Drug development is expanding in ever-increasing directions as the human race seeks to find new ways to deal with disease in ever more precise and targeted ways. Drug side effects aim to be minimized as more is learnt about the effects of certain drugs on the body, and we look to enhance the positive drug attributes and remove the negative features. Along with growth in medicinal knowledge we are also expanding our capabilities at the chemical level to engineer more complex and better-designed drugs. Chemical synthesis technologies continue to evolve and one just needs to look at the use of click chemistry for biocompatible conjugation to see how this has opened the door to novel ways of joining chemicals to biochemicals.
Examples of drug conjugations that immediately spring to mind are antibody-drug conjugates, where a specific monoclonal antibody is chemically modified to carry a number of smaller drug units that are the active payload and are attached via chemical linker units to the side chains of specific amino acids, Cysteine or Lysine most commonly. Examples of approved drugs of this nature are Kadcyla and Mylotarg, used in the treatment of relapsed acute myelogenous leukemia and as continuation treatment for HER-2positive metastatic breast cancer respectively.
The linker units, along with the drug itself, can vary in nature considerably depending on the overall function that is intended for the molecule. PEGylation of proteins is another obvious form of conjugation with Neulasta and Esperoct (used in the treatment of Neutropenia and Haemophilia A respectively) being examples of this class of conjugated drug.
More recently antibody-oligonucleotide conjugates have been developed, where nucleic acid (either double or single stranded) is conjugated to a monoclonal antibody. As with ADCs, various specific chemical cross-linkers can be used to allow conjugation to take place in the manner required. Currently these products are still very much a developing area of research but they give a glimpse into a medical future where this form of monoclonal antibody targeting is used for nucleic acid delivery.
Outside of the sphere of monoclonal antibody focused chemistries, advances are also being made in the localized chemical modification of proteins and glycoproteins which can then serve as substrates in their own right for further reactions and crosslinking with other proteins. This has been demonstrated through the use of azidogluconolactone to crosslink SAR-CoV-2 receptor binding domain with a virus-like particle to give a product that has been shown to act as a COVID-19 vaccine.
With the variety of protein modification chemistries that are in use in research labs, how can we assess the structures of the protein products that are created to confirm their veracity and examine samples for any unwanted impurities or reaction by-products? We need analytical techniques that are completely impartial to the type of chemistry performed and that can give meaningful, structural information on the nature and molecular location within the protein chain of the chemical modifications that have taken place. The ideal technique for such analysis is mass spectrometry, with its ability to dissect protein molecular structure at the level of individual amino acids in the chain and thus give precise mass and structural information across the protein backbone.
The modern breed of mass spectrometer, of the Q-TOF type of geometry, is able to generate peptide mass information from a proteolytic digest of the protein conjugate at the same time as it is able to produce higher energy sequence derived fragment ions. These ions are produced through peptide backbone cleavage, generating fragment ions from which the sequence of the peptide can be read. Since most amino acids have unique masses, they can be precisely identified through these fragment ions. Any mass variation, such as the addition of a chemical unit through the drug-conjugate synthesis pathway can be readily identified and located within the protein chain. This same approach also means that any unwanted modifications or side-reactions on non-targeted amino acids can also be detected.
As well as giving fine structural detail, the high mass accuracy of Q-TOF type instruments in conjunction with the use of electrospray as their mode of sample ionization, means intact mass analyses can also be performed which can provide initial information on the success (or otherwise) of the chemistry and the number of conjugations that have taken place through a determination of the mass observed relative to the expected mass of the protein.
It is also worth pointing out that many biomolecules that are considered as targets for conjugation chemistry are glycoproteins, so it is also important to confirm that the glycan chains themselves have not been adversely impacted by the conjugation chemistry process (e.g. desialylation under acidic conditions). This case study describes a project where the objective was to provide analytical data to show that the chemistry used to attach the drug did not impact the glycosylation pattern of the mAb.
As advances in chemistry allow us to produce tailored drugs with ever more chemical creativity, we need analytical tools that can probe the structures of these products in fine molecular detail and give precise information on what exactly has taken place chemically and where. It is a requirement from the regulatory authorities that detailed molecular structural information is generated to precisely define the product and mass spectrometry is a very powerful tool in the armory for this type of work.
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