An efficient route to bispecific antibody production using single-reactor mammalian co-culture

Abstract

Bispecific antibodies have shown promise in the clinic as medicines with novel mechanisms of action. Lack of efficient production of bispecific IgGs, however, has limited their rapid advancement. Here, we describe a single-reactor process using mammalian cell co-culture production to efficiently produce a bispecific IgG with 4 distinct polypeptide chains without the need for parallel processing of each half-antibody or additional framework mutations. This method resembles a conventional process, and the quality and yield of the monoclonal antibodies are equal to those produced using parallel processing methods. We demonstrate the application of the approach to diverse bispecific antibodies, and its suitability for production of a tissue specific molecule targeting fibroblast growth factor receptor 1 and klotho β that is being developed for type 2 diabetes and other obesity-linked disorders.

Keywords: Antibody purification; CHO co-culture; bispecific antibodies; diabetes; knobs-into-holes.

Figure 1.

Comparison of different methods for producing bispecific antibodies (A) With transient transfection, the heavy chain (HC) and light chain (LC) plasmids for each H-L fragment are transfected into Chinese hamster ovary (CHO) cells and cultured separately. Antibodies are captured from the secreted media as a mixture of half-antibodies and homodimers. Bispecific assembly is performed by combining both pools together in vitro at a one-to-one molar ratio followed by addition of reduced glutathione (GSH) to catalyze disulfide formation. The result is fully oxidized bispecific antibody with very little homodimer impurities. (B) Using co-culturing in E.coli, cells are transfected with either plasmid and grown together in the same bioreactor, targeting a one-to-one molar ratio of H-L fragments based on historical data. During cell lysis, the H-L fragments dimerize and interchain disulfides are formed. Assembled bispecific antibodies are then captured from the supernatant after cell lysis with very little homodimer impurities. (C) With co-culturing of 2 stable cell lines, both cell types are grown in the same production flask targeting a one-to-one molar ratio of H-L fragments based on historical data. During cell culture, protein is secreted from each cell line as a mixture of half-antibody and homodimer. Addition of GSH favors correct pairing and disulfide formation in the co-culture media. Assembled bispecific antibodies are then captured from the secreted media with very little homodimer impurities.

Figure 2.

Relative bispecific antibody content (% abundance) from different co-culture conditions (A) Anti-C/D (top panel) and anti-E/F (bottom panel) stable cell lines were co-cultured together in a 40 L bioreactor with varying concentrations of glutathione (GSH) added 15 h prior to harvest. For each GSH concentration, cell viability was measured and is reported here as relative abundance (%) of total cell concentration. Protein titers were also measured and are presented as values relative (%) to individual cell titers when no GSH is added. Finally, the cation-exchanger (CEX) method was used to quantitate relative abundance (%) of bispecific antibody formation as a function of GSH concentration. As is demonstrated, addition of GSH did not affect titer or cell viability while in both cases, increasing concentrations of GSH increased the abundance of covalent bispecific. (B) Quantitating the relative abundance of bispecific antibody for all 5 co-cultured pairs demonstrates efficient bispecific antibody formation across a range of cell-to-cell ratios. The CEX method was used to quantitate abundance of assembled bispecific antibody content in each protein pool. Optimized knobs and holes cell ratios (%) have been plotted with the corresponding relative abundance of bispecific antibody (%).

Figure 3.

Schematic of CHO stable cell co-culture processes Overview of the different steps involved in co-culturing 2 stable cell lines. With unknown titers, scale up of seed train flask production of each cell line to 1 L and individual 35 mL shake flask cell cultures are done concurrently. At both day 7 and day 10 day, titers are measured from each 35 mL culture and the averages of these titers inform the cell-to-cell ratio used for mixing the 2 seed train flask productions in the third step. The cell mixture is then successively scaled up from 3 L to 40 L over 18 d. With known titers, individual seed train flask production is scaled up to 1 L shake flask as with unknown titers, however 35 mL shake flasks cultures are not grown concurrently. At day 10, mixing of both 1 L seed trains is done using historical titers.

Figure 4.

Comparison of anti-E/F bispecific antibody pairs harvested from small scale shake flask (0.04 L) and large scale bioreactor (40 L) CHO co-cultures (A) Top panel shows analysis of anti-E/F bispecific antibody from 0.04 L shake flask co-culture while the bottom panel shows the same analysis for anti-E/F bispecific antibody from 40 L bioreactor co-culture. Bispecific antibody produced from each culture volume was captured using MabSURE SELECT and subsequently loaded onto the CEX column. (B) Main peaks (Peak 2) from each analytical run were analyzed by size exclusion chromatography (SEC) and ESI-TOF mass spectrometry (MS) as described in Methods. No discernable differences in bispecific antibody abundance, aggregate or homodimer content were detected.

Figure 5.

Characterization of anti-FGFR1/βKL bispecific antibody pairs assembled in vitro and captured directly from co-culture media (A) SEC chromatograms of bispecific antibodies produced from separate cultures with assembly in vitro (top panel), and from assembly in co-culture media (bottom panel). The insets show a zoomed-in view indicating both assembly methods produce bispecific antibody with less than 1% aggregate and very minimal residual H-L fragments. (B) Bispecific antibodies from both assembly methods were analyzed by ESI-TOF MS as described in Methods. The top panel shows the deconvoluted spectra over a mass range of 25,000–160,000 amu, indicating the overall purity of the bispecific antibody. The bottom panel shows a zoomed in view around the bispecific antibody and the arrows point to the theoretical locations of each knob/knob (K/K) and hole/hole (H/H) homodimer.

Figure 6.

Comparison of anti-FGFR1/βKL bispecific antibody activity derived from different cell culture processes reveals no functional differences. (A) Anti-FGFR1/βKL bispecific antibodies produced from separate cultures with in vitro assembly and assembly in co-culture media were tested for activation of FGFR1 receptor in the absence of βKL expression. The lack of signaling activity is an indication of non-detectable anti-FGFR1-related impurities in the bispecific antibody pools using both strategies. (B) In cells expressing both FGFR1 and βKL, cell-specific activation of FGFR1/βKL complex with the bispecific antibodies produced from both methods was tested. Equivalent activity was observed with antibodies from both production methods, where the error bars indicate standard error of the mean. Included in both assays is a reference standard (Ref. STD) of anti-FGFR1/βKL, where the bispecific antibody was produced from assembly in vitro and polished to a high purity, confirming expected activity of the assembled bispecifics, in addition to full length, parental anti-FGFR1 (R1Mab3) and anti-βKL (KLBMab1) antibodies.