Continuous chromatography beyond affinity capture of monoclonal antibodies

The focus on process intensification and increased process control continues in the biopharmaceutical industry. The key driver is to reduce production costs, while maintaining product quality and throughput in the manufacturing of biopharmaceuticals. The introduction of continuous processing technologies has supported the industry in evaluating different approaches for continuous and/or hybrid solutions for up- and downstream processing. Continuous chromatography has the potential to increase chromatography resin capacity utilization, eliminate or minimize the need for intermediate hold-up steps, reduce equipment footprint and buffer consumption as well as introducing a higher degree of automation. The benefits from this can in turn have a positive impact on the process economy. The efforts in continuous chromatography in the industry so far have mainly been focusing on affinity capture of monoclonal antibodies (mAb) but the interest in exploring other applications is now increasing. In this poster, we will show the usage of periodic counter-current chromatography (PCC) in and beyond affinity chromatography mAb capture applications, for example in purification processes for viral vectors as well as plasma proteins. We will show examples of flow through applications, ion exchange- and size exclusion chromatography in a continuous mode

Efficient approaches for perfusion medium development

Here, we present a fast and convenient strategy for developing a high-cell density perfusion process for antibody-producing Chinese hamster ovary (CHO) cells based on the commercially available ActiCHO™ Media System. ActiCHO P base medium was used as a starting point and ActiCHO Feed-A and Feed-B were added in various concentrations as supplements. The resulting perfusion medium prototypes were first evaluated in batch cultures, applying a design of experiment (DoE) strategy (Figure 1), and then tested in small-scale perfusion cultures in rocking single-use WAVE bioreactor™ systems (Figure 2). The medium optimization resulted in a final process with a cell-specific perfusion rate (CSPR) of less than 50 pL/cell/d, which is a more than 45% decrease compared with the starting process conditions. The performance of the perfusion process was further validated in lab-scale single-use stirred-tank bioreactor systems. Productivity and product quality of the perfusion process were compared with a standard fed-batch culture process

Functional Significance of Multiple Poly(A) Polymerases (PAPs)

3’ end cleavage and polyadenylation are important steps in the maturation of eukaryotic mRNAs. Poly(A) polymerase (PAP), the enzyme catalysing the addition of adenosine residues, exists in multiple isoforms. In this study the functional significance of multiple poly(A) polymerases have been investigated. It is concluded (i) that at least three mechanisms generate the multiple isoforms i.e. gene duplication, post-translational modification and alternative mRNA processing and (ii) that the different isoforms of poly(A) polymerases have different catalytic properties. The study highlights regulation of poly(A) polymerase activity through modulation of its affinity for the substrate as visualised by the KM parameter. We suggest that trans-acting factors modulating the KM of poly(A) polymerase will play important roles in regulating its activity. A new human poly(A) polymerase (PAPγ) encoded by the PAPOLG gene was identified. PAPγ is 65% homologous to the previously identified PAP. In human cells three isoforms of poly(A) polymerases being 90, 100 and 106 kDa in sizes are present. These native isoforms were purified. The PAPOLA gene encoded the 100 and 106 kDa isoforms while the 90 kDa isoform was encoded by the PAPOLG gene. Native PAPγ was found to be more active than 100 kDa PAP while the hyperphosphorylated 106 kDa PAP isoform was comparably inactive due to a 500-fold decrease in affinity for the RNA substrate. The PAPOLG gene was shown to encode one unique mRNA while the PAPOLA gene generated five different PAP mRNAs by alternative splicing of the last three exons. The PAPOLA encoded mRNAs were divided into two classes based on the composition of the last three exons. Poly(A) polymerases from the two classes were shown to differ in polyadenylation activities. These differences revealed two novel regulatory motifs in the extreme C-terminal end of PAP, one being inactivating and the other activating for polyadenylation activity