Capto adhere

Human TIMP3, modified TIMP3 with glycosylation mutations (v2, v82), and fusion constructs (HSA, Fc, or Ab) were expressed in a Chinese hamster ovary cell line, whereby conditioned media were concentrated by tangential flow filtration (TFF; Millipore,10 kDa MWCO) and filtered. The TIMP3 and fusion constructs were next purified through a three‐column chromatography procedure, each utilizing a specific capture column; TIMP3 and glycosylation mutants with Capto MMC (GE Healthcare, Freiburg, Germany): HSA fusions with Cibacron Blue (Merck KGaA, Darmstadt, Germany): and Fc or Ab fusions with MabSelect Sure (GE Healthcare, Pittsburg, PA). Each of these was then followed by Capto Adhere and SP HP (GE Healthcare, Freiburg, Germany) chromatography steps. The TIMP3 containing SP HP fractions were finally concentrated and buffer exchanged (10 mmol/L sodium acetate pH 5.2, 9% sucrose) by TFF. N‐TIMP3 (13.9 kDa N‐domain; aa 1‐120) was expressed as inclusion bodies in an E. coli system with a subtilisin prodomain fusion. After refolding and prodomain cleavage, the N‐TIMP3 was purified by a three‐column procedure using SP HP, CHT, and Butyl HP chromatography. The N‐TIMP3 containing Butyl HP fractions were also concentrated and buffer exchanged (10 mmol/L sodium acetate pH 5.2, 9% sucrose) by TFF (Pall, 5 kDa MWCO).

All chemicals were obtained from Sigma-Aldrich (St. Louis, MO). WorkBeads™ 40 TREN^high was purchased from BioWorks (Uppsala, Sweden). Eshmuno^® HCX was purchased from Merck Millipore (Merck KGaA, Darmstadt, Germany). UNOsphere™ Q was purchased from Bio-Rad Laboratories (Hercules, CA). Toyopearl AF-rProtein A-650 was purchased from Tosoh Bioscience (Tokyo, Japan). Capto™ adhere was purchased from GE Healthcare (Uppsala, Sweden). Chromatography media were packed in XK or Tricorn™ series columns (GE Healthcare). Chromatography experiments were conducted on an ÄKTA™ Explorer 100 or Avant 25 (GE Healthcare).