The structural characterisation of biotechnology products presents a challenge for modern analytical techniques. In contrast to small molecule drugs, biopharmaceuticals can be extremely large, complex entities, often comprising of multiple disulphide-bridged proteins or glycoproteins.In particular, these products demand the use of methods capable of determining the primary structure of a recombinant or engineered molecule and of detecting and assigning post-translational modifications and microheterogeneities. By Dr Fiona M Greer.
Mass spectrometry (MS) has now emerged as an essential tool for the analysis of awell-characterised' biomolecules. Complementary to aclassical' Edman degradation protein sequencing techniques, MS has the advantage of being able to probe areas inaccessible to conventional sequencing.
Its use can be extended to not only synthetic peptide products and recombinant proteins, but also large glycoproteins such as monoclonal antibodies which pose a particular problem for structure analysis.
Indeed, the same MS instruments which deduce protein structure can also provide valuable information on the glycan moiety when combined with particular carbohydrate chemistries.
Mass spectrometry methods
Leading on from the development of fast ion bombardment (FAB) ionisation in the early eighties, recent advances in instrumentation including electrospray (ES) and matrix assisted laser desorption (MALDI) have firmly established MS as an essential method for biopharmaceutical product characterisation.
Electrospray ionisation allows the mass range of ionisable molecules to encompass intact proteins up to approximately 150kDA. This technique is widely applied using small or benchtop quadrupole instruments, often triple-quadrupoles, capable of MS/MS experiments to produce sequence or fragment data as well as molecular weight information.
One advantage of ES-MS is that it can be directly coupled to a liquid on-line sample introduction method such as HPLC, allowing separation and simultaneous detection of the components of complex mixtures.
MALDI ionisation, usually coupled with a time-of-flight (TOF) analyser, and delayed extraction (DE) technology is also capable of intact mass measurement on extremely large molecules-up to about 500kDA, together with sequencing applications.
A new generation of instruments has emerged, composed of a quadrupole analyser followed by a collision cell with an orthogonal injection of ions into a TOF analyser. These instruments (for example, Q-TOF and Q-STAR) are currently the most powerful tandem MS/MS sequencing instruments available. Further information on mass spectrometry techniques for biopolymers can be found in references 1 and 2.
Performed on specific enzymic digest mixtures of any protein or glycoprotein, MALDI-TOF aMapping' of the unseparated mixture, or on-line LC-MS provides a reliable method of confirming the overall amino-acid structure of the molecule and can pinpoint areas for further investigation. In short, modern MS methods have the capability to provide molecular weight and sequence data, detect errors of translation etc, identify post-translational modifications, identify sites of glycosylation (including carbohydrate structure and sequence), identify heterogeneity at the N- and C- termini and assign disulphide bridges amongst other things!
MS procedures have been developed, applied and validated to address the problems of characterisation for regulatory body purposes (3). These techniques will be further refined to keep pace with the demands of the biopharmaceutical industry.
ENQUIRY No 52
Dr Fiona M Greer is director, Biochemical Services,M-Scan Ltd, Wokingham, UK. www.m-scan.com
