The production of recombinant proteins using mammalian cell culture has become an increasingly important process in the biopharmaceutical industry. The N-1 perfusion method has emerged as a promising approach for improving protein yields and reducing production costs. The aim of this discussion is to explore the advantages and disadvantages of this method compared to traditional batch and fed-batch processes. However, this technology also presents other challenges compared to traditional bioprocessing. Therefore, we will also examine other aspects such as facility design, equipment design, and process reproducibility.

A high-seed density upstream fed-batch process was developed through the implementation of N-1 perfusion scale-down models in shake flasks and bench-scale bioreactors. A greater than 50% increase in harvest titer yields with comparable product quality was achieved as proof-of-concept for a bifunctional fusion protein using this intensified seed train process. By adding only a few additional days to the seed train duration, a significant gain in harvest titer can be achieved for increasing overall manufacturing throughput while also reducing the associated costs of goods

Scaling up a bioprocess for manufacturing is complex and the impact of cell culture parameters influence manufacturing modalities. BioSolve Process incorporating Multi-objective Bayesian Optimization is used to analyse the complex design space to help identify optimal solutions. This case study identifies optimal configurations in terms of Fed Batch, Perfusion, or Intensified Fed Batch. The outcomes of the optimisation studies identify those factors that maximise economic, and sustainable benefits.

Bleed recycling is an innovative method to enhance yield in steady-state perfusion processes by concentrating process bleed, selectively removing biomass, and recycling the liquid fraction. This saves significant product otherwise wasted. Inclined gravity settling was compared to acoustic separation as bleed recycling technologies. With similar efficiency and no negative impact on cell viability, nutrient levels, or product quality, it emerged as the preferred technology due to its reduced complexity and scalability. A 3.5-fold bleed reduction with a 19% average harvest rate increase was achieved during a 42-day perfusion process, making it an attractive option to improve process sustainability and yield.

Sieving efficiency decline caused by foulants in therapeutic biologics production using continuous manufacturing (CM) is a significant challenge. It is undesirable to pass large quantities of foulants through the filter as it may complicate the downstream purification process. Moreover, membrane fouling may lead to filter failure and eventually batch failure. Adding expensive media additives to improve sieving efficiency might affect cell culture performance, increase cost of goods and may not be as effective across clones or programs. Here, we evaluated three commercially available, commonly utilized membrane chemistries from multiple manufacturers and compared their sieving efficiency performance. 

Continuous centrifugation is commonly used as the initial clarifying stage in the recovery of biopharmaceuticals from cell culture. The benefit of a low shear environment and suitability for manufacturing scale makes the technology a great choice over other methods like depth filtration. However, lack of a proper scale-down model make the implementation of continuous centrifugation usually a try-and-error operation directly at large scale. In this study, with the intention to develop a proper scale down model, we side-by-side compared a single-use pilot-scale centrifuge to a bench-top centrifuge. Turbidity, lactate dehydrogenase (LDH), and host cell protein were all evaluated for comparison. Successful harvesting was accomplished with high yield, great filterability, and low additional cell lysis. The process will be scaled up to a stainless steel continuous centrifuge at manufacturing scale based on the outcomes of the pilot scale conditions.

In the Integrated Continuous Biomanufacturing project, CPI and its partners have produced an end-to-end continuous mAb production and purification system that demonstrates the possibilities of Advanced Process Control, where CQAs are measured in real-time and controlled in a flexible process. The system also utilises a flexible digital architecture to enable model-based control while maintaining robustness, and a novel flow-balancing architecture to greatly simplify continuous processing.

Intensified and continuous processes require fast and robust methods for in-line titer monitoring. FTIR and chemometric-based multivariate modeling are promising tools for real time titer monitoring. This presentation demonstrates an adaptive modeling strategy: the model was initially built using a calibration set of available CB samples and then updated by augmenting spiking samples of the new molecules to the calibration set to improve the model robustness.

• Lessons learned from mAb intensified and continuous process, pros and cons
• What are your opinions to adopt this technology to other modalities such as vaccines, AAV, and plasmid purification process development?
• ICH5QA (R2) addressed viral clearance for continuous manufacturing. What are current and future industrial approaches to align with regulatory requirements?
  

Processes can be intensified at all scales and at all dimensions. However, that requires implementing approaches to achieve “more, with less efforts, faster” already at the beginning. This presentation gives a comprehensive overview on strategies how to integrate process intensification through the whole product life cycle and when you switch scales and facilities. The opportunities from early development to continuous process improvements will be summarized in this talk.

Sustainability of biologics manufacturing processes is critical in ensuring the efficient production of these life-saving therapies in a resource constrained world. This presentation will provide an overview of the current state of biologics sustainability for different modalities and discuss the findings from process mass intensity (PMI) and life cycle assessments (LCA). The work highlights the need for a comprehensive metric(s) that will drive innovations in sustainability of biologics manufacturing. Future directions to assess and improve the sustainability of biologics manufacturing will be discussed. 

Affinity Chromatography operational time is directly related to affinity load volume. Implementing an ultrafiltration step to concentrate AAV Clarified Cell Lysate (CCL) prior to Affinity loading can reduce overall operational time, maintain product quality, reduce cost of goods, and simplify the manufacturing procedure. Three commercially available membranes were evaluated over a range of conditions to show proof of concept, reproducibility, scalability, maintained or improved product quality and high product recovery.

Continuous manufacturing for mAbs, involving multi-column capture, has demonstrably improved productivity. Process characterization of multi-column ProA, requires substantially large volumes of material, long run duration to achieve steady state, and operational complexity of a closed, sterile system. This talk will explore the opportunities of utilizing a single column as a scale-down model for continuous chromatography process characterization of a multi-column capture step, and share the lessons learned using this approach.

The aging of chromatography columns impacts process economics intensively, including productivity, resin utilization, and buffer consumption. Our online optimization approach employs a residence time gradient during the loading step to balance these demands. Using an extended Kalman filter and model predictive controller, the approach can forecast optimal conditions to maximize productivity and resin utilization. Results showed a savings of up to 43% in buffer consumption and increased productivity and resin utilization beyond the feasible range with classic chromatography.