Polymorphic distribution of proteins in solution by mass spectrometry: The analysis of insulin analogues.
The characterization of conformational and oligomeric distribution of proteins is of paramount importance for the understanding of the correlation between structure and function. Among the bioanalytical approaches currently available, the electrospray ionization-mass spectrometry (ESI-MS) coupled to ion mobility spectrometry (IMS) is the best suited for high resolution identification with high sensitivity, allowing the in situ separation of oligomeric and conformational species. We tested the performance of the ESI-MS technique along with the IMS separation approach on a broad variety of insulin and insulin analogues with distinct oligomeric distribution pattern. The measurement of commercial insulin allowed the identification of species ranging from monomers to hexamers and their complexes with zinc ions. Dissimilar distribution profile for regular insulin as a function of formulation component and among the insulin analogues were observed by ESI-IMS-MS but not by ESI-MS along, crystallographic assays or size-exclusion chromatography. These data suggest the additional suitability of ESI-IMS-MS in conformational and oligomeric profiling of biomacromolecules and biopharmaceuticals. The easiness of the technique provides further motivation for its application in the characterization of both raw and formulated protein biopharmaceuticals in routine and comparability exercises.
Federal University of Rio de Janeiro - UFRJ, Av. Carlos Chagas Filho 373, CCS, Bss24, Ilha do Fundão, 21941-902, Rio de Janeiro, RJ, Brazil; Brazilian National Cancer Institute (INCA), 20230-014, Rio de Janeiro, RJ, Brazil.
Maely P
MP
Fávero-Retto
Federal University of Rio de Janeiro - UFRJ, Av. Carlos Chagas Filho 373, CCS, Bss24, Ilha do Fundão, 21941-902, Rio de Janeiro, RJ, Brazil; Department of Chemistry, Institute of Exact Sciences, Rural Federal University of Rio de Janeiro - UFRRJ, Rodovia BR 465, km 7, CEP: 23890-000, Seropédica, RJ, Brazil.
Luiz Henrique
LH
Guerreiro
Federal University of Rio de Janeiro - UFRJ, Av. Carlos Chagas Filho 373, CCS, Bss24, Ilha do Fundão, 21941-902, Rio de Janeiro, RJ, Brazil.
Cássio M
CM
Pessanha
Federal University of Rio de Janeiro - UFRJ, Av. Carlos Chagas Filho 373, CCS, Bss24, Ilha do Fundão, 21941-902, Rio de Janeiro, RJ, Brazil.
Leonardo C
LC
Palmieri
Federal University of Rio de Janeiro - UFRJ, Av. Carlos Chagas Filho 373, CCS, Bss24, Ilha do Fundão, 21941-902, Rio de Janeiro, RJ, Brazil; Laboratory for Macromolecules (LAMAC-DIMAV), Brazilian National Institute of Metrology, Quality and Technology - INMETRO, Av. N. Sa. das Graças, 50 - Xerém, Duque de Caxias-RJ, 25250-020, Rio de Janeiro, Brazil; National Institute of Science and Technology for Structural Biology and Bioimaging (INBEB-INCT), Federal University of Rio de Janeiro, Rio de Janeiro, 21941-590, Brazil. Electronic address: Mauricio@pharma.ufrj.br.
Luís Maurício T R
LMTR
Lima
D002850:Chromatography, Gel / Q000379:methods; D002942:Circular Dichroism; D003460:Crystallization; D018360:Crystallography, X-Ray; D030562:Databases, Protein; D006801:Humans; D007328:Insulin / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D011487:Protein Conformation; D055503:Protein Multimerization; D011506:Proteins / Q000737:chemistry*; D012996:Solutions; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D015032:Zinc / Q000737:chemistry
D007328:Insulin; D011506:Proteins; D012996:Solutions; D015032:Zinc
Biopharmaceuticals; Biosimilar; Human insulin; Ion mobility spectrometry; Mass spectrometry; Oligomer