Anuj Goel

Influence of cell specific productivity on product quality.

The micro heterogeneity or quality of a protein has been shown to have a significant impact on its physical, chemical and biological properties both in vitro and in vivo [1]. Micro heterogeneity is evaluated in terms of post translational modifications such as glycosylation, charge variants, aggregates and fragments profile. The biggest challenge in process development is to find a balance between increasing productivity while maintaining product quality. In our study we focus on protein glycosylation, which is a process in which oligosaccharides are added to the protein during synthesis. There are multiple possible reactions in the pathway and it takes a long time for a glycosylated protein to be fully processed. If some protein molecules have a shorter residence time in the ER and Golgi, the glycan may be only at an intermediate stage [1]. For recombinant glycoprotein, increase in cell specific productivity (amount of product produced per cell per unit time) which may result in shorter residence time in the ER and Golgi, must be weighed against possible changes in product quality attributes like glycosylation [2]. Our study concludes that it is possible to produce a protein with desired product quality profile with high specific productivity. Two different clones with the same productivity can have different product quality profiles; alternatively, the same clone with different specific productivity can be manipulated to produce the same desired product quality by altering the cell culture parameters or addition of supplements. This observation also influences the acknowledged methodology for selecting clones with higher productivity while still maintaining their product quality profile. Various process manipulations were evaluated as an attempt to improve on the product quality profiles without compromising the productivity.

Applications of biomass probe in PAT

In the current study, effective use of biomass probe was demonstrated in applications ranging from direct measurement of VCC to indirect applications during perfusion. The probe can be used for these and similar applications as an effective PAT tool to improve process consistency and robustness.

Platform process will give platform product - Can we afford it?

Case 1: The use of platform process enabled accelerated PD from cell culture perspective. However, accommodating the specific PQ requirements resulted in extended process development, affecting timelines. Case 2: Change in clone selection criteria was observed to significantly impact culture performance while applying platform process. This almost resulted in rejection of these clones, thus extending PD timelines. This was prevented by modifying the platform process. Case 3: Clones developed using different cloning technologies when run with the platform process resulted in different cell culture and PQ profiles. Therefore, the type of cloning technique forms an integral part of the platform process. Though platform process was not suitable in most of the cases discussed here, it still offers advantages like expedited project timelines and established work flows. These benefits were achieved by establishing four versions of the platform process to meet the varied cell culture and PQ requirements. Based on the cell line characteristics and target PQ profiles, the appropriate version is chosen to initiate PD. These versions retained the major advantages of the platform process such as having common media and feeds with only changes in their concentrations and set point of main process parameters to achieve desired PQ.

Are Clones Really Unstable

Selection of a stable clone is one of the most essential criteria for the successful production of any therapeutic protein. The stability of the clone needs to be evaluated in terms of various parameters like cell growth, specific cell productivity (PCD) and product characteristics with increasing generations. To generate sufficient amount of inoculum for the production bioreactor, cells from cell bank are periodically sub-cultured which in turn increases the generation number. Thus the clone needs to be stable for multiple generation numbers. In this study a stability programme has been designed which is based on the use of a scale down model of inoculum generation and the manufacturing process. The stability of the clones also showed a correlation with the diameter of the cells during sequential passaging. It was shown that choice of appropriate medium used for cell passaging make these clones stable.

Manipulation of a Perfusion Process by Medium Optimization

A NS0 cell line was used for the production of monoclonal antibody using a perfusion process based on spin filter technology. The process involves a batch phase followed by perfusion for building up the cell counts. The duration of the perfusion phase is usually restricted by the clogging of the spin filter. Perfusion rate was found to be a very important factor influencing the performance of the spin filter. Higher perfusion rates resulted in higher cell loss from the bioreactor and early clogging of the filter. The loss of cells also resulted in switching of the cells to growth phase which lowered the cell productivity. This observation was confirmed by using other perfusion devices like hollow fiber cartridges and perfusion wave bags which allow complete cell retention and hence increase cell productivity. Medium screening program resulted in finding a medium which gave more than double the cell count with half the perfusion rate of the original process. A further improvement by the spent medium analysis with supplementation of deficient medium components and perfusion rates optimization also resulted in a significant increase in IgG titre. With the new process there was a three-fold increase in the total product per day from the bioreactor and from the increase in cell count.

Challenges in scaling up a perfusion process

Development and scale-up of a perfusion process has challenges due to the complex nature of the process and unavailability of direct scale-up of the perfusion equipment. The initial scale-up to production scale resulted in poor cell growth profile. Upon investigation, the reason for low cell concentration was attributed to poor cell retention by the perfusion device. Changes were introduced in the type of mesh used for filter construction and perfusion pumps to improve retention. These modifications helped in better cell culture profiles and yields. However, the product quality was impacted because of these changes. Further changes in the perfusion flow rates were done to address the product quality differences.