Geoffrey Rule - Principal Scientist, MilliporeSigma, Bellefonte, PA - A business of Merck KGaA, Darmstadt, Germany
Martin De Cecco, Ph.D. - Global Scientific Lead for Product Characterization, BioReliance Ltd., Glasgow, UK - An affiliate of Merck KGaA, Darmstadt, Germany
In general, can you tell us about the products and services you provide for the research and development of therapeutic monoclonal antibodies (mAbs)?
Yes, within MilliporeSigma, an affiliate of Merck KGaA, Darmstadt, Germany, we have both products and services offered for mAb development, characterization, and quality control. On the product side we offer a host of reagents, enzymes, solvents, chromatography, and sample preparation products to cover almost every aspect of mAb characterization. For example, we have recently prepared application notes for mAb peptide mapping, intact and middle-up mass analysis, and release and labelling of glycans, all using products and columns that we offer. Enzymes that we offer are such things as PNGase for release of N-linked glycans, SoluTrypsin for digestion of proteins, and IdeS for cleavage of mAbs into subunits.
In addition, we have laboratories specialized in the structural and functional characterization of mAbs, where we provide testing services for our clients. As well as standard analytical techniques like HPLC and capillary electrophoresis, we also employ more complex methods involving mass spectrometry or cell-based assays, for example. These assays can be used for characterization purposes to support R&D activities or validated for use in GMP lot release and stability testing.
What are peptides, and what is peptide mapping in relation to characterization of mAbs?
A peptide is simply a short chain of amino acids, connected by peptide bonds (a type of covalent bond formed between a carboxyl group on one side and an amine on the other).
A monoclonal antibody is a large protein comprised of 1300 or so amino acids connected by peptide bonds. (Note, mAbs are actually comprised of four separate polypeptide chains, which are then connected through separate disulfide bonds to form the complete mAb.)
In peptide mapping, we wish to confirm the complete amino acid sequence of the protein by breaking the protein into smaller pieces (peptides). We can separate the peptides using HPLC and then use the mass spectrometer to break these peptides into even smaller pieces to tell us the exact sequence. Software processing of the mass spectra does a lot of the work for us and reports what we call the sequence coverage, or the percentage of all the individual amino acids that can be accounted for by the data.
MilliporeSigma recently developed a peptide analysis workflow. Can you briefly describe how this process works and what are its benefits to researchers developing mAbs?
Yes, as mentioned disulfide bonds generally link different sections of mAbs so in the workflow we start by breaking these bonds and then capping them so they can’t reform. Next, we add a proteolytic enzyme, such as tryspin, that cleaves certain peptide bonds specifically so that the protein is “digested” into smaller pieces. Then we introduce the digested protein onto our HPLC-MS system after removing the trypsin, which remains as a large protein, using a molecular weight cut-off filter. The latter is commonly performed with something called filter-assisted sample preparation (FASP). Using a long chromatographic run, we are able to separate many of the individual smaller protein fragments, or peptides, and introduce them individually to the mass spectrometer. Once in the mass spectrometer, the peptides are fragmented using one of a number of specialized techniques to provide the amino acid sequence information. Often, we can obtain additional information about the mAb, such as sites and types of glycosylation, in addition to other types of protein modification.
In essence then, researchers use this technique to characterize mAb products that are either being newly developed or are being tested for release in pharmaceutical formulations. In both cases, confirming the precise nature and quality of the protein is invaluable.
Regarding the analysis of peptides – does MilliporeSigma offer customized service and support for client’s specific protein research?
Absolutely. We have a number of platform analytical methods in place that are applicable to a wide range of monoclonal antibodies. However, these can be further optimized as necessary for a client’s particular product. For example, in peptide mapping, differences in the amino acid composition of proteins may necessitate the use of an alternative proteolytic enzyme or require optimization of the chromatography of the resultant peptides.
Beyond peptide mapping, some methods – notably bioassays to assess binding and function – are very custom in nature and we have extensive experience in the development and validation of such methods.
Looking ahead how will MilliporeSigma continue to support the development of mAbs and other future treatments for both its current and future clients?
With evolving regulatory guidance and advances in technology, we continue to develop new services for the characterization and quality control of biotherapeutics.
One example of this would be the characterization of host cell proteins (HCPs) by LC-MS. For recombinant proteins such as mAbs, there is a need to measure the level of host cell proteins that remain in the final product. Generally, this is done by ELISA, which is used to quantify the total amount of HCPs that produce an immunological response.
However, this technique doesn’t distinguish between the different HCPs present – some of which might be of more concern than others. For that reason, we are establishing an LC-MS method to use as an orthogonal approach for the characterization of HCPs.
A key benefit of this approach is the ability to identify, as well as quantify, individual HCPs. This knowledge should provide clients with a better understanding of their manufacturing process, enabling them to evaluate the potential risk of the specific HCPs present.
Beyond mAbs, we see a growing number of novel modalities – particularly in the cell and gene therapy space – and so we are developing a comprehensive service offering for the analysis of these products also.
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