Mapping and partial characterization of proteases expressed by a CHO production cell line

Abstract

The proteolytic activities expressed by a Chinese hamster ovary (CHO) cell line in the cultivation supernatant during the production of recombinant factor VIII were mapped with a broad spectrum protease assay and a series of different types of protease inhibitors. The destabilizing effect on the product emanated from a metalloproteinase, which could be effectively blocked by chelating agents to lead to product stabilization. Amino acid sequences of the isolated metalloproteinase were found to have sequence homology with matrix metalloproteinases (MMPs) MMP3, MMP10, and MMP12. Several species with metalloproteinase activity were characterized and found to be related to each other. The results indicate that an MMP pro-enzyme of >/=200 kDa was released from the CHO cells during the production phase. The enzyme expressed collagenase/gelatinase activity when activated. Due to autoproteolysis, a number of smaller, less specific MMPs were formed with the smallest form, a 19.4 kDa protein, being the most active. These results may be of particular relevance for other production processes using CHO cells for the expression of recombinant proteins.

Proteomic Profiling of Secreted Proteins from CHO Cells Using Surface-Enhanced Laser Desorption Ionization Time-of-Flight Mass Spectrometry

“…In manufacturing recombinant proteins, the use of serum-free media for cell cultivation is preferred in order to meet quality and regulatory requirements but this may increase the exposure of the expressed product to proteolytic attacks that can result in product heterogeneity, denaturation, and loss of protein function. For example, an MMP pro-enzyme has been found to be released from CHO cells during the production phase of recombinant factor VIII, and as a result of autoproteolysis, a number of smaller, less specific MMPs were also detected (18). In another study, pro-MMP9 (gelatinase B) was identified from conditioned media of CHO-K1 cells grown in serum-free conditions (19).…”

Degradation of an Fc‐fusion recombinant protein by host cell proteases: Identification of a CHO cathepsin D protease

“…Thus, the other protease showing optimal activity at pH 7.5 needs co-factors that are likely divalent cations, sensitive to chelation by EDTA. As demonstrated by Sandberg et al (2006), metalloproteases are present in the CHO cell culture supernatant. As the post-capture material is stored at pH lower that 6, this proteolytic activity is less likely to be responsible for the observed degradation of our therapeutic product.…”

Tailoring recombinant protein quality by rational media design

“…Even though the protein backbone is extremely resistant to nonenzymatic hydrolysis under physiological conditions, certain sites may become prone to fragmentation as a function of the presence of specific side-chains residues, such as Asp, Gly, Ser, Thr, Cys or Asn, which may facilitate cleavage due to increased flexibility of the local structure, solvent conditions (pH, temperature) and the presence of metals or radicals 179 . Furthermore, clipping may be observed as a result of the activity of proteases released by cells into the cell culture supernatant during the protein production process [180][181][182] . Adverse effects of fragmentation are various and potentially include reduced biological activity, shorter half-life and immunogenicity reactions and hence provoke patient safety issues 68 .…”