BioProcessing 2019

Development of novel types of therapeutics require adaptation of traditional bioprocessing applications to include the use of primary cells, such as stem cells, fibroblasts, astrocytes and neurons. Culture of these cells requires significant optimization at the level of assay development and scalability. Detection of changes in cell viability can have a major impact on yield and cost efficiency of bioprocessing applications. Therefore, the use of cell viability assays during early assay development is critical for scalability and implementation success.  In this presentation, I will discuss the development of a custom lactate dehydrogenase assay for the use in both cell lines and primary cells and highlight its use for optimization of bioprocessing applications. 

Learning Objectives:

1. Critically evaluate the need for cell viability assays in the development of bioprocessing applications 
2. Design and troubleshoot cell viability assays for culture of both cell lines and primary cells

Multi-mode chromatography has come to prominence in recent years due to the general utility of multi-mode ligands for product capture and polishing. In particular, their application in flow-through polishing has significantly improved process economy. However, significant optimisation of binding and elution conditions is often required to achieve the desired level of purity.

To overcome the deficiencies of individual multi-mode ligands, we have developed a diverse library of Multi-Mode Mimetic Ligands™  incorporating both ionic and hydrophobic groups, which offer significant flexibility in use. The Multi-Mode Mimetic Ligand™ Library is organised into a 12 x 8 array micro-column block and comprises a diverse array of ligands with a broad spread of charge, hydrophobic and hydrophilic functionalities. The adsorbents are produced using an agarose support matrix and incorporate triazine attachment chemistry for ease of synthesis and superior stability, Screening can be performed in high-throughput mode using robotic liquid handling systems or by use of conventional hand-held multi-channel pipettes. This approach allows for rapid screening, and identification of potential ligands for a specific target. Furthermore, adjustment of the starting protein fraction to suit individual ligands is not required as the diversity of the library enables the identification of binding ligands which are best-suited to the existing conditions and impurities.

This approach has been evaluated in a variety of applications, including development of ligands for the removal of specific contaminants in a flow through mode.

Learning Objectives: 

1. Understand how you can use a diverse library of synthetic ligands to screen for adsorbents for your molecules that are difficult to purify using standard adsorbents. The screening library is fast becoming a must-have technology in the tool-box of a process development scientist.
2. A case study of using the Multi-Mode Mimetic Ligand™ Library to deliver a flow-through polishing step to remove FXI from human blood plasma.

The biologics development process is fraught with risks from pre-clinical studies through clinical evaluation. Prominent among these risks are changes in the critical quality attributes of the biologic during the drug development process.  Here, we present data on the use of the ExpiCHO expression system for high titer transient expression of candidate therapeutics through creation of stable cell lines for large scale manufacturing.  By using the same cell line from transient to stable production, the risk of product quality drift can be reduced during the transition from pre-clinical to clinical development.

Learning Objectives: 

1. Learn how the ExpiCHO system can improve your processes
2. How to lower risks during the biologics development process

