Control of column loading in Protein A chromatography is a crucial part of development of robust and flexible process platforms for continuous production of monoclonal antibody (mAb) products. In this paper, we propose a control system that uses near infrared spectroscopy (NIRS) flow cells to accomplish the above. Two applications have been demonstrated using a periodic counter-current continuous chromatography setup. The first application involves use of single NIR flow cell before the inlet of the loading column to measure the concentration of mAb in the harvested broth. Measurement was in real-time (every 3 s) and within ±0.05 mg/ml, significantly better than making UV-based concentration estimations. The second application involved use of an additional NIR flow cell at the outlet of the loading column to measure column breakthrough in real time. The concentration data was transferred to a Python-based monitoring and control algorithm layered over a Cadence BioSMB system. The program could successfully run a three-column periodic counter current method on the BioSMB whereas controlling loading to ensure optimal resin utilization in each loading cycle phase based on precharacterized dynamic binding capacity models, whereas maintaining periodic elutions. The system was tested with multiple perturbations in harvest concentration, modeled after deviations that could arise downstream of a perfusion cell culture system. The resultsshow that the proposed control is a spectroscopy-based process analytical technology tool that facilitates real time monitoring and control of loading in process chromatography. It is adaptable to any continuous chromatography equipment and is very well suited for implementation in a continuous mAb production train.

Affordability of biopharmaceuticals continues to be a challenge, particularly in developing economies. This has fuelled advancements in manufacturing that can offer higher productivity and better economics without sacrificing product quality in the form of an integrated continuous manufacturing platform. While platform processes for monoclonal antibodies have existed for more than a decade, development of an integrated continuous manufacturing process for bacterial proteins has received relatively scant attention. In this study, we propose an end-to-end integrated continuous downstream process (from inclusion bodies to unformulated drug substance) for a therapeutic protein expressed in Escherichia coli as inclusion body. The final process consisted of a continuous refolding in a coiled flow inverter reactor directly coupled to a three-column periodic counter-current chromatography for capture of the product followed by a three-column con-current chromatography for polishing. The continuous bioprocessing train was run uninterrupted for 26 h to demonstrate its capability and the resulting output was analyzed for the various critical quality attributes, namely product purity (>99%), high molecular weight impurities (<0.5%), host cell proteins (<100 ppm), and host cell DNA (<10 ppb). All attributes were found to be consistent over the period of operation. The developed assembly offers smaller facility footprint, higher productivity, fewer hold steps, and significantly higher equipment and resin utilization. The complexities of process integration in the context of continuous processing have been highlighted. We hope that the study presented here will promote development of highly efficient, universal, end-to-end, fully continuous platforms for manufacturing of biotherapeutics. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:998-1009, 2017.

Process analytical technology is gaining interest in the biopharmaceutical industry as a means to enable consistency in processing and thereby in product quality via process control. Protein refolding is known to be significantly impacted by critical process parameters and feed material attributes including composition and pH of the solubilisation and refolding buffers. Hence, to achieve robust process control and product quality, these attributes and parameters need to be monitored. This paper presents an approach towards statistical process control and monitoring of protein refolding, from buffer preparation to refold quenching, during manufacturing of therapeutic proteins from Escherichia coli based systems. The proposed approach utilises measurements of online redox potential, temperature, and pH for development of a statistical model. The model has then been integrated with LabView to permit real‐time monitoring of the refolding process. The proposed system has been demonstrated to successfully identify process deviations and thereby enable process control for manufacturing product of consistent quality.

Dead end filtration is a critical unit operation that is used for primary and secondary clarification during manufacturing of both microbial and mammalian cell based biotherapeutics. Dead end filtration is conventionally done in batch mode and requires filter pre-sizing using extensive scouting studies, along with filter over-sizing before deployment to handle potential variability. However, continuous manufacturing processes require consistent use of dead-end filtration over weeks or months, with potential unpredictable variations in feed stream attributes, which is a challenge currently facing the industry. In this work, a dead-end filtration skid is designed for continuous depth filtration, incorporating multiple small-sized filters along with turbidity, and pressure sensors with immediate switching to a fresh filter whenever turbidity or pressure breakthrough above a predetermined cutoff is detected in real time. The skid has been successfully tested for manufacturing of granulocyte colony stimulating factor from Escherichia coli, human serum albumin from Pichia pastoris, and a monoclonal antibody therapeutic from CHO cells. The proposed skid can be directly applied for any dead-end filtration application with minimal prior scouting studies or sizing calculations for scale-up. It is a useful solution for continuous processing trains where the nature of the feed, such as its turbidity or host cell proteins content, may change over long continuous campaigns, rendering previous sizing calculations inaccurate. The skid also allows significant cost savings by eliminating the sizing safety factor of 1.5-2x which is generally added before filter deployment at manufacturing scale.

The developed process is able to produce purified rLTNF with 78±2% recovery. The study shows that recombinant technology can be used to produce rLTNF cost effectively and shows potential as a substitute for currently available antivenoms against snakebite.