Single-batch production of recombinant human polyclonal antibodies

Abstract

We have previously described the development and implementation of a strategy for production of recombinant polyclonal antibodies (rpAb) in single batches employing CHO cells generated by site-specific integration, the Sympress I technology. The Sympress I technology is implemented at industrial scale, supporting a phase II clinical development program. Production of recombinant proteins by site-specific integration, which is based on incorporation of a single copy of the gene of interest, makes the Sympress I technology best suited to support niche indications. To improve titers while maintaining a cost-efficient, highly reproducible single-batch manufacturing mode, we have evaluated a number of different approaches. The most successful results were obtained using random integration in a new producer cell termed ECHO, a CHO DG44 cell derivative engineered for improved productivity at Symphogen. This new expression process is termed the Sympress II technology. Here we describe proof-of-principle data demonstrating the feasibility of the Sympress II technology for single-batch rpAb manufacturing using two model systems each composed of six target-specific antibodies. The compositional stability and the batch-to-batch reproducibility of rpAb produced by the ECHO cells were at least as good as observed previously using site-specific integration technology. Furthermore, the new process had a significant titer increase.

Fig. 1

a Mammalian expression vector used for expression of the anti-Vaccinia and anti-RSV antibodies. CMV human CMV promoters, VH antibody heavy chain variable region, IgG1 constant genomic DNA encoding the human IgG1 constant region, IRES + DHFR ECMV internal ribosome entry sequence + mouse DHFR cDNA, SV40 poly A the SV40 polyadenylation sequence, bla and Amp ampicillin resistance gene, pUC ori pUC origin of replication, bGH poly A bovine growth hormone polyadenylation sequence, LC antibody light chain cDNA. b Comparison of specific productivities in selected pools of DG44 and ECHO cells. DG44 cells and ECHO cells were transfected with three different anti-Vaccinia virus antibody constructs (Ab numbers refer to the numbers used in Fig. 2). Specific productivities were measured in selected pools

Fig. 2

Relative content of six anti-Vaccinia antibodies in nine compositions maintained in co-culture in 50 ml culture tubes for 6 weeks. The nine compositions containing six different anti-Vaccinia antibodies (I–VI) with one or more clonal ECHO cell line(s) expressing each antibody were prepared as described in “Materials and Methods” section. Supernatants were analyzed for content of the individual antibodies by CIEX at the start (dark gray columns) and end (light gray columns) of the cultures. The relative content of the six antibodies (along the horizontal axis) is given as percentage of total antibody (vertical axis)

Fig. 3

Relative content of six anti-Vaccinia antibodies from two independent fed batch runs of each of the rpAb compositions 1A, 2A, 3A, and 4A, performed in DASGIP bioreactors at 500 ml scale as described in “Materials and Methods” section. The compositions 1A–3A contained one ECHO clone per antibody and 4A contained three ECHO clones per antibody. Samples for CIEX profile analysis were taken on day 16 of the bioreactor run

Fig. 4

Experimental setup of the compositional stability studies with compositions expressing the anti-RSV rpAb. Suspension ECHO cell clones expressing each of the six monoclonal anti-RSV antibodies were banked individually. Selected ECHO clones, one for each of the six anti-RSV antibodies, were thawed and mixed. The mixed compositions were immediately frozen as polyclonal master cell banks (pMCB). One ampoule of the pMCB was subsequently thawed, expanded, and frozen as polyclonal working cell bank (pWCB). pWCB cells were then thawed and seed-train expanded prior to a fed batch production run in bioreactors

Fig. 5

CIEX profile of an anti-RSV rpAb composition containing six different anti-RSV specific antibodies (Ab1–Ab6). The borders of the six individual peaks are marked by the vertical lines. Integration of the area of the individual peaks is used for relative quantification of the antibodies in the rpAb

Fig. 6

Relative concentrations of the six different anti-RSV antibodies after single-batch manufacturing in fed batch mode. Three different combinations of stable ECHO clones expressing the anti-RSV rpAb were prepared and tested for compositional stability in a fed batch setup at 500 ml scale designed to simulate production at 5,000–10,000 l scale. The supernatant harvested after single-batch manufacturing was purified by protein A capture and the antibody compositions were analyzed by CIEX. The CIEX peaks were quantified by integration to obtain the relative distribution of the individual antibodies in the compositions. a Composition I, b composition II, c composition III. All runs were performed in duplicate

Fig. 7

Comparison of the relative area of the individual anti-RSV antibodies from two independent sets of fed batch production runs performed with rpAb composition I. The dark bars represent the average antibody concentration of four parallel consistency runs. The light bars represent the average antibody concentration of the duplicate runs initially performed with composition I (see Fig. 6a). Samples for CIEX profiles were taken at day 10 (right bars) and day 12 (left bars). The standard deviation is indicated as error bars