Prodrugs are harmless in their native state, as they are not targeted by human enzymes. But they can be converted into highly toxic compounds (the “drug”) by viral or bacterial enzymes. Thus, provided that the delivery of the gene encoding the activating enzyme is confined to the cancer, DDEPTs hold the promise of avoiding the general and severe toxicity of conventional chemotherapeutic approaches, most of which lack specificity and besides killing the cancer cells, also damage normal tissues and organs. The prodrug regimen Herpes simplex virus type 1 (TK1) thymidine kinase and ganciclovir (HSV-tk/GCV) was tested in phase III clinical trial; the TK1 gene from a retroviral vector was injected by craniotomy. No benefits resulted. The reasons included low-level and short-lived gene expression, lack of enzyme potency, insufficient attention to generate a strong bystander effect; also, there are concerns about the use of viruses: immune recognition, insertional mutagenesis, and inflammatory toxicity. Similar reasons account for the failure to proceed beyond phase I/II stages of clinical trials of CB1954 (tretazicar) + Escherichia coli nitroreductase (NTR) GDEPT for treating prostate cancer – the NTR encoding adenovirus was injected directly into the localized cancer.  Our GDEPT employs a new prodrug, CNOB, and a new bacterial enzyme, which we discovered, improved and humanized (“HChrR6”). This enzyme converts CNOB into the drug MCHB. The latter is not only highly lethal, it is strongly fluorescent and can be visualized in living mice. This greatly helped in ensuring that the activation of CNOB was confined to orthotopic implanted HER2 xenografts in mice. We utilized extracellular vesicles (EVs) as vehicles for gene delivery, which, being nature’s own antigen delivery system, circumvent the issues of viral use. Further, we utilized mRNA for gene delivery instead of DNA used in the above trials; mRNA is superior to DNA for gene delivery and for rapid expression. We also quantified the degree of transfection with the gene (as mRNA) of tumor cells for an effective BE. mRNA was loaded in HEK293 generated EVs by a novel plasmid that we generated; this was the first time that functionally competent exogenous mRNA loading into the EVs was accomplished. The EVs displayed a novel protein (EVHB) that binds to the EV surface and displays a high affinity anti-HER2 scFv, thus targeting the EVs specifically to the HER2 receptor; these loaded and directed EVs are termed “EXO-DEPTs. Ip injection of EXO-DEPTs into mice with implanted HER2+ breast cancer xenografts, along with iv CNOB resulted in near-complete arrest of tumor growth. Thus, GDEPT was accomplished using systemic delivery, eliminating the limitation imposed by the current approaches that necessitate gene injection directly into the tumor site. To enhance translatability of this approach we have accomplished this with the prodrug CB1954 (paper in submission), for which a safe dose in humans has been established, and with 220-fold less EVs than before. Further, we have used in vitro synthesized mRNA to load the EVs; this eliminates the potential toxicity that inclusion in the EVs of the plasmid genetic material might cause. No notable off-target toxicity was seen. Work is in progress to test this regimen in immune-competent mice, and so are IND-enabling studies. We hope to conduct first-in-human, phase 1 clinical trials using EVs obtained from patients own dendritic cells; we have successfully done this before.

Learning Objectives: 

1. Use of extracellular vesicles in cancer treatment
2. How to make cancer treatment directed to the tumor so as to avoid off-target toxicity of conventional chemotherapy.

Ambrx’s mammalian expression platform (EuCODE™) enables non-native amino acids (nnAAs) through an expanded genetic code to both generate novel bio-therapeutics and to optimize the performance of antibody drug conjugate (ADC), therapeutic proteins, monoclonal antibodies (mAbs), and, bi- and multi-specific medicines. While the ability to control the defined Drug-to-Antibody Ratio (DAR) and payload site can provide an advantage to an ADC, the site-specific incorporation of the nnAAs into the antibody heavy chain introduces a unique challenge for antibody production.

To enable higher performance benchmarks in time and resources for process development with stringent product quality requirements, a proprietary cell culture platform is being developed and demonstrated fast-track development of high-quality, high-titer processes for producing recombinant proteins from CHO cells. 

We successfully generated a CHO-K1 cell line, stably expressing engineered amber suppressor tRNA and its cognate tRNA synthetase specific for nnAA para-acetyl phenylalanine (pAF), to achieve high production of monoclonal antibodies (mAbs) containing nnAAs. The stable cell lines were further evolved using CRISPR/Cas9 genome editing technology to sequentially knock out selected genes in glutamine synthesis, and, apoptosis pathways to improve selection efficiency and prevent loss of viable cell mass in production cultures, respectively.  Inhibition of apoptosis pathway leads to dramatic increase in viable cell mass and results in extended production time and increased productivity. 

In this presentation, we will discuss overcoming the challenges in cell culture platform development including cell line engineering, systematic DoE-based approaches on optimal chemically defined media and cell culture processes, and, strategies for scale up to clinical and commercial scales.

Learning Objectives: 

1. Learn what is cell culture platform and how to development your own cell culture platform
2. Learn the strategy to develop your own chemically-defined media