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In this study, eight commercially available, chemically defined Chinese hamster ovary (CHO) cell culture media from different vendors were evaluated in batch culture using an IgG-producing CHO DG44 cell line as a model. Medium adaptation revealed that the occurrence of even small aggregates might be a good indicator of cell growth performance in subsequent high cell density cultures. Batch experiments confirmed that the culture medium has a significant impact on bioprocess performance, but high amino acid concentrations alone were not sufficient to ensure superior cell growth and high antibody production. However, some key amino acids that were limiting in most media could be identified. Unbalanced glucose and amino acids led to high cell-specific lactate and ammonium production rates. In some media, persistently high glucose concentrations probably induced the suppression of respiration and oxidative phosphorylation, known as Crabtree effect, which resulted in high cell-specific glycolysis rates along with a continuous and high lactate production. In additional experiments, two of the eight basal media were supplemented with feeds from two different manufacturers in six combinations, in order to understand the combined impact of media and feeds on cell metabolism in a CHO fed-batch process. Cell growth, nutrient consumption and metabolite production rates, antibody production, and IgG quality were evaluated in detail. Concentrated feed supplements boosted cell concentrations almost threefold and antibody titers up to sevenfold. Depending on the fed-batch strategy, fourfold higher peak cell concentrations and eightfold increased IgG titers (up to 5.8 g/L) were achieved. The glycolytic flux was remarkably similar among the fed-batches; however, substantially different specific lactate production rates were observed in the different media and feed combinations. Further analysis revealed that in addition to the feed additives, the basal medium can make a considerable contribution to the ammonium metabolism of the cells. The glycosylation of the recombinant antibody was influenced by the selection of basal medium and feeds. Differences of up to 50 % in the monogalacto-fucosylated (G1F) and high mannose fraction of the IgG were observed.

Background and aims Anti-idiotypic antibodies (Abs) represent an alternative vaccination approach in human therapy. This approach is based on the idiotype (Id) network theory postulated by Jerne describing the Ab (Ab1) – anti-idiotypic Ab (Ab2) – anti-anti-idiotypic Ab (Ab3) cascade stimulation. Specific anti-Id Abs serve as an “internal image” of the target antigen and can be used to induce Abs able to bind to the cognate antigen [1]. The anti-Id Ab Ab2/ 3H6 [2], developed at our Institute was generated in mouse and is directed against the human monoclonal Ab (mAb) 2F5, which broadly and potently neutralizes primary HIV-1 isolates [3]. Ab2/3H6 which has been characterized previously [4,5] is able to mimic the antigen recognition site of 2F5 and therefore it is suggested as a putative candidate for an HIV-1 vaccine. We investigated the potential of Ab2/3H6 by immunization of Fab fragments and fusion proteins with interleukin 15 (IL15) and tetanus toxin (TT) tags as immune modulators. After three prime/boost administrations rabbit sera were purified and analyzed for 2F5-like specific Abs. Further, the 2F5-like Abs from the sera were enriched by affinity purification and characterized for their binding affinity to 2F5. In an additional approach we applied different humanization methods to reduce the immunogenicity of the originally mouse derived Ab2/3H6. The mouse variable regions of Ab2/3H6 were subjected to three different humanization methods, namely resurfacing, CDR-grafting and superhumanization. Four differently humanized Ab2/3H6 variants were characterized for their binding affinity to 2F5 in comparison to the original Ab2/3H6. Results To evaluate the humoral immune response of Ab2/3H6 we designed Ab2/3H6 fusion proteins with IL15 and TT. Recombinant CHO cell lines were established and after protein purification New Zealand white rabbits were immunized with the Ab2/3H6 variants. Ten days after the final boost sera were collected and analyzed for total rabbit IgG levels. After proteinA affinity purification of the sera the isolated rabbit IgGs were tested for Ab2/3H6 and recombinant gp140 (UG37) specificity (Figure 1A). Further an affinity enrichment step using a UG37/ELDKWA column was performed and the obtained Ab3 fraction was tested on UG37 (Figure 1B) and additionally on the original 2F5 epitope ELDKWA (Figure 1C). Finally the Ab3 fraction was tested for binding affinity to the UG37 in a bio-layer interferometry assay which showed that the Ab3 fraction has a 6.6 fold reduced affinity towards UG37 compared to the mAb 2F5 (Table 1). For the humanization approach three different methods were chosen. The “resurfaced” variant (RS3H6) was developed by a computer model and surface exposed amino acids in the murine framework (FR) were substituted by residues usually found at equivalent positions in human Abs. The “superhumanized” form (SH3H6) was designed by structural homologies between the murine Ab2/3H6 CDRs and human germline CDRs. The most homologous human germline Ab was then used as acceptor FR. For the “CDR-grafted” variant two versions were expressed. An “aggressive” graft (GA3H6) harbouring less backmutations, making the grafted Ab more human-like and a “conservative” graft (GC3H6) with more backmutations. The different “reshaped” variable regions of Ab2/3H6 were expressed in CHO-cells as IgG1 molecules. The obtained “humanized” Ab variants were further characterized by competition ELISA (Figure 1D). * Correspondence: renate.kunert@boku.ac.at Department of Biotechnology, Institute for Applied Microbiology, BOKU – University of Natural Resources and Life Sciences, A-1190 Vienna, Austria Kunert and Mader BMC Proceedings 2011, 5(Suppl 8):P64 http://www.biomedcentral.com/1753-6561/5/S8/P64

Address: 1AIDS/Viral Immunology, Torrey Pines Institute, San Diego, CA, USA, 2MonoGramBio, San Francisco, CA, USA, 3Simon Fraser University, Vancouver, Canada, 4Los Alamos National Laboratory, Los Alamos, NM, USA, 5National Institute for Communicable Diseases, Johannesburg, South Africa, 6Beth Israel Medical Center, Boston, MA, USA, 7University of Agricultural Sciences, Vienna, Austria, 8University of California, San Diego, La Jolla, CA, USA, 9HIV Bioinformatic Center, Bangkok, Thailand and 10The Scripps Research Institute, La Jolla, CA, USA * Corresponding author