Performance Liquid Chromatography) methods are invaluable for identity testing and can be developed as stability-indicating methods during biologic drug development.

All HPLC and UPLC analyses at Emery Pharma are performed on top-tier analytical systems from Thermo Scientific, Waters, and Agilent, ensuring precise and reliable results in biologics testing.

Sample Enrichment

Sample enrichment is a pivotal step in biologics research and development, enabling deeper and more sensitive biological analysis by selectively increasing the concentration of targeted components. This process significantly enhances analytical sensitivity, which is crucial for detecting low-abundance analytes during early-stage biologics development.

By isolating specific molecular targets, sample enrichment techniques allow for a more comprehensive view of complex biological systems. This is especially important when addressing sample heterogeneity and biological matrix complexity. Enrichment strategies are selected based on the analytical target: for example, phosphorylated peptides may be enriched using an iMAC (immobilized metal affinity chromatography) column, cell organelles via ultracentrifugation, and activity-based enrichment through the use of chemical probes.

This strategic process not only accelerates the development of innovative biologic therapies but also provides a customizable approach to decoding molecular complexity, tailored to specific enrichment objectives in biopharmaceutical research.

If you have any questions regarding biologics characterization please reach out to us through our Contact Us page, or call us at +1 (510) 899-8814!

About the Author

Authored by Dr. Prajita Pandey, Associate Director of Chemistry