Characterization of Protein Structure: The Value of Intact Mass Analysis

Biologics Characterization

Mar 02, 2021 | 3 min read

Intact mass analysis is the assessment of a protein’s total molecular weight by mass spectrometry (MS) without prior digestion or fragmentation of the molecule of interest. Molecular weight determination forms part of the ICH Q6B guidelines for physicochemical analysis of biological products: what use is this information in the analytical characterization of biopharmaceuticals?

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By: Dr Martin De Cecco - Senior Scientist

High-resolution MS allows the average molecular weight of large proteins, for example monoclonal antibodies (mAbs), to be measured with accuracy better than ± 0.01%. The observed mass can then be compared to the expected mass for a given amino acid sequence. If there is a match, what does this tell us?

All 4 polypeptide chains (2 light chains, 2 heavy chains) are joined together
The signal peptide of each chain has been cleaved correctly
We have preliminary assurance of protein identity

Note the use of the phrase “preliminary assurance of protein identity” since confirmation of the primary structure will require further analysis, such as peptide mapping. Nevertheless, the fact the observed mass is consistent with the expected amino acid sequence provides some confidence in that regard.

In fact, there’s more information we can garner from intact mass analysis. Let’s look at a typical mass spectrum of a therapeutic mAb (Figure 1).

Several peaks are observed, each corresponding to the same protein with different combinations of post-translational modifications (PTMs). In this case, the major peaks correspond to the most abundant glycoforms of the mAb. Depending on the sample, other modifications such as C-terminal lysine clipping, N-terminal pyroglutamic acid formation and glycation, may also be apparent. The intensity of each peak gives an indication of the relative abundance the corresponding PTM. Therefore, to our list above, we can also add:

An overview of the molecule’s glycan profile
An assessment of other PTMs

Not bad for one measurement! Thus, intact mass analysis can be a valuable method for protein characterization throughout the development process. It is useful when evaluating different batches of product – for example during process optimization – or for comparing a candidate biosimilar to the reference molecule.

Analysis of Protein Subunits

Further detail can be obtained by employing different enzymes during preparation of the sample before analysis by MS. A glycosidase, such as PNGase F, is often used to remove the heterogeneity associated with N-glycans. Alternatively, proteases can be used cleave the protein into a small number of protein fragments. Similarly, a reducing agent can be utilized to break inter-chain disulfide bonds. For mAbs this yields the separate light and heavy chains (Figure 2).

^{Figure 2: LC-MS data for a reduced monoclonal antibody. The light and heavy chains are separated chromatographically, with on-line MS detection. Inset: Deconvoluted mass spectra for the peaks corresponding to the light and heavy chains.}

Breaking the protein into smaller fragments generally improves the sensitivity, resolution and accuracy of the method, allowing low mass modifications to be detected at lower levels. Does this negate the need for analysis of the intact protein? Not entirely: just because we have all the right pieces doesn’t mean that they are joined together!

In practice a combination of the above strategies – intact, deglycosylated and reduced protein structure analysis – provides the most informative approach to molecular weight analysis.