Speed to regtox and clinical trials has become a common paradigm in the pharmaceutical industry. Pfizer’s targeted integration CHO host provides the platform for developing robust cell lines on accelerated timelines. The platform utilizes an engineered dual-landing pad and multiple-copy vectors to develop cell lines with predictable and stable genotype, growth characteristics, and production of standard mAbs, multi-specifics, and fusion proteins, which is critical to enable acceleration.

Proteomic methods and Sanger sequencing have been used in cell line development for over a decade, but the advent of next-generation sequencing (NGS) has revolutionized the screening and characterization of cell lines for biologics production. IGM Biosciences uses NGS to identify sequence variations and evaluate cell line genotypes relating to their growth and productivity. We will discuss the benefits of utilizing these sequencing technologies when selecting lead clones.

Process intensification, including implementation of highly concentrated media, is widely pursued to improve process yield and reduce footprint for monoclonal antibody production. Media preparation parameters contribute significantly to media quality, cell culture performance, and productivity. Thus, it is important to understand and develop proper media preparation procedure for robust CHO cell culture process. Here we evaluated the key parameters on media stability by cell culture and different process analytical technologies.

This talk will give an overview of the development of a next-generation upstream manufacturing process for a CHO-derived therapeutic protein to significantly improve yield, reduce cost of goods, and ultimately serve more patients with rare diseases. Newly accessible AstraZeneca proprietary media and feeds were leveraged to achieve this goal, with modifications to the platform feeding strategy leading to an increase in titer of ~33%.

A non-platform CHO process using proprietary commercial medium was internalized, and initial results in our internal platform process showed a 3-fold decrease in productivity. Using quality-by-design principles, we identified key medium components and process parameters that significantly increased cell-specific productivity, and after optimization a 5-fold increase in titer was achieved in an internal medium platform. The final optimized process was scaled from AMBR250 and benchtop bioreactors to pilot-scale single-use bioreactors to demonstrate process and scale-up robustness.

This case-study will address the methodologies for cell culture optimization to generate a process-specific HCP reagent, recommended for late-stage product characterization. A combination of screening DoE and OFAT experimentation was implemented to identify optimal process conditions in the ambr15 miniature bioreactor system. These conditions were then implemented in a 2 L single-use bioreactor system to show comparable growth attributes and HCP production to that of the original producer cell line.

Developing cellular models of disease have the potential to facilitate and/or accelerate the drug discovery and development. At REGENXBIO, we are evaluating the use of genome engineering and cell line development approaches, to generate mammalian disease cell line models that have utility in the screening of drug candidates. These models should facilitate the robust advancement of gene therapy products from the lab bench to the clinic.

Manufacturing cell lines can benefit from host cell engineering to improve performance and product quality. MAD7 is an engineered class 2 type V-A CRISPR-Cas nuclease (Cas12a/Cpf1) shown to function in a variety of systems, recently including CHO cells. MAD7 can successfully facilitate site-specific indels and knock-ins in CHO, making it a useful tool for engineering in cell line development.

CHO cell lines have significant phenotypic variability though derived from a single cell progenitor. This variability may lead to or be indicative of the propensity of a cell line to exhibit production instability over time. Here we characterize RNA expression from stable and unstable cell lines using single-cell RNA sequencing. Our work shows that clonally derived cell lines are complex metapopulations with distinct subpopulations that change differently with time. We explore how this changing metapopulation may influence production stability and search for stability signals in those subpopulations that drive changes within the metapopulation.

• ​How relevant are isogenic cell lines in drug discovery and development today? 
• What are the factors to consider in choosing a cell line for isogenic cell line development? 
• What are some strategies to evaluate long term stability of clones?  
• What new technologies can we employ to predict clone stability?
  

• Tackling novel molecular formats
• Aligning clone screening with next generation manufacturing platform
• Advancement in single cell cloning​
  

Synthetic biology based on the “Design-Build-Test-Learn” cycle offers a new paradigm for genetic vector design, where it is possible to engineer a host cell factory in a product or cell type specific manner via combinatorial tuning of discrete cellular synthetic processes. This approach permits “one-size fits-all” genetic parts and vectorology to be replaced with tailored design and construction of specifically fit-for purpose genetic systems. Utilizing a platform of genome-scale mining and informatic tools, we generate libraries of engineered/synthetic parts with user-defined functionality that can boost biologic manufacturability and specificity via context-specific control of primary cellular processes.

The insect cell lines widely used as hosts in the baculovirus-insect cell system are contaminated with adventitous viruses. We assessed the infectivity of Sf-rhabdoviruses for mammalian cell lines and immunocompromised mice to determine their impact on the biosafety profile of this biologics manufacturing platform.

Developing high-yielding, scalable and commercially viable processes for AAV manufacture, with associated analytical methods for assessing the CQAs, presents challenges. Here we present work being performed at CPI to address some of the upstream challenges, including development of an intensified N-1 seed train step to reduce inoculum requirements. Further, we discuss some of the approaches we have been working on for full and empty capsid determination, including mass photometry.

To improve AAV transient titers in HEK293 system, REGENXBIO had made significant efforts in host cell line development and engineering. In addition, we generated new plasmid expression systems by several rounds of plasmid optimization. Combining the new cell lines and plasmids, we increased our overall transient yield significantly while improving the product quality.

Regulatory approvals of Sf9 cell-derived rAAV products, Glybera, Hemgenix, and Roctavian, demonstrated a higher success rate than other cell types. However, there are two major issues with using Sf9 cells: 1) inconsistency in rAAV production with over passages; 2) persistent carrying Sf rhabdovirus (Sf-RhV) which is noninfectious to human or mammalian cells, may have a potential risk of inducing inflammation or immunogenicity using an Sf-RhV containing Sf9 cell line for producing biologicals. To this end, we developed several Sf9 clones free of Sf-RhV, consistently producing rAAV of all tested serotypes with high yield and activity. 

Using a CDMO for GMP cell banking is mainstream in the biopharmaceutical industry. There are potential challenges to successfully manage the tech transfer process. Key factors to generate a successful GMP cell bank include 1) sourcing appropriate raw materials, 2) identifying the correct equipment and its setting, and 3) defining growth parameters, freezing condition. Application of a systematic approach can help ensure the successful generation of a GMP cell bank.

Advantageous in its high throughput and process automation degree, the AMBR250 platform is gaining industrial adaptation. Here we report a successful ‘fit-for-purpose’ AMBR250 scale-down model case-study for a vaccine bioprocess. This gas-transfer-guided model development not only demonstrates representative process attributes, but also produced statistically equivalent productivity of target product, enabling a faster process evaluation platform to match an accelerated project timeline.