Heteronuclear NMR provides an accurate assessment of therapeutic insulin's quality

An NMR-Based Similarity Metric for Higher Order Structure Quality Assessment Among U.S. Marketed Insulin Therapeutics

“…12 In recent years, one-or two-dimensional (1D or 2D) NMR methods have been used for protein HOS profiling. 4,[13][14][15][16][17][18][19][20][21][22] These studies support the use of NMR as a robust, reproducible, and sensitive method in identifying HOS changes. 18 Importantly, for HOS assessment, principal component analysis (PCA) on NMR spectral data has been increasingly used as a chemometric approach for spectral comparison.…”

Protecting group free radical C–H trifluoromethylation of peptides

“…Since isoleucine A2 is in close proximity to tyrosine A19, we reasoned that the CF 3 -labeling on Tyr-A19 possibly induced a small change in the local conformation, altering the chemical shifts of the isoleucine methyl groups. 28 Finally, comparison of 19 F-NMR traces of 18 and the mixed fraction obtained by semi-preparative HPLC containing the other three CF 3 -Tyr insulin regioisomers showed well-resolved 19 F-NMR signals for each CF 3 -modified insulin regioisomer. 18 This observation validated the notion that CF 3 -labeling of aromatic sidechains provides a sensitive 19 F-NMR spectroscopic probe of the local environment in complex peptides while being minimally perturbing to the overall structure of the biomacromolecule.…”

Simple NMR methods for evaluating higher order structures of monoclonal antibody therapeutics with quinary structure

“…, the chemical shift depends on amino acid type, secondary and tertiary structure and the peak line-width is affected by quaternary and quinary structure and exchange kinetics between forms. Recently, several laboratories have applied 1D 1 H and 2D heteronuclear NMR profiling protocols for structure evaluation of protein therapeutics [10,13,14,24-29], including the proposed biosimilar rituximab at 22 mg/mL [15] and the NIST mAb at 40 mg/mL [12]. Previously Poppe et al [9] developed a 1D- 1 H diffusion filtering method, which reduced the sucrose peaks in the spectrum and was applied on Amgen’s proprietary mAb samples concentrated at 30 mg/mL in 9% sucrose or phosphate buffer [30].…”