03472nas a2200181 4500008004100000245013100041210006900172260000800241300001100249490000800260520278500268100001903053700002803072700002403100700002503124700001403149856012703163 2021 eng d00aComparison of glycoprofiles of rituximab versions licensed for sale in India and an analytical approach for quality assessment0 aComparison of glycoprofiles of rituximab versions licensed for s cJUL a1042670 v2443 a

Glycosylation affects clinical efficacy and safety; therefore, is a critical quality attribute of therapeutic monoclonal antibodies. Glycans are often labile and complex in patterns, giving rise to macro- and micro-heterogeneity. Recombinant production, diverse geographical locations, associated transportation and storage conditions further compound the problem. Two-way studies comparing glycoprofile of the originator and its given biosimilar are aplenty. However, the extent of analytical variation and similarity in glycoprofile across all approved versions of a drug is hardly explored. Using UHPLC and mass spectrometry, we compared the glycoprofiles of eight rituximab drug samples licensed for sale in India. While the types of glycans were found identical, the abundance of some glycans varied significantly within the tested population. The quality range of glycosylation parameters of the tested sample population differed significantly from the previously established values for US/EU licensed rituximab. As the mean abundance of the 90% of identified glycans falls within ±3SD, the extent of mutual variations amongst tested lots is less significant compared to the extreme deviation from previously established QR limits. Thus, we propose this approach as an orthogonal method to capture glycan variations in licensed versions of mAbs for quality surveillance and in cases where originator samples' are limiting. SIGNIFICANCE: As fluctuation in glycosylation may be of clinical significance, we identify that a one-to-one comparison with originator alone is insufficient in sensing the extent of variations in glycosylation parameters in licensed biosimilars of a given therapeutic mAb. Here we propose that future biosimilarity analysis may include an orthogonal approach of generating an additional combined QR range representing variations across the originator and its biosimilars. The glycosylation profiles of eight rituximab drug samples of different make obtained from the point of sale in India were found identical amongst the tested rituximab versions. However, the QR limits corresponding to important glycosylation parameters differed significantly across all tested samples from the previously established QR limits of US- and EU-licensed rituximab in statistical terms. Such an approach may be useful in defining the true range of glycan variations in licensed versions of therapeutic mAbs.

1 aKaur, Tejinder1 aShukla, Bhupendra, Nath1 aYadav, Vinay, Kumar1 aKulkarni, Mahesh, J.1 aRao, Alka uhttps://library.ncl.res.in/content/comparison-glycoprofiles-rituximab-versions-licensed-sale-india-and-analytical-approach02995nas a2200265 4500008004100000022001400041245015900055210006900214260000800283300001100291490000800302520207900310653001302389653000802402653001402410653003802424653001602462100001902478700001802497700002702515700002102542700001402563700002502577856012702602 2021 eng d a1874-391900aMultiple-parallel-protease digestion coupled with high-resolution mass spectrometry: an approach towards comprehensive peptide mapping of therapeutic mAbs0 aMultipleparallelprotease digestion coupled with highresolution m cFEB a1040530 v2323 a

Therapeutic monoclonal antibodies (mAbs) are structurally large and complex molecules. To be safe and efficacious, a biosimilar mAb must show high similarity to its reference product in Critical Quality Attributes (CQA). mAbs are highly sensitive to protein expression, production, manufacturing, supply chain, and storage conditions. All these factors make biosimilar mAbs intrinsically susceptible for variability during production. Accordingly, several lots of references and tests are required to establish the biosimilarity of a test mAb. The primary structure is a CQA of a mAb affecting its safety and efficacy. Here, we apply peptide mapping as an analytical method to decipher the primary structure and associated modifications for a quick quality assessment of TrastuzumAb and RituximAb innovator and biosimilar. A multiple-parallel-protease digestion strategy followed by high-resolution mass spectrometric analysis consistently achieved 100% sequence coverage along with reliable detection of post-translational modifications. Additionally, the use of supporting methods such as intact mass analysis and circular dichroism helped us to decipher the primary and higher order structures of these mAbs. We identify discernible variations in the profile of the innovator and biosimilar mAbs and validate the method for quick yet deep comparability analysis of the primary structure of biosimilar mAbs sold in the market. Significance: Peptide mapping using bottom-up approach is one of the most common methods for the characterization of therapeutic monoclonal antibodies. Herein, we describe a multi-parallel-protease digestion strategy using a combination of five different proteases followed by high-resolution mass spectrometric analysis with TrastuzumAb and RituximAb as an example. This resulted in a comprehensive identification of peptides with increased reliability and identification of different PTMs. Additional supporting orthogonal methods like intact mass and higher-order structure analysis helped evaluate broader conformational properties.

10aLC-MS/MS10aPTM10aRituximAb10aTherapeutic monoclonal antibodies10aTrastuzumAb1 aPradhan, Gauri1 aSneha, J., M.1 aSonwane, Babasaheb, P.1 aSanthakumari, B.1 aRao, Alka1 aKulkarni, Mahesh, J. uhttps://library.ncl.res.in/content/multiple-parallel-protease-digestion-coupled-high-resolution-mass-spectrometry-approach