The early specification of bioprocesses often has to be achieved with small (tens of millilitres) quantities of process material. If extensive process discovery is to be avoided at pilot or industrial scale, it is necessary that scale-down methods be created that not only examine the conditions of process stages but also allows production of realistic output streams (i.e., streams truly representative of the large scale). These output streams can then be used in the development of subsequent purification operations. The traditional approach to predicting filtration operations is via a bench-scale pressure filter using constant pressure tests to examine the effect of pressure on the filtrate flux rate and filter cake dewatering. Interpretation of the results into cake resistance at unit applied pressure (alpha) and compressibility (n) is used to predict the pressure profile required to maintain the filtrate flux rate at a constant predetermined value. This article reports on the operation of a continuous mode laboratory filter in such a way as to prepare filter cakes and filtrate similar to what may be achieved at the industrial scale. Analysis of the filtration rate profile indicated the filter cake to have changing properties (compressibility) with time. Using the insight gained from the new scale-down methodology gave predictions of the flux profile in a pilot-scale candle filter superior to those obtained from the traditional batch filter used for laboratory development.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is defective in cystic fibrosis. The most common mutation, DeltaF508 CFTR, is retained in the endoplasmic reticulum, retrotranslocated into the cytosol, and degraded by the proteasome. In a proteomics screen to identify DeltaF508 CFTR interacting proteins, we found that valosin-containing protein (VCP)/p97, a Type II AAA ATPase that is a component of the retrotranslocation machinery, binds DeltaF508 CFTR, and this interaction is stabilized by proteasomal inhibition. Since wild-type (WT) CFTR has been reported to be inefficiently processed during biogenesis with as much as 75% of the newly synthesized protein degraded by the proteasome, we examined the VCP interaction in Calu-3, T-84, and 16HBE, three epithelial cell lines that endogenously express WT CFTR. The results indicate that when WT CFTR processing is efficient, as demonstrated in Calu-3 cells, VCP does not interact. Interestingly, overexpression of recombinant WT CFTR in Calu-3 cells results in inefficient processing and VCP interaction, demonstrating that CFTR processing efficiency and the VCP interaction are tightly coupled. Furthermore, induction of ER stress and activation of the unfolded protein response result in inefficient processing of WT CFTR in Calu-3 cells and promote the WT CFTR-VCP interaction. The results support the hypothesis that components of the retrotranslocation machinery such as VCP do not interact with CFTR in epithelial cells that endogenously express WT CFTR, since under normal conditions the processing of the WT protein is efficient.

No abstract

Viral vectors are a new class of biologics which facilitate gene transfer and modification in living cells, potentially treating a multitude of conditions with genetic causes. Scalable manufacturing technologies are critical to ensuring these cutting-edge medicines can be produced in sufficient quantities to meet the needs of process development, clinical trials, and ultimately commercial manufacturing [1]. As viral vector-based products have only relatively recently received regulatory approval, public information on scalable optimization of these processes is very limited. Abeona Therapeutics is a gene therapy company developing novel gene replacement therapies for rare inherited diseases. These conditions can impact development and limit both quality of life and/or life expectancy [2]. These transformative medicines can be used to replace a defective gene with a functional copy, silence a defective gene or even directly edit genes [3,4]. We evaluated the Pall Allegro™ STR bioreactor family as an rAAV vector production platform and evaluated the scalability of the PEI-mediated transfection manufacturing process for rAAV at the 50 L and 500 L working volume. Process scalability was evaluated based on cell growth, metabolic profile, and vector production. This testing demonstrates that control of key process parameters enables a scalable vector production process between the 50 L and 500 L scale using Allegro STR single use bioreactors.

No abstract

No abstract

No abstract

No abstract