With the UK government debating the introduction of a tax on sugar, recent Easter celebrations with two young children in the house and the burgeoning biotherapeutic market, I’ve got sugar on my mind. With respect to the sugar tax and the heady mix of chocolate and children, they’re topical, it makes sense for me to thinking about them...but why did the biotherapeutic market also spring to mind? Glycosylation. The attachment of sugars (glycans) to proteins, via a glycosidic link. Glycosylation is one of the most regulated aspects of protein therapeutic production, and rightly so. Unlike the government’s decisions on the price of sugar and my young children’s reaction to an Easter egg binge, I feel like I might have some control over glycans...do you?
Control Over Glycans
The millennials of the protein biologics scene are the antibody and enzyme-based therapeutics. Mammalian cells (NS0 murine myeloma cells, PER.C6 human cells, and Chinese hamster ovary (CHO) cells) are often required for the production of these complex proteins, due, in part, to their sophisticated post translational modifications (PTMs). Glycosylation represents a single type of PTM which is commonly found in the Fc region of a typical IgG antibody glycoprotein.
The bioproduction process can be controlled by fine-tuning the choice of cells used, the culture media and the conditions within the bioreactor, to produce the protein amino acid sequence and precise mix of PTMs required in the end product. Successful bioproduction requires rigorous protocols and involves maintaining the desired quality attributes while reducing time to market, preserving profitability, and providing manufacturing flexibility. The biotherapeutic market is at fever pitch and several companies are now working on therapies for similar targets and clinical indications. It is fair to say that this market could not successfully be addressed by setting up a small-scale operation in your garden shed, as Dr. Timothy Cross recently (thankfully) concluded. #shedibodies...it’ll never catch on...
The Importance of Glycosylation
Glycosylation is an enzyme-directed site-specific reaction, with the covalent addition of a carbohydrate to asparagine and serine/threonine amino acid residues (N-linked and O-linked, respectively). Small changes in the type and level of glycosylation can influence the safety, efficacy and, in the case of biosimilars, the similarity between proposed biogenerics and their reference products. Biosimilar production calls for specific attention to process control, with similitude being the key. FDA regulatory guidelines require full analytical testing and comparison of innovator and biosimilar molecule, inclusive of full glycosylation profile. In this particular case a difficult compromise of selecting a host cell which provides the highest quality product, rather than the highest product yield is not uncommon.
The FDA, EMA and others have guidelines recommending glycoprofiling is conducted throughout the drug life cycle. These rules are being harmonised under the ICH programme (ICH Q6B and Q5E guidelines detail the requirements). Although they don’t hang their hat on any specific test procedures or acceptance criteria for glycosylation profiling, they do suggest technical approaches which are considered to provide sufficient information. Luckily, there is a broad portfolio of solutions for glycan analysis, at both the macro- and micro- heterogeneity levels. One such example, cited in USP <129>, official from May 1st 2016, is the use of high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) for sialic acid monitoring. Sialic acids are located terminally on the oligosaccharide chains and are more likely to interact with other endogenous proteins and receptors. They have a direct link to both drug safety and efficacy, playing a part in immunomodulation and protein half-life, respectively. HPAE-PAD offers advantages in the rapid analysis of monosaccharides, sialic acids and both charged and neutral glycans, without the need for prior derivatization.
The use of UHPLC with fluorescence detection and/or universal charged aerosol detection is also well documented for the routine high-throughput released N- and O-linked glycan analysis. Add to this a suite of mass spectrometric tools for glycan characterization and we’ve got glycans covered.
Back to the uncontrollable – I’d better get my kids to brush their teeth after all that chocolate and start weaning myself off Coca Cola before this sugar tax kicks in!
Do you analyze glycans? Which analytical techniques do you use and why?
Need more sugar? Check out these additional resources:
eBook covering the Fundamental UHPLC Workflows for Biotherapeutic Characterization