nSMOL - A Novel Approach to Quantification of Therapeutic Monoclonal Antibodies!

- Ashutosh Shelar
Senior Application Specialist
Shimadzu Analytical India

nSMOL proteolysis (nano-surface and molecular orientation limited proteolysis), is the innovative proposal to the antibody analysis by mass spectrometry focused on the size of antibody and its orientation. Immobilized antibody on the resin with 100 nm pore has been digested by immobilized protease on 200 nm nano particles, and detected and quantified by liquid chromatography/mass spectrometry (LC/MS). This experimental design successfully performed limited proteolysis on complementarity-determining region of the antibody variable region by limiting protease access to the substrate. This nSMOL method is made possible therapeutic drug monitoring (TDM) independent of a variety of monoclonal antibodies, and accelerated the development of TDM system with MS.

There has been no dearth of data and statistics all over the web and print stating trends of different sorts in Pharma market. As various market reports and sales data suggest, biologics is way forward for every pharmaceutical organization, inclined towards high profitability. Last few years, majority of the top 10 blockbuster drugs have been large molecules and essentially proteins. Many of these biologics are nearing the end of the exclusivity period granted by their respective patents. This steers way clear for follow-on biologics business. The global biosimilars market is expected to reach USD 6.22 Billion by 2020 from USD 2.29 Billion in 2015, at a CAGR of 22.1 per cent from 2015 to 2020.

Monoclonal antibody (mAb) based drug form the biggest pipeline of both innovator and biosimilar drugs. Hence they also form the biggest lot of molecules to be studied for pre-clinical and clinical studies.

Traditionally, measurement of antibodies is based on antigen and antibody interaction, and detecting antibody by treating it as an antigen against an anti-antibody. This also is basic underlying principal of Ligand Binding Assay (LBA). However, the major drawbacks are prolonged assay development times, reagent optimization and procurement, and matrix effects. There have been some developments in countering this through automation which not only improves throughput, but also reduces matrix effects and assay development time.

Currently, LC-MS/MS based methods are emerging as an alternative approach to quantify mAbs using stable isotope labelled signature proteins or peptides as internal standards (IS). In comparison to conventional LBAs, LC/MS/MS based approach affords many advantages in terms of selectivity by MRM, improved throughput by multiplexed detection of targets, wide dynamic range, short development time lowering cost of analysis etc.

Conventional proteolysis methods can make identifying the signature peptide amongst gamut of peptides very difficult, thereby decreasing the quantitative limits.

Another workflow for antibody quantification is affinity enrichment of sample, followed by reduction and alkylation and then trypsin digestion to release the tryptic fragments that can either be analysed by Immunoassay or LC-MS/MS.

There have been efforts by technology providers to address the affinity step by providing streptavidin magnetic beads through which one could capture the specific binding partner that is biotynylated resulting in the enrichment of the desired population while yet others have concentrated on the trypsin digestion step optimizing the trypsin concentration and time of digestion.

Due to extent of similarity between mAb and IgG sequence, it becomes very difficult to identify and quantitate the signature peptide meant for designated mAb amongst high concentration of Fc peptides, which is more or less common. Hence in approaches where all Immunoglobulins are captured doesnot really serve the purpose. While one thinks of increasing signal of signature peptide, reduction in production and injection of background constant peptides is a must.

A simple yet elegant way to address this would be to have a selective trypsin digestion restricted to the variable portion of the mAbs contributing to the CDRs thereby reducing the number of samples to be screened and quantified.

Shimadzu Life Science Research Center has been relentlessly working on establishing universal bioanalytical pretreatment method for IgG derived MAbs, which is easy and more selective. Current proteolysis methods can make identifying the signature peptide amongst gamut of peptides very difficult, thereby decreasing the quantitative limits. To simplify this process, Shimadzu has devised a novel technique - nSMOL (nano Surface and Molecular Orientation Limited proteolysis) which can be applied to all MAbs.

nSMOL works on selective proteolysis of Fab by making use of the difference in size of the protease nanoparticle diameter (200 nm) and the antibody resin pore size (100 nm. To achieve limited proteolysis of the antibody Fab region, the antibody is immobilized in such a way that only Fab region of antibody is spatially available for selective cleavage with protease (trypsin) immobilized on beads. Moreover, effective Fab proteolysis is possible under non-denaturing physiological condition (as demonstrated by Iwamoto et al, Analytical Metods, 2015). Thus there is a considerable reduction of the digest peptides that are formed with enhanced selectivity and also time required for method setup and optimization is drastically reduced. In other words, using nSMOL, one can maintain the specificity of the antibody sequences while minimizing the sample complexity as well as the elimination of extra protease. ).

(Development of the validated LCMS bioanalysis of Trastuzumab in human plasma using selective detection method for complementarity-determining regions of monoclonal antibody: nano-surface and molecular orientation limited (nSMOL) proteolysis- Iwamoto et al, Analytical Methods, 2015)

This approach leads to the shortening of analytical time, LCMS robustness, wide dynamic range, and considerable improvement in sensitivity. This technique can already boast of ready to use completely validated methodology for bioanalysis of Trastuzumab, Bevacizumab and other MAbs in human plasma in accordance with the Japan Guideline on Bioanalytical Method Validation in Pharmaceutical Development from Notification 0711-1(2013) of the Evaluation and Licensing Division, Pharmaceutical and Food Safety Bureau, the Ministry of Health, Labour and Welfare, dated July 11, 2013.
Establishing correct measurement range based on dosage, plasma concentration etc are key to determining swiftness and robustness of pre-clincal and clinical bioanalysis of mAb based biosimilar drugs. nSMOL can play a pivotal role in reducing the turn around time for these studies by providing a generic method for mAB analysis which can be a biosimilar, bio-better or an innovator drug.