Step-by-Step Intact Mass & Subunit Analyses of a Therapeutic Antibody by Hi Res Orbitrap Mass Spec

5 Minutes Read:
Dr. Rodney Bannwart, a principal scientist at Emery Pharma, discusses LC-MS intact and subunit analysis of biologic molecules. He has over 20 years of experience in LC-MS, previously working with biologic molecules at Alcon, Novartis, and small molecules at Pfizer. Biologics, like monoclonal antibodies, require multiple analytical methods due to their much larger size and complexity compared to small molecule pharmaceuticals.

LC-MS intact mass determination is a straightforward analysis used throughout the product development cycle. LC-MS intact mass analysis provides information on primary sequence verification, major glycosylations, N-terminal modifications, glycation, GMP release characterization, and comparability studies. The protein sample is diluted and injected into Emery Pharma's Orbitrap UPLC-MS system using different chromatographic methods. Subunit analysis reveals more detailed modifications by treating the mAb with enzymes and/or reduction to produce subunits with specific molecular masses.

A case study of the NIST mAb demonstrates modifications such as glycation, lysine modifications, and glycosylation. Detailed information on modifications, including glycation of the light chain, can be obtained through subunit LC-MS analysis. More detailed N-glycosylated information is obtained by treating the sample with the PNGase F enzyme. Native intact LC-MS analyses can also provide information on the drug-to-antibody ratio (DAR) for antibody-drug conjugates (ADCs). Intact and subunit analyses help characterize complex molecules and are valuable throughout the product development cycle, including GMP product release, comparability, and characterization studies.

The LC-MS intact method can be transferred to affordable low-cost mass spectrometers in QC labs, enabling its use in ID tests and commercial release testing. For further inquiries, contact Emery Pharma at emerypharma.com.


About Emery Pharma:
Emery Pharma is a full-service contract research laboratory, supporting the end-to-end development of small molecule drugs, biologics, biosimilars, gene therapy, botanicals, and more.

Emery Pharma's premium services include analytical, bioanalytical solutions, microbiology & cell biology services, drug and impurity characterization, and general R&D and cGMP/GLP support.

At Emery Pharma, our focus is to find innovative solutions for our Clients’ toughest challenges and tailoring those solutions to fit each Client’s unique needs. Our experienced scientific team will manage the process: from drug discovery, proof-of-concept, data management, data analysis, to gathering insights. The team excels at solving scientific challenges, and addressing any issues early in the process, so Clients don’t experience unnecessary setbacks.

Emery Pharma’s experienced chemists and biologists deploy state-of-the-art technology. Instrumentation ranges from high-resolution mass spec (Orbitrap), Triple quad LC-MS/MS, GC-MS, Preparative HPLC, and 400 MHz multinuclear NMR.

Contact us: [email protected]
https://emerypharma.com/

keyword:
LCMS intact
Subunit analysis
Biologic molecules
Monoclonal antibodies
Chemical properties
Molecular weights
Small molecule pharmaceuticals
HPLC method
Product development cycle
Primary sequence verification
Major glycosylations
N-terminal modifications
Glycation
GMP release characterization
Comparability studies
Protein sample
Emery Pharma
Orbitrap UPLC-MS system
Chromatographic methods
Case study
NIST mab
Detailed modifications
Glycosylation
Subunit LCMS analysis
D-glycosylated map
PNGase F enzyme
Drug-to-antibody ratio
Low-cost mass spectrometers
QC labs
ID tests
Commercial release testing