We describe the development and characterization of a precipitation step using sodium caprylate for the reduction of host cell proteins in the commercial manufacturing scale purification of a monoclonal antibody.  Development and process characterization using Quality by Design principles was performed to gain understanding of process parameter impacts and robustness.  A two-step approach, in which the first step focuses on product quality and the second focuses on process performance, provided an efficient, streamlined strategy for characterization.  Data is presented to illustrate the development, characterization and resulting robust reduction of host cell proteins using sodium caprylate precipitation.

We aim to develop highly productive integrated continuous downstream processes to realize our envisioned next-generation factory concept where extremely low manufacturing cost would be achieved along with environmental friendliness. In this presentation, our future vision and recent highlighted progress will be reported: (1) next-generation factory concept and facility design status, (2) long-duration capture MCC operation connected with perfusion upstream, (3) scale-down demonstration run of integrated filtration-polishing.

The AMECRYS project is an EU-funded consortium with the ambitious goal of producing a disruptive alternative to traditional chromatography-based mAb purification, by forming mAb crystals directly from cell culture material.  This presentation will cover the background and later stages of the project, focusing on CPI’s role in developing mAb crystallization at process scale.

Full convergence of Pirani to capacitance manometer pressure was traditionally used to determine the endpoint of primary drying, which is usually inefficient due to lacking key understanding of the correlation between Pirani pressure (or moisture history) and the underlying vapor flow rate. The present study uses computational fluid dynamics (CFD) modeling to build a soft-sensor PAT tool to predict this correlation and enable safe reduction of the lyophilization process time.

Viral vector processes are still maturing. Here we study adenovirus and lentivirus. The labile nature of these products is a concern when using adsorption-based separation. Downstream processing sequences involving hollow fibre UF/DF and nanofiber based ion exchange chromatography were investigated. Minimising the time spent in the adsorbed state was shown to be beneficial in retaining high yield. While resolution is highly sensitive to the ion exchange ligand density.

Adeno-Associated Viruses (AAVs) for in vivo gene therapy are a dominant viral vector platform in the gene therapy landscape. A key challenge in viral vector manufacturing, particularly with AAVs, is effective and consistent separation of full capsids. Monolith-based ion exchange chromatography (IEX) is a promising tool to achieve this separation but poses challenges with consistency upon scale-up. This paper outlines some of the scale-up challenges and potential mitigations to achieve consistent separation of full and empty particles. IEX is evolving to become the industry standard for downstream separations as industrialization of gene therapy manufacturing continues.

In a typical monoclonal antibody process (mAb), process-related impurities (e.g., host cell proteins (HCP) and residual DNA) are well controlled due to the specificity of Protein A chromatography and other well-defined downstream unit operations. To further reduce HCP, the protein A immobilization can be exploited by exposing the bound mAb to various washing additives to disrupt the protein-protein interactions. HCP content is typically reduced from >500,000 ng/mg to <1000 ng/mg in this single-step.For non-antibody processes, the commercial use of custom affinity ligands is not typically used in early phase development. Due to significant development time and a high cost of goods of custom affinity resins, conventional chromatography techniques (CEX, HIC, and Mixed-mode) are typically employed to purify the target protein from process-related impurities. In this case study, host cell protein reduction challenges were encountered with a recombinant protein conjugate expressed in CHO cells. A baseline process using conventional chromatography and filtration techniques was rapidly developed to support Toxicology and Phase 1 production. However, HCP levels of 10,000-20,000 ng/mg were measured at the intermediate stage (pre-conjugation) of the process. Here we discuss the systematic approach to reduce HCP levels to within specification using strategies amenable to manufacturing.

Purification processes of oligonucleotides are often developed through comprehensive small-scale studies which relies highly on experimental design and interpretation of the generated data. Design of experiments (DoE) is a statistical tool that provides valuable information for study design, and screening and optimization via analyzing the impacts of individual and interactive effects of process parameters on the process performance.

In this presentation, case studies will be discussed where DoE models were used for process intensification and simplification by identifying optimum operating conditions and using model’s predictability for process outputs under various conditions.

Mersana Therapeutics is a clinical-stage biopharmaceutical company using its differentiated and proprietary ADC platforms to rapidly develop novel ADCs with optimal efficacy, safety and tolerability to meaningfully improve the lives of people fighting cancer. Its DolaLock technology delivers the proprietary auristatin-based payload utilizing state-of-the-art bioconjugation strategies that enables an enhanced therapeutic window for the treatment of multiple cancers. This presentation will provide an overview of Mersana’s novel ADC platforms and discuss the QbD-based process development, scale-up and process intensification strategies, leveraging the synergies between synthetic chemistry, small molecule and antibody production, and bioconjugation processes.

With the constant improvements to genetic engineering the production of recombinant Fabs has become a viable alternative to antibodies. We previously established a single IMAC step, generating His-Tag rFab with 97% monomer. The IMAC method, however, causes aggregation of another His-Tag rFab and reduces assay potency. The goal is to generate stable purified His-Tag rFab meeting the assay potency requirement.  Design of Experiments (DOE) is used to establish the manufacturing purification method including steps in the order of cation exchange chromatography, CH1-XL affinity chromatography, and TFF.

The promise of high flow rates offered by affinity chromatography membranes has been limited by insufficient binding capacity, inconsistency, and unproven scalability for process development and manufacturing. Data will highlight flexibility of binding capacity versus residence time and demonstrate consistent scalability at low pressure drop aligning with existing systems and providing a path to developing rapid cycle protocols for GMP clinical manufacturing

Our laboratory has developed the use of fiber-based chromatographic stationary phases that allow rapid (<10 min) separations of exosomes and lentivirus particles from cell culture media using standard laboratory HPLC instrumentation. Fibers allow isolation from culture media components and host cell proteins at >95% efficiencies on sample volumes from 10-1000 microliters. Scale-up is also anticipated.

During the early stage development of downstream processes for non-mAb therapeutics, tools available for unpurified and partially purified product are usually very limited, especially for rapid estimates of titer, purity, and yield. The lack of analytical tools greatly limits the capability of designing an excellent process that delivers both high yield and purity under constrained development timelines. Rapid process monitoring also facilitates the selection of highly orthogonal process steps that ensure that critical quality attribute requirements are met. In this presentation, two 3-minute, rapid HPLC methods were developed for their utility in designing downstream processes. The analytics described herein were optimized and employed to develop a purification process for a non-mAb related product. As an added benefit the development timeline was greatly streamlined. This strategy for purification development can be applied to other projects as well.

A significant proportion of the cost of manufacturing recombinant protein subunit vaccines is the purification which occurs in multiple chromatography steps. Crystallization has the potential to reduce purification costs and provide new product storage modality, improved operational flexibility, and reduced facility footprints. This presentation describes an automated controlled microdrop-based crystallization system for the collection of thermodynamic and kinetic data and its application to a rotavirus vaccine candidate. These results support the promise of using crystallization in the production of recombinant protein subunit vaccines.

Combining a continuous recombinant antibody precipitation with tangential flow filtration allows for a constant mass flow capture process. Deciding the conditions for the process is an important step to ensure process key parameters that has a direct impact on the purity and recovery rates. We describe what to account for when designing such a process showing a case study with different recombinant antibodies.