Abstract
The presence of leachables in biopharmaceutical processes using single-use technologies (SUT) is well known. For the detection and quantification of the latter, extractable studies of SUT are very common nowadays. Although a mixture of compounds is regularly found in extractable studies, research has only been carried out regarding the effect of individual compounds on cell culture and the cumulative effect of a mix of leachables has not been investigated yet. In this study, a set of leachable model compounds (LMCs) was chosen and the effect of the LMCs on a Chinese hamster ovary DG44 cell line producing an IgG antibody was investigated concerning cell growth, cell cycle distribution and productivity. It was shown that even if worst-case concentrations were used, the LMCs solely impact cell growth. Additionally, interaction studies revealed that the inhibiting effect of the mix is lower than the expected cumulative effect. A strong antagonism between the antioxidant butylated hydroxytoluene and the plasticizer Tris(2-ethylhexyl)trimellitate was found using an isobologram analysis.
Highlights
The fast growing market of biopharmaceuticals has increased the pressure to develop cost-effective and flexible production processes
In order to classify the toxicity of the leachable model compounds (LMCs)-mix compared to the individual compounds, Chinese hamster ovary (CHO) DG44 cultures were spiked with bDtBPP, acetophenone, Butylated Hydroxytoluene (BHT) and TOTM each, in the respective concentrations of the mix (Figure 1, bottom)
The concentrations of the leachables in LMC-mix 3, which were found in extractables and leachables studies (Table 1), cause a decreased cell growth (71%), while the viability remains above 97%
Summary
The fast growing market of biopharmaceuticals has increased the pressure to develop cost-effective and flexible production processes. We have examined the impact of a set of leachables on an IgG production process that potentially migrate out of different SU materials. Thereafter, a fed-batch cultivation was performed in shake flasks and the effect of the LMC-mix on cell growth, viability and productivity was examined. A subset of leachables was chosen as model compounds that represent typical substances commonly detected in extractable analysis of polymeric material and are commercially available. These selected LMCs (Table 1) are different in terms of chemical characteristics (i.e., molecule size and lipophilicity expressed as the log of the partition in an octanol/water system, log KO/W). The cell-specific productivity qP was calculated according to Equation (1), with Δc being the concentration difference between two time points and the IVCC the integral viable cell concentration between these two time points.[29]
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