Molecular Weight Analysis

Protein therapeutics and monoclonal antibodies (mAbs) are the fastest growing therapeutic class of drug candidates and products developed by biopharmaceutical companies around the world. During the cell culture, purification, formulation, and storage processes, variants with altered primary sequences and post-translational modifications (PTMs) can be produced. This heterogeneity can impact the overall stability and biological activity of mAbs, making it necessary to perform a comprehensive characterization of the preparation.

Creative Biolabs offers antibody molecular weight analysis, which is part of the ICH Q6B guidelines for physicochemical analysis of mAb products, using our mass spectrometry (MS)-based techniques. More specifically, we offer intact mass analysis that provides information on sequence integrity and PTMs, and subunit mass analysis using reduced or enzymatically digested samples.

mAb Intact Mass Analysis

The molecular weight of a protein is an important parameter in the physicochemical properties of the protein. Intact mass analysis of mAbs is the process of determining its molecular weight. It can be a valuable method for mAb characterization throughout the development process. Applying intact mass analysis, it can give us the following information:

• The molecular mass of the intact protein for comparison with the theoretical mass, giving preliminary assurance of amino acid composition;
• Quick information about primary structure of protein or sequence variants, such as isoforms, truncation, mutations, the addition of signal sequences, or PTMs including glycosylations;
• Provide information in evaluating different batches of mAb product.

The major advantages of this approach are that the sample preparation is minimal and the data analysis is straightforward.

Fig.1 MS and IM-MS analysis of intact trastuzumab. (Beck, 2012)

mAb Subunit Analysis

Alternatively, subunits or fragments of mAbs can be obtained by reduction of disulfide bonds or proteolysis with enzymes such as pepsin, papain, or IdeS, to reduce complexities for large size mAbs. More specifically, antibodies can be treated with PNGase F to remove the N-Glycans, digested with proteases such as IdeS to generate antibody fragments, or reduced to generate light and heavy chains prior to mass measurement. Subunits resulting from these treatments are smaller in size, allowing from improved mass accuracy and resolution as well as subunit-specific localization of PTMs, allowing low mass modifications to be detected at lower levels.

Molecular Weight Analysis Services Provided by Creative Biolabs

MS-based approaches for analyzing intact and/or subunit-level species can be very informative regarding the primary structure of a given protein, as well as some of the micro-heterogeneity present. Creative Biolabs offers several MS-based technologies for molecular weight analysis, including matrix-assisted laser desorption/ionization (MALDI-TOF), electrospray ionization (ESI-MS), and on mobility mass spectrometry (IM-MS). Our experts are familiar with the advantages and limitations of these techniques, which enables us to choose the optimal method and customize the services to fit your exact requirements.

Besides, peptide mapping can also be provided to further confirm the primary structure (amino acid sequence) of mAb products.

If you are interested in our services, please do not hesitate to contact us for more details.

Reference

1. Beck, A.; et al. Biosimilar, biobetter, and next-generation antibody characterization by mass spectrometry. 2012.

For Research Use Only.