The challenges of performing high density perfusion processes in single use bioreactors

Single use technologies (SUT) have become embedded in the biopharmaceutical industry over the last 20 years. An important recent trend in the industry is a pivot towards continuous processing. SUT are being deployed for continuous perfusion cultures where expected viable cell concentrations are higher than 1 x 108 /mL. Although SUT provides many advantages, there are also some challenges associated with its application to intensified perfusion processes. In particular, the single use bioreactor has to provide enough oxygen to the culture to maintain aerobic respiration. The main challenges of adapting single use systems for perfusion culture are limited power dissipation (P/V) and limited gas flow rates. These problems arise because plastic is a weak material of construction limiting torque on the simpler shaft and back pressure on the gas outlet filters. These problems are compounded at larger scales due to unfavourable cross-sectional area to volume ratios. Lonza have characterised a number of single use bioreactor systems for their ability to achieve sufficient mass transfer to support perfusion culture. In this poster we will present results from one such system. Oxygen mass transfer was characterised for different sparger and impeller configurations over a range of power dissipations and superficial gas velocities at various levels of oxygen enrichment. A DoE approach was used to characterise the design space for oxygen mass transfer in two different bioreactor scales. Response surface models were used to quantify the contributions of different factors on the overall oxygen mass transfer. Whilst the response of oxygen mass transfer was broadly in line with expectations certain interesting observations were made. For example, an interaction between impeller type and power dissipation was observed. This caused us to look more closely at the impact of impeller design on the distribution of the gas phase. The experiments we have carried out so far enabled us to design a new approach to manipulate the oxygen transfer rate via multiple spargers. It was concluded that the single use bioreactor system under evaluation was capable of supporting between 0.4 and 1 x 108 viable cells/mL depending on the cell specific oxygen consumption rate

Challenges of mass transfer for perfusion cultures in single use bioreactors part 1:Oxygen

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Controlling pCO2 in high density perfusion cultures

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TP53 polymorphism in plasma cell myeloma

Introduction. Significant and accessible predictive factors for bortezomib treatment in plasma cell myeloma (PCM) are still lacking. TP53 codon 72 polymorphism (P72R) results in proline (P) or arginine (R) at 72 amino acid position, which causes synthesis of proteins with distinct functions. The aims of our study were to: 1) analyze whether this polymorphism is associated with an increased risk of PCM; 2) study whether the P72R polymorphism affects overall survival (OS) among PCM patients; 3) assess the possible association of the P72R polymorphism with sensitivity to bortezomib in cell cultures derived from PCM patients. Material and methods. Genomic DNA from newly diagnosed 59 patients (without IgVH gene rearrangements and TP53 deletions) and 50 healthy blood donors were analyzed by RFLP-PCR to identify TP53 polymorphism. Chromosomal aberrations were detected by use of cIg-FISH. The lymphocyte cell cultures from a subgroup of 40 PCM patients were treated with bortezomib (1, 2 and 4 nM). Results. The P allele of the P72R polymorphism was more common than the R allele in PMC patients compared to controls (39% vs. 24%), and the difference was significant (p = 0.02). The PP and PR genotypes (in combina­tion) were more frequent among cases than in controls (65% vs. 42%, OR = 2.32, p = 0.04). At the cell culture level and 2 nM bortezomib concentration the PP genotype was associated with higher necrosis rates (10.5%) compared to the PR genotype (5.7%, p = 0.006) or the RR genotype (6.3%, p = 0.02); however, no effect of genotypes was observed at bortezomib concentrations of 1 and 4 nM. The shortest OS (12 months) was observed in patients with the PP genotype compared to patients with the PR or RR genotypes (20 months) (p = 0.04). Conclusions. The results suggest that P72R polymorphisms may be associated with an increased PCM risk and may affect OS of PCM patients. However, we saw no consistent results of the polymorphism effect on apoptosis and necrosis in cell cultures derived from PCM patients. Further studies are need in this regard