title: Structural meta-analysis of regular human insulin in pharmaceutical formulations.
abstract: We have studied regular acting, wild-type human insulin at potency of 100 U/mL from four different pharmaceutical products directly from their final finished formulation by the combined use of mass spectrometry (MS), dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR), and single-crystal protein crystallography (PX). All products showed similar oligomeric assembly in solution as judged by DLS and SAXS measurements. The NMR spectra were compatible with well folded proteins, showing close conformational identity for the human insulin in the four products. Crystallographic assays conducted with the final formulated products resulted in all insulin crystals belonging to the R3 space group with two a dimer in the asymmetric unit, both with the B-chain in the T configuration. Meta-analysis of the 24 crystal structures solved from the four distinct insulin products revealed close similarity between them regardless of variables such as biological origin, product batch, country origin of the product, and analytical approach, revealing a low conformational variability for the converging insulin structural ensemble. We propose the use of MS, SAXS, NMR fingerprint, and PX as a precise chemical and structural proof of folding identity of regular insulin in the final, formulated product.
authors.affiliation: School of Pharmacy, Federal University of Rio de Janeiro - UFRJ, Rio de Janeiro, Brazil; Brazilian National Cancer Institute (INCA), Rio de Janeiro, Brazil; Brazilian National Institute of Traumatology and Orthopedics (INTO), Rio de Janeiro, Brazil.
authors.forename: Maely P
authors.initials: MP
authors.lastname: Fávero-Retto
authors.forename: Leonardo C
authors.initials: LC
authors.lastname: Palmieri
authors.forename: Tatiana A C B
authors.initials: TA
authors.lastname: Souza
authors.forename: Fábio C L
authors.initials: FC
authors.lastname: Almeida
authors.forename: Luís Mauricio T R
authors.initials: LM
authors.lastname: Lima
mesh_terms: D018360:Crystallography, X-Ray; D006801:Humans; D007328:Insulin / Q000737:chemistry*; D008027:Light; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D004364:Pharmaceutical Preparations / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D015203:Reproducibility of Results; D053838:Scattering, Small Angle; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013678:Technology, Pharmaceutical / Q000379:methods*
chemical_list: D007328:Insulin; D004364:Pharmaceutical Preparations
keywords: Biosimilar; Crystallography; Human insulin; MALDI-TOF; Mass spectra; Meta-analysis; NMR; Nuclear magnetic resonance; SAXS; Small-angle X-ray scattering; hINS; human insulin; matrix-assisted laser desorption/ionization – time-of-flight; nuclear magnetic resonance; small-angle X-ray scattering