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A novel rapid analysis using mass spectrometry to evaluate downstream refolding of recombinant human insulin‐like growth factor‐1 (mecasermin)
Author(s) -
Furuki Kenichiro,
Toyo'oka Toshimasa,
Yamaguchi Hideto
Publication year - 2017
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.7906
Subject(s) - chemistry , downstream (manufacturing) , mass spectrometry , recombinant dna , chromatography , downstream processing , biochemistry , operations management , economics , gene
Rationale Mecasermin is used to treat elevated blood sugar as well as growth‐hormone‐resistant Laron‐type dwarfism. Mecasermin isolated from inclusion bodies in extracts of E. coli must be refolded to acquire sufficient activity. However, there is no rapid analytical method for monitoring refolding during the purification process. Methods We prepared mecasermin drug product, in‐process samples during the oxidation of mecasermin, forced‐reduced mecasermin, and aerially oxidized mecasermin after forced reduction. Desalted mecasermin samples were analyzed using MALDI‐ISD. The peak intensity ratio of product to precursor ion was determined. The charge‐state distribution (CSD) of mecasermin ions was evaluated using ESI‐MS coupled with SEC‐mode HPLC. The drift time and collision cross‐sectional area (CCS) of mecasermin ions were evaluated using ESI‐IMS‐MS coupled with SEC‐mode HPLC. Results MALDI‐ISD data, CSD values determined using ESI‐MS, and the CCS acquired using ESI‐IMS‐MS revealed the relationship between the folded and unfolded proteoforms of forced‐reduced mecasermin and aerially oxidized mecasermin with the free‐SH:protein ratio of mecasermin drug product. The CCS area, which is determined using ESI‐IMS‐MS, provided proteoform information through rapid monitoring (<2 min) of in‐process samples during the manufacture of mecasermin. Conclusions ESI‐IMS‐MS coupled with SEC‐mode HPLC is a rapid and robust method for analyzing the free‐SH:protein ratio of mecasermin that allows proteoform changes to be evaluated and monitored during the oxidation of mecasermin. ESI‐IMS‐MS is applicable as a process analytical technology tool for identifying the “critical quality attributes” and implementing “quality by design” for manufacturing mecasermin.