Analytical challenges when characterising complex molecules

Michael-John O’Connor, Director of Biochemical Services at BioPharmaSpec, discusses analysing biosimilars, glycosylated mAbs and PEGylated proteins with Lu Rahman. 

LR: Outline the importance of bio-molecules in drug discovery. What is their potential within therapeutic development?   

MJOC: The FDA’s recent approval of Biocon’s Semglee (long acting Insulin glargine) is the first example of an interchangeable biosimilar that can be substituted for its reference medicinal product, Lantus, by pharmacists without requiring a doctor’s permission. This is because it has been shown through rigorous testing that there are no clinically meaningful differences between Semglee and Lantus. Hopefully biosimilar development and approval continues to promote competition in the drug market, leading to more affordable healthcare for diseases such as diabetes and cancer. 

Another area with potential is the development of innovative drug delivery mechanisms. Included in this would be gene therapy, where recombinant viruses such as adeno-associated viruses (rAAVs) are used as a delivery vehicle for transgenes that will replace a faulty or mutated gene, or deliver a new gene where it is missing. I would also include drug conjugation, where a payload drug is attached to a delivery molecule such as an antibody, which targets the site of action. These innovations have led to antibody drug conjugates (ADCs) such as Kadcyla and the developing technology of antibody oligonucleotide conjugates (AOCs). 

LR: What are the challenges associated with characterising biopharmaceuticals and what are the analytical considerations? 

MJOC: The largest challenge within the characterisation of biopharmaceuticals is ‘heterogeneity’ within the product. Biopharmaceuticals are manufactured in natural systems, such as mammalian, plant or insect cell lines, unlike traditional small molecule therapeutics which are chemically synthesised. These cell systems will naturally modify the protein chains with protein specific post translational modifications such as glycosylation, modifications to the protein termini or possible misincorporations of amino acids during the translation process. 

On top of these cell synthesis derived modifications there is also heterogeneity introduced through the manufacturing process itself such as oxidation and deamidation of specific amino acid residues. This natural and process-derived heterogeneity presents specific analytical challenges by increasing the range of molecular species present in the sample. Nonetheless the analytical guidelines require that heterogeneity in the sample is characterised and so the techniques employed must be able to identify and characterise the various modifications giving rise to the heterogeneity.   

LR: How can these challenges be overcome? 

MJOC: We overcome these challenges by employing instrumentation and analytical techniques to assess different aspects of molecular structure, from the primary structure of the protein backbone right through to higher order structure (ie. the 3D shape of the biopharmaceutical), including aggregation. Mass spectrometry (MS) plays a pivotal role in analyses, providing precise mass and fragment ion information on different aspects of primary structure and associated modifications. The techniques we use give information on structural attributes but we also work to use procedures that are orthogonal to one another. This is important in corroborating findings and presenting a robust analytical data package to regulators. 

Using these structural analytical methods from the earliest stage of development allows you to identify positive and negative structural features of your molecule. This allows key development decisions to be made based on quality analytical data and through this, the control of process related heterogeneity.  

LR: How can researchers create efficiencies and what is the role of analytical instrumentation? 

MJOC: Drug development companies are focussed on creativity, discovery and R&D and do not have the means, man-power or knowledge to set-up an analytical laboratory with expensive instrumentation that covers the structural analysis mentioned above. Partnering with Contract Research Organisation (CROs), with expertise in specific analytical areas, can bring about efficiencies and advantages such as access to their knowledge base (analytical, regulatory and product knowledge), access to better instrumentation and having an independent assessment of your molecule.  

LR: In relation to the development of therapeutics how can quality, purity, identity and potency be ensured? What type of methods/tools can help?  

MJOC: It is important to start understanding the structure of the biologic from the earliest stages with consideration given to the identified critical quality attributes. Using techniques such as MS-based peptide mapping to confirm amino acid sequence, some basic glycosylation analysis (if the protein is glycosylated), icIEF to assess the charge isoforms, Circular Dichroism to look at higher order structure and an assessment of aggregation will provide good initial information, in terms of the primary and higher order structure of the protein. Functionality is also important to assess, and the relationship between the structural and the functional data is highly informative as well as being a key component in the development and acceptance of products as biosimilars. 

As development proceeds and more batches are manufactured, this initial analyses should be expanded to include ICH Q6B guideline recommendations. A robust, orthogonal data package containing data from different batches of biopharmaceutical shows consistent quality, identity, purity etc. 

LR: What are the challenges analysing a PEGylated protein? 

MJOC: Polyethylene glycol (PEG) units are large polymeric structures often used in drug development to enhance product half-life and efficacy or increase solubility. This type of chemical modification poses analytical challenges. Methods must be developed for analysis of the protein-PEG conjugate, the site(s) of PEG attachment, the degree of PEGylation at these sites and assessment of the PEG unit is important. We rely on MS to obtain high resolution data on the intact mass and extent of heterogeneity of the PEGylated biopharmaceutical and associated PEG moiety. The aim of the data for the drug development company is to show proper control of the PEGylation process.  

LR: How challenging is the analysis of highly glycosylated proteins? What methods are employed in glycan analysis? 

MJOC: The plant, insect and mammalian cell lines that are used to produce biologics will produce glycans which are a reflection of the cellular machinery. As most biopharmaceuticals are derived from non-human cell lines, there is a risk that the glycoproteins produced could be immunogenic to humans. The Galα1-3Gal epitope from murine cell lines is an example of such an immunogenic glycan structure. Safety considerations and regulatory advice are the main reasons why thorough characterisation of the glycosylation pattern of your development molecule is important. 

We use many techniques to assess glycosylation in our labs. This is important since glycans are complex and potentially very heterogeneous. Using different techniques to look at aspects of structure means we can draw data from these techniques to provide overall structural information even in the midst of significant complexity. Each technique is specific to the aspect of glycosylation it has been developed to assess. Eg. for identification and quantitation of monosaccharides we use gas chromatography with mass spectrometric detection (GC-MS) which allows us to detect neutral sugars (most commonly Fucose, Mannose and Galactose) and amino sugars (N-Acetylglucosamine, N-Acetylgalactosamine) or liquid chromatographic procedures for the identification and assessment of levels of sialic acid species. For N-linked glycan population assessment in terms of identification of components and determination of relative abundance we use liquid chromatography with mass spectrometric detection (LC-MS) of fluorescently tagged glycans. For O-linked glycans, which are more challenging to analyse, we employ matrix assisted laser desorption ionisation mass spectrometry (MALDI-MS) of the permethylated glycans. We use GC-MS procedures to determine linkages between monosaccharides since glycans are non-linear structures. How the monosaccharides are linked to each other can impact on functionality. 

Volume 22, Issue 4 – Fall 2021 

About the author 

Michael-John O’Connor is Director of Biochemical Services, BioPharmaSpec. Since obtaining a degree in Chemistry at the University of Southampton, O’Connor has spent his career focused on Protein and Glycoprotein structural characterisation. He is the Director of Biochemical Services and oversees the development and utilisation of new techniques and instrumentation at BioPharmaSpec, as well as managing the on-going laboratory science programme. 

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