Mass Spectrometry Based Mechanistic Insights into Formation of Tris Conjugates: Implications on Protein Biopharmaceutics

Abstract

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.

Soluble SARS‐CoV‐2 RBD and human ACE2 peptidase domain produced in DrosophilaS2 cells show functions evoking virus–cell interface

“…However, as deamidation frequency was not correlated with charges, we could not solely attribute micro‐heterogeneity to its occurrence. In addition, Tris adducts on deamidated asparagine (and glutamine) residues (which could be further modifying deamidated asparagine residues [Kabadi et al, 2016] and affecting its detection) remained as a very marginal fraction (0.1%–1.3%) (Supporting Information Figure S8A; Supporting Information Table S9). Asparagine deamidation to aspartate (or iso‐aspartate) entails changes in overall charge (and protein backbone) and has been associated with aging in betacoronaviruses spike protein, with RBM described as a deamidation hotspot (Lorenzo et al, 2021).…”

Enzymatic Fluoromethylation as a Tool for ATP‐Independent Ligation

“…One type of technique is based on the idea that PIMT‐treated proteins could be trapped with a high concentration (>1 M) of synthetic nucleophile such as hydrazine or Tris (2‐Amino‐2‐(hydroxymethyl)‐1,3‐propanediol) to facilitate enrichment and identification of conjugated PIMT substrates [15a,c,16–17] . This Scheme is elegant, but the protocols published so far are not efficient enough to detect isoAsp‐ or succinimide‐containing peptides in complex samples.…”

PIMT-Mediated Labeling of l-Isoaspartic Acid with Tris Facilitates Identification of Isomerization Sites in Long-Lived Proteins

“…The succinimide ring appears to be amine reactive in general, as it has also been found that Tris can react in solution with succinimide rings formed during deamidation of IgG peptides. 34 While the covalent modification of peptides with Tris during deamidation is potentially undesirable, in the context of L-isoAsp discovery, it presents an opportunity for a convenient labeling technique requiring nothing more than concentrated Tris buffer and PIMT.…”

“…Hydrazine can also react with the succinimide ring, resulting in a mass shift of +14 Da at the modified aspartic acid and allowing for affinity enrichment via aldehyde resins. , In this case, reaction of l -isoAsp with PIMT in the presence of 1 M hydrazine was able to compete with the ongoing hydrolysis of the succinimide intermediate, resulting in some aminolysis product formation. The succinimide ring appears to be amine reactive in general, as it has also been found that Tris can react in solution with succinimide rings formed during deamidation of IgG peptides . While the covalent modification of peptides with Tris during deamidation is potentially undesirable, in the context of l -isoAsp discovery, it presents an opportunity for a convenient labeling technique requiring nothing more than concentrated Tris buffer and PIMT.…”