Recombinant Antibody-Producing Stable CHOK1 Pool Stability Study.

Mammalian cell line stability is an important consideration when establishing a biologics manufacturing process in the biopharmaceutical and in vitro diagnostics (IVD) industries. Traditional Chinese hamster ovary (CHO) cell line development methods use a random integration approach that requires transfection, selection, optional amplification, screenings, and single-cell cloning to select clones with acceptable productivity, product quality, and genetic stability. Site-specific integration reduces these disadvantages, and new technologies have been developed to mitigate risks associated with genetic instability. In this study, we applied the Leap-In® transposase-mediated expression system from ATUM to generate stable CHOK1 pools for the production of four recombinant antibody reagents for IVD immunoassays. CHO cell line stability is defined by consistent antibody production over time. Three of the CHOK1 pools maintained productivity suitable for manufacturing, with high antibody yields. The productivity of the remaining CHOK1 pool decreased over time; however, derivative clones showed acceptable stability. l-glutamine had variable effects on CHOK1 cell line or stable pool stability and significantly affected antibody product titer. Compared with traditional random integration methods, the ATUM Leap-In system can reduce the time needed to develop new immunoassays by using semi site-specific integration to generate high-yield stable pools that meet manufacturing stability requirements.

Differential regulation of lipopolysaccharide-induced monocyte matrix metalloproteinase (MMP)-1 and MMP-9 by p38 and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases.

Signal transduction events in monocyte matrix metalloproteinase (MMP) production have been shown to include a PGE(2)-cAMP-dependent step. To determine earlier pathway components, we examined the role of mitogen-activated protein kinases (MAPKs) in the regulation of monocyte MMP-1 and MMP-9, two major MMPs induced by LPS. Stimulation with LPS resulted in the activation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) and mitogen-activated kinase p38. The p38-specific inhibitor SB203580 suppressed p38 activity and MMP-1 mRNA and protein, but increased ERK activity and MMP-9 mRNA and protein. In contrast, the MAPK kinase 1/2-specific inhibitor PD98059 inhibited MMP-1 and MMP-9. However, both MAPK inhibitors decreased the production of cyclooxygenase-2 and PGE(2), but only the inhibition of MMP-1 by SB203580 was reversed by PGE(2) or dibutyryl cAMP. Examination of the effect of these MAPK inhibitors on the promoters of MMP-1 and MMP-9 revealed that PD98059 inhibited the binding of transcription factors to all of the MMP promoter-specific complementary oligonucleotides tested. However, SB203580 only inhibited the binding of MMP-1-specific CREB and SP 1 oligonucleotides, which was reversed by PGE(2). Additionally, SB203580 enhanced transcription factor binding to the oligonucleotides complementary to a NF-kappaB site in the promoter of MMP-9. Thus, LPS induction of MMP-1 production by monocytes is regulated by both ERK1/2 and p38, whereas MMP-9 stimulation occurred mainly through the ERK1/2 pathway. Moreover, p38 regulates MMP-1 mainly through a PGE(2)-dependent pathway, whereas ERK1/2-mediated MMP-1 and MMP-9 production involves the activation of additional MMP promoter sites through a PGE(2)-independent mechanism.