Multiplex secretome engineering enhances recombinant protein production and purity

Host cell proteins (HCPs) are process-related impurities generated during biotherapeutic protein production. HCPs can be problematic if they pose a significant metabolic demand, degrade product quality, or contaminate the final product. Here, we present an effort to create a “clean” Chinese hamster ovary (CHO) cell by disrupting multiple genes to eliminate HCPs. Using a model of CHO cell protein secretion, we predicted the elimination of unnecessary HCPs could have a non-negligible impact on protein production. We analyzed the total HCP content of 6-protein, 11-protein, and 14-protein knockout clones and characterized their growth in shake flasks and bioreactors. These cell lines exhibited a substantial reduction in total HCP content (40%-70%). We also observed higher productivity and improved growth characteristics, in specific clones. With the reduced HCP content, protein A and ion exchange chromatography more efficiently purified a monoclonal antibody (mAb) produced in these cells during a three-step purification process. Thus, substantial improvements can be made in protein titer and purity through large-scale HCP deletion, providing an avenue to increased quality and affordability of high-value biopharmaceuticals. Overall design: CHO-S cells were subjected to 4 cycles of CRISPR/Cas9-mediated multiplex gene disruption to produce 6-protein (BGN, TIMP1, LGALS3BP, NID1.1, NID1.2, and CTSD), 11-protein (BGN, TIMP1, LGALS3BP, NID1.1, NID1.2, CTSD, Aga, Erp29, Gpr56, Tinagl1, and Lgmn), and 14 protein (BGN, TIMP1, LGALS3BP, NID1.1, NID1.2, CTSD, Aga, Erp29, Gpr56, Tinagl1, Lgmn, Yeats2, Sparc, and Lpl) knockout strains. WT CHO-S and knockout cell lines (6x, 11x, and 14x) were transacted with a plasmid encoding the Rituximab antibody and the zeocin selection gene using Freestyle MAX reagent. The WT CHO-S, 6xKO and 11xKO strains were subjected to RNA-Seq at 3 different time points (4 days, 6 days, and 8 days). Library preparation was performed with Illumina's TruSeq Stranded mRNA Library Prep Kit High Throughput (Catalog ID: RS-122-2103).