title: Mass Spectrometry Based Mechanistic Insights into Formation of Tris Conjugates: Implications on Protein Biopharmaceutics.
abstract: We present here extensive mass spectrometric studies on the formation of a Tris conjugate with a therapeutic monoclonal antibody. The results not only demonstrate the reactive nature of the Tris molecule but also the sequence and reaction conditions that trigger this reactivity. The results corroborate the fact that proteins are, in general, prone to conjugation and/or adduct formation reactions and any modification due to this essentially leads to formation of impurities in a protein sample. Further, the results demonstrate that the conjugation reaction happens via a succinimide intermediate and has sequence specificity. Additionally, the data presented in this study also shows that the Tris formation is produced in-solution and is not an in-source phenomenon. We believe that the facts given here will open further avenues on exploration of Tris as a conjugating agent as well as ensure that the use of Tris or any ionic buffer in the process of producing a biopharmaceutical drug is monitored closely for the presence of such conjugate formation. Graphical Abstract ᅟ.
authors.affiliation: Molecular Characterization Laboratory, Biocon Research Limited, Biocon Limited, Biocon Park, Bommasandra - Jigani Link Road, Bommasandra Industrial Area Phase IV, Bangalore, 560099, India.
authors.forename: Pradeep G
authors.initials: PG
authors.lastname: Kabadi
authors.affiliation: Molecular Characterization Laboratory, Biocon Research Limited, Biocon Limited, Biocon Park, Bommasandra - Jigani Link Road, Bommasandra Industrial Area Phase IV, Bangalore, 560099, India.
authors.forename: Praveen Kallamvalliillam
authors.initials: PK
authors.lastname: Sankaran
authors.affiliation: Molecular Characterization Laboratory, Biocon Research Limited, Biocon Limited, Biocon Park, Bommasandra - Jigani Link Road, Bommasandra Industrial Area Phase IV, Bangalore, 560099, India.
authors.forename: Dinesh V
authors.initials: DV
authors.lastname: Palanivelu
authors.affiliation: Molecular Characterization Laboratory, Biocon Research Limited, Biocon Limited, Biocon Park, Bommasandra - Jigani Link Road, Bommasandra Industrial Area Phase IV, Bangalore, 560099, India.
authors.forename: Laxmi
authors.initials: L
authors.lastname: Adhikary
authors.affiliation: Molecular Characterization Laboratory, Biocon Research Limited, Biocon Limited, Biocon Park, Bommasandra - Jigani Link Road, Bommasandra Industrial Area Phase IV, Bangalore, 560099, India.
authors.forename: Anand
authors.initials: A
authors.lastname: Khedkar
authors.affiliation: Molecular Characterization Laboratory, Biocon Research Limited, Biocon Limited, Biocon Park, Bommasandra - Jigani Link Road, Bommasandra Industrial Area Phase IV, Bangalore, 560099, India. Amarnath.Chatterjee@gmail.com.
authors.forename: Amarnath
authors.initials: A
authors.lastname: Chatterjee
mesh_terms: D000911:Antibodies, Monoclonal; D001702:Biopharmaceutics; D002021:Buffers; D013058:Mass Spectrometry
chemical_list: D000911:Antibodies, Monoclonal; D002021:Buffers
keywords: Deamidation; Mass spectrometry; Monoclonal antibody; Succinimide intermediate; Tris conjugation