Production of anti TNF-α antibodies in eukaryotic cells using different combinations of vectors carrying heavy and light chains

Abstract

Tumor necrosis factor-a (TNF-a) plays a key role in rheumatoid arthritis and some other autoimmune diseases. Therapy with anti-TNF-a recombinant antibodies (Ab) appears to be highly effective. Production of new hyper-producing eukaryotic cell lines can decrease the treatment cost, which currently is very high. However, due to the complexity of protein transcription, translation, processing, and secretion in mammalian cells, the stages at which antibody expression is affected are still poorly determined. The aim of this work was to compare the productivity of two cell lines developed in CHO DG44 cells, deficient in dihydrofolate reductase, transfected with vectors carrying either heavy (H) or light (L) chains of chimeric antibody under different combinations of selective elements. Both H and L chains were cloned either in pOptiVEC or pcDNA3.3 vectors and different combinations were used to produce HL and LH cell lines. We have shown that Ab production has been low and comparable between HL and LH cells until selection on methotrexate (MTX) when LH but not HL cells have responded with 3.5 times increased productivity. Flow cytometry analysis has demonstrated that intracellular concentration of full size Abs in LH cells was 5.6 times higher than in HL ones due to higher amount of H chain synthesis. No differences in viability between HL and LH cells have been found. We have concluded that the expression of H chain in the pOptiVEC vector, which is responsible for MTX resistance, has led to the suppression of H chain synthesis and limitation in full Ab assembly.

A BioDesign Approach to Obtain High Yields of Biosimilars by Anti-apoptotic Cell Engineering: a Case Study to Increase the Production Yield of Anti-TNF Alpha Producing Recombinant CHO Cells

“…Tumor necrosis factor alpha (TNF-α), which has both proinflammatory and immunoregulatory functions, is an important target in biopharmaceutical industry [7][8][9]. Commercial TNF antagonists comprise of engineered monoclonal antibodies and fusion proteins that have been successfully used for the treatment of the chronic inflammatory diseases such as RA, Crohn's disease, and psoriasis [10,11]. There are currently five TNF-α antibodies approved by the US Food and Drug Administration (FDA) for clinical applications on the market either chimeric, humanized, or fully human (i.e., etanercept, infliximab, adalimumab, certolizumab, and golimumab) [12,13].…”

“…Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, murine myeloma cells (NS0 and Sp2/0), and baby hamster kidney (BHK21) cells are the most prevalent non-human, mammalian cell lines used for recombinant protein production [18]. Recombinant CHO (rCHO) cells frequently used mammalian expression systems for large-scale production of biopharmaceuticals to satisfy the huge market demand due to their adaptation capability to suspension culture, high cell growth rate, high protein production yield, and resistance to mechanical stress [10][11][12][13][14][15][16][17][18][19], mostly when they are used for stable expression [20,21].…”