Econo pac 14 cm high 1.5 12 cm polypropylene columns

Frag-It kits containing immobilized IdeS protease (Immunoglobulin G-degrading enzyme of Streptococcus pyogene) columns designed for cleavage of up to 0.5 mg mAb were purchased from Genovis (cat. # A2-FR2-025). Tributylphosphine (TBP) (a 200 mM solution in N-methyl-2-pyrrolidinone (cat. # T-7567)) and iodoacetamide (IAM, cat. # I-6125) were from Sigma. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP, cat # 20490) was obtained from Thermo Scientific Pierce. The Vivaspin 500 μL concentrators (cat. # 28-9322-25) were from GE Healthcare. Guanidine hydrochloride (Gu-HCl, cat. # A1449) was from AppliChem. To separate TBP and IAM from the fragmented antibody after reduction and alkylation, Sephadex G-25-80 size exclusion beads were from Sigma, and disposable columns were from BioRad (Econo-Pac 14 cm–high 1.5 × 12 cm polypropylene columns, cat. # 7321010EDU). Tris base, NaCl, and concentrated formic acid, sequencing grade, were from Fisher Scientific.

Tris(2-carboxyethyl)phosphine hydrochloride (TCEP, cat # 20490) and agarose bound Tris(2-carboxyethyl)phosphine hydrochloride (TCEP-agarose, cat # 77712), were obtained from Thermo-Scientific Pierce. Iodoacetamide (IAM, cat. # I-6125) was from Sigma. 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (ABD-F, cat # A5597) was from TCI America, while fluorescein 5-maleimide (F-MAL, cat # F-150) was purchased from Molecular Probes, and dibromobimane (DBB) was from Anaspec, Inc. (cat # AS81448). Maleimide–polyethylene glycol (5000 Da PEG chain, MPEG-MAL-5000) was from Nektar Transforming Therapeutics. The 0.22 µm 500 µL spin filters were from EMD Millipore (cat. # UFC30GV0S), and the 10,000 MWCO Vivaspin 500 µL concentrators (cat. # 28-9322-25) were from GE Healthcare. To separate iodoacetamide from the Fab fragment after reduction and alkylation for mass spectral analysis, Sephadex G-25-80 size exclusion beads were from Sigma, and disposable columns were from BioRad (Econo-Pac 14 cm–high 1.5 × 12 cm polypropylene columns, cat. # 7321010EDU), equilibrated with 0.1% formic acid just before use, as previously described [8 (link)]. Tris base, NaCl, EDTA, and other buffer components, as well as sequencing grade concentrated formic acid, were from Fisher Scientific.