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European Biopharmaceutical Review

Molecular Weight

Monoclonal antibodies, particularly immunoglobulin G (IgG), are one of the most common types of biopharmaceuticals. Knowledge of an antibody’s molecular weight is vital to confirm that the correct structure is present and essentially pure, in order to be confident that the drug is safe and effective for medicinal use.

Analysis of antibodies is more challenging than that of small molecules due to their size, which is around 150kDa. IgG molecules are comprised of two light and two heavy chains, complexed via several disulphide bridges. In addition, there are a number of possible posttranslational modifications (PTMs), such as N-linked glycosylation, on the heavy chains that have an impact on immunogenicity and half-life (1). Other modifications, such as deamidations and oxidations, may also affect function (2,3). Antibody molecular weight may be determined through electrophoretic techniques or, increasingly, by liquid chromatography-mass spectrometry (LC-MS).

A murine IgG monoclonal antibody of known molecular weight, amino acid sequence and glycan structure was used as a model in the following study to compare and contrast electrophoresis and LC-MS. The antibody was reduced with dithiothreitol (DTT), and the intact and reduced samples were analysed by both sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Thermo QExactive LC-MS. An additional LC-MS analysis was performed on a reduced and non-reduced sample, which was deglycosylated with Peptide-NGlycosidase F (PNGase F).

SDS-PAGE

SDS-PAGE is a technique used to denature proteins that removes all secondary and tertiary structure. The SDS coats the protein with a negative charge and enables separation through a gel on the basis of apparent molecular weight.

Two aliquots of the antibody were analysed: an intact and a reduced sample. The reduced sample had been treated with DTT in order to break the disulphide bonds, thereby separating the heavy and light chains.

It can be seen that the intact antibody had the expected mass of around 150kDa, and the heavy and light chains had the expected masses of approximately 50kDa and 24kDa, respectively. There were also no other protein components detectable. However, any mass differences attributable to a single missing amino acid – for example, the C-terminal lysine of the heavy chain, a common modification on monoclonal antibodies – would not be easily discernible. This technique is relatively simple and will easily show major truncation products and other protein impurities, but suffers from a lack of resolution, so small differences in mass may not be noticeable.

LC-MS

Intact mass LC-MS is a technique used to separate proteins chromatographically, then determine their masses accurately by ionisation of the proteins generating multiply-charged molecules, before finally measuring their mass-to-charge ratios (m/z).

It can be seen that the mass spectrum has what is known as a charge envelope and a typical bell-shaped distribution. Each peak represents the same molecule, but with an increasing number of protons as one moves down the m/z axis. When the spectrum is examined in more detail, it is clear that each peak in the mass spectrum is, in fact, a family of ions comprising at least five significant members. These members represented the different glycoforms of the antibody. The process used to determine the mass of the neutral molecule from the charge envelope is known as deconvolution, during which software will attempt to match an ion to a neutral mass trying different charge states. When the spectrum is deconvoluted to obtain the mass of the neutral molecule, this results in a mass of 148,221.9Da. This is in close agreement with the predicted value of 148,220.4Da. The other isoforms are attributable to a differing number of glycan units. Two glycoform structures are shown for each peak, since the intact molecule contains two glycosylated heavy chains. The glycofamily profile disappeara in the mass spectrum of the deglycosylated antibody, compared to the intact antibody. This confirms that the glycosylation seen on the previous non-treated sample was N-linked.

When the reduced antibody and reduced, deglycosylated antibody were analysed, two peaks were resolved chromatographically from each sample: one being the light chain, and the other being the heavy chain – each with their own charge envelope. Analysis of the mass spectra of the heavy and light chains in the same way as shown for the intact antibody gave the expected molecular weights. The heavy chain spectrum also confirmed the glyocoprofile that was observed in the spectrum of the intact molecule.

Summary of Results


All masses were within 1.5Da of the expected amount, confirming that there were no major unexpected truncations, extensions or modifications. The glycosylation assignments made were based on the known structures.

Intact mass analysis by mass spectrometry can be used to confirm the molecular mass of a given protein complex, as well as its sub-units to a degree of accuracy not achievable with other techniques – although care still needs to be exercised when interpreting results (5). Expected glycan structures can also be confirmed, but since many oligosaccharides are isobaric (same mass) it cannot be used for a definitive assignment of glycan structures on its own. Experimental error can be up to 2Da when looking at such large molecules; therefore, intact mass analysis is not suitable for detecting very small differences, such as a single deamidation (+0.98Da mass shift) or a single disulphide bridge reduction/ formation (+/- 2.02Da mass shift). It is, however, very good for detecting larger mass differences – for example, C-terminal lysine clipping of the heavy chain or cyclisation of the N-terminal glutamine.

Mass spectrometry can give a much greater level of precision and accuracy than electrophoretic techniques such as SDS-PAGE. Intact mass analysis can be used for the characterisation and monitoring of PTMs with the advantages of minimal sample preparation, high speed of analysis and relatively minor methodinduced sample modifi cations.

References

1. Ha S et al, Isolation and characterization of IgG1 with asymmetrical Fc glycosylation, Glycobiology 21(8): pp1,087-1,096, 2011
2. Gaza-Bulseco G et al, Effect of methionine oxidation of a recombinant monoclonal antibody on the binding affi nity to protein A and protein G, J Chromatogr B Analyt Technol Biomed Life Sci 870(1): pp55-62, 2008
3. Timm V et al, Identification and characterization of oxidation and deamidation sites in monoclonal rat/mouse hybrid antibodies, J Chromatogr B Analyt Technol Biomed Life Sci 878(9-10): pp777-784, 2010
4. Konermann L and Douglas DJ, Unfolding of proteins monitored by electrospray ionization mass spectrometry: A comparison of positive and negative ion modes, J Am Soc Mass Spectrom 9(12): pp1,248-1,254, 1998
5. Michael N and Easton P, Considerations when performing an intact mass analysis of a monoclonal antibody by LC-MS, RSSL white paper


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Nicholas Michael is a Protein Technical Specialist at Reading Scientific Services Ltd (RSSL). He studied with the Department of Pharmacy at the University of Brighton and School of Crystallography at Birkbeck, London. Nicholas has significant experience identifying unknown proteins and performing post-translational modifi cation analysis in the elucidation of cancer pathways. He has also used his LC-MS skills to characterise manufactured monoclonal antibodies to current Good Manufacturing Practice guidelines (cGMP).

Pat Easton
is Biomolecular Analysis Manager at RSSL. Following her PhD involving analysis of leukotrienes by mass spectrometry, Pat gained seventeen years’ experience within a biotechnology environment at Amersham International (later GE Healthcare), focusing on protein and genomic arrays, protein labelling and detection. She then moved into a Quality Control Manager role at GE Healthcare where she stayed for six years. After this, Pat joined RSSL as the Pharmaceutical Chemistry Laboratory Manager. Her combination of protein chemistry and cGMP experience puts her in a good position to lead and manage the cGMP services that RSSL offers.
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