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A bench scale cell culture model representative of manufacturing scale (2,000 L) was developed based on oxygen mass transfer principles, for a CHO-based process producing a recombinant human protein. Cell culture performance differences across scales are characterized most often by sub-optimal performance in manufacturing scale bioreactors. By contrast in this study, reduced growth rates were observed at bench scale during the initial model development. Bioreactor models based on power per unit volume (P/V), volumetric mass transfer coefficient (kL a), and oxygen transfer rate (OTR) were evaluated to address this scale performance difference. Lower viable cell densities observed for the P/V model were attributed to higher sparge rates and reduced oxygen mass transfer efficiency (kL a) of the small scale hole spargers. Increasing the sparger kL a by decreasing the pore size resulted in a further decrease in growth at bench scale. Due to sensitivity of the cell line to gas sparge rate and bubble size that was revealed by the P/V and kL a models, an OTR model based on oxygen enrichment and increased P/V was selected that generated endpoint sparge rates representative of 2,000 L scale. This final bench scale model generated similar growth rates as manufacturing. In order to take into account other routinely monitored process parameters besides growth, a multivariate statistical approach was applied to demonstrate validity of the small scale model. After the model was selected based on univariate and multivariate analysis, product quality was generated and verified to fall within the 95% confidence limit of the multivariate model.

Interactions between vascular endothelial cells and inflammatory leukocytes are intermediated via cell adhesion molecules and they become one of the key events for vascular cell injury and development of atherosclerosis. This study evaluated the effects of MTX-peptide conjugates as anti-inflammatory agents on human coronary artery endothelial cells (HCAEC) and Molt-3 T cells. Cyclic peptides, cLABL and cLBEL, were derived from the a- and b-subunits of leukocyte function-associated antigen-1 (LFA-1), respectively. They interact with intercellular adhesion molecule-1 (ICAM-1) to inhibit LFA-1/ICAM-1-mediated homotypic or heterotypic T-cell adhesion. cLABL and cLBEL were linked to the anti-inflammatory drug, methotrexate (MTX), to produce MTX-cLABL and MTX-cLBEL conjugates. This study showed that peptides and MTX-peptide conjugates inhibited T cell adhesion to HCAEC monolayers while MTX alone did not. The conjugates, but not MTX, inhibited binding of anti-ICAM-1 monoclonal antibody (mAb) to ICAM-1 on the HCAEC. This indicates that conjugation of MTX to cLABL and cLBEL peptides did not dramatically change their binding properties to ICAM-1. The conjugates had relatively lower toxicity to cells compared to MTX alone, while they were more toxic than the parent peptides. At low concentrations, MTX, MTX-cLABL and MTX-cLBEL decreased production of IL-6 and IL-8 as inflammatory cytokines. In contrast, higher concentrations of the parent peptides compared to the conjugates were required to inhibit IL-6 and IL-8 productions. Overall, both MTX-cLABL and MTX-cLBEL were more active than both free-peptides. In addition, the conjugates were less toxic than MTX alone. In conclusion, the conjugate can selectively target MTX to ICAM-1-expressing cells to increase cell targeting and to lower MTX toxicity.