Technical Director for Structural Analysis
BioPharmaSpec
Dr. Richard Easton is the Technical Director for Structural Analysis at BioPharmaSpec, an independent analytical CRO with labs in the UK & US. He has over 20 years’ experience in glycoprotein structural characterization using mass spectrometry and is well acquainted with the various regulatory requirements for biopharmaceutical development.
Independent Consultant, Dr. Fiona Greer, caught up with Richard to ask him more about what inspires and motivates him in his role.
RE: Well Fiona, that’s a tough question to give a short answer to! Glycosylation is not a passive piece of glycoprotein architecture, and it is not templated in the same way as protein structure is through DNA. However, it can be a critical to conferring biological activity or serving to modulate that biological activity in some manner, like control of half-life in the bloodstream.
Glycans rely not just on the nature of the monosaccharides for specific activities but also on the linkages between the monosaccharides. This exquisite control of function through structural precision is delicately controlled at the biosynthesis level, meaning any change in the production system away from the natural source of a glycoprotein (or innovator product), could easily result in a change in glycosylation through, for example, a change in cellular expression of glycan biosynthesis enzymes, different transition times through the biosynthetic pathway resulting in different exposure time to the glycan biosynthetic enzymes of the cell, or cell culture conditions which could affect the nature of the glycan structures produced.
If differences in glycosylation profiles are detected then, depending on the degree or nature of these differences, efforts need to be made to adjust production conditions to better represent the innovator profile of the glycans, since aspects of the drug product could be directly or indirectly affected by this changed glycan population.
If glycan profile differences are relatively minor compared to the innovator, then use of appropriate functional analysis can serve to demonstrate if these differences have biological significance or are not “clinically meaningful”.
RE: There are a variety of methods that need to be employed for glycan characterization. If we just think about N-glycans for a moment, which are the most common type of glycosylation we encounter, then one of the most useful techniques is on-line liquid chromatography coupled with mass spectrometric analysis of fluorescently derivatized glycans.
During this analysis at BioPharmaSpec, we enzymically release the glycans from the protein backbone and then tag them with a fluorescent label. We can then separate and detect the derivatized glycans chromatographically. Since the fluorescent label is in a 1:1 ratio with the glycan, we can use the strength of the fluorescent response to determine the relative abundances of the glycan structures. The nature of what those structures actually are comes from the masses observed as the labelled glycans are analyzed by the mass spectrometer. Structurally related species which have the same mass but different arrangements of monosaccharides can be separated, so profile information can be very detailed.
Data obtained from LC-analysis of 2-AB labeled N-linked oligosaccharides. The N-linked oligosaccharides were released from a monoclonal antibody using PNGase-F.
This is really valuable information, but is just compositional. We also need to know the linkages of the monosaccharides, since this is a key part of assessment of biosimilarity and can have functional significance. We do this through some beautiful chemistry, where we carry out sequential labelling of monosaccharides derived from the glycan species. The labelled monosaccharides are analyzed by gas chromatography mass spectrometry and the fragment ion profiles we see are unique to the individual linkages.
This is my favorite glycan analytical technique – it really is lovely to see the data work so perfectly with the theory. Indeed, you can predict the fragment ions you will see for the different linkages when you know how the chemistry and fragmentation pathways work.
Summed Extracted Ion Chromatogram from GC-MS analysis of a mixture of PMAAs released from a mixture of N-linked oligosaccharides.
Yes, orthogonality is really important and we can use a different derivatization and mass spectrometry strategy to generate data orthogonal to the fluorescent profile I just mentioned. This derivatization MS data does not have a chromatographic component so does not allow separation of different isomeric species like the fluorescent data does, but it does give us good supportive mass data and fragment ion information useful for supporting structural assignments. It works with O-glycans too, which is not something that can easily be achieved in the fluorescent labeling sense.
We can also use peptide mapping and identification of glycopeptides to support the information we have on glycan structures, but it should be remembered that in this case glycans can fragment in the source of the mass spectrometer as they ionize. This means we can lose some structural information from larger components and detect some smaller structures due to this fragmentation. For this reason, glycans should be removed from the protein backbone and characterized in their own right, rather than relying on peptide mapping as a mechanism for assessing a glycan population profile.
RE: With regard to the techniques that we commonly use for our glycan analytics, I would say that having the ability to relatively easily generate data-rich chromatographic profiles has really helped to open up our ability to define glycan populations in a way that we could never easily do before.
This required developments in glycan chemistry, with regard to MS compatibility of fluorescent dyes, to get the best sensitivity out of a sample which can be very heterogenous, in terms of the number of glycan species present, and thus have some glycans present at very low relative abundances. The advancement of column technologies for optimum separation of these components was also crucial and of course the development of high sensitivity mass spectrometers such as the Q-TOF geometry instruments.
All of these areas of scientific innovation draw together to give us the techniques and technologies we have available to us today for the best structural assessments of glycan profiles that we can deliver. What will we be able to achieve in the future?
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