Immortalization of Human Cells
At the University of California, San Francisco, Dieter Gruenert, Ph.D., has developed a standard approach to establish immortalized human cells that maintain the differentiated characteristics of the primary cells from which they are derived. These cell lines enable the study of normal cell function and disease mechanisms, and serve as screening platforms for therapeutic intervention. They are preferred over primary cell systems which have a limited lifespan and can be difficult to manipulate.
“We use an origin-defective SV40 plasmid in a standard transfection protocol to isolate clones of human epithelial cells with the desired phenotypes. We take specific care to capture the cells of interest at a particular stage of differentiation and then lock the cells in that stage under prescribed culture conditions,” said Dr. Gruenert.
“We have 15–20 different cell lines that are available to scientists for their research efforts. We also have hundreds of clones that have been established but not yet fully characterized prior to freezing them down.”
The key here is that the cell lines are of human origin and not animal or insect cells. Essentially each species has similarities to humans, but there are often significant molecular and metabolic differences that can influence the actions of pharmacological agents. Dr. Gruenert is adamant: “If you are focused on human disease, then use cell lines of human origin.”
Dr. Gruenert is also very sensitive to the issues of establishing immortalized cell lines. The issue of de-differentiation of cells in culture is taken seriously, and cell-line provenance is monitored on a regular basis by comparison to the primary cells and evaluation of the epithelial endpoints: keratin expression, tight junction formation and polarity, and finally neoplastic progression.
As long as the cells are maintained under the culture conditions in which they’ve been isolated, they will maintain their proper character. To that point, the lab has a workhorse cell line that has been in culture for over 20 years.
The development of human-induced pluripotent stem (iPS) cells has opened a new arena of possibilities for immortalized cells. Since the iPS cells are immortal and retain their diploid karyotype, they can potentially be directed to differentiate into any cell type within the body. This is not only significant in terms of cellular therapy through tissue repair and regeneration, it is a significant tool for the development of pharmacological and genetic therapies.
Biogen Idec has a dedicated drug development focus on production of biotherapeutic proteins for use in the clinic for neurodegenerative and autoimmune disease states. Brian Majors, Ph.D., a scientist in the cell engineering group at Biogen Idec shared the process by which his group has been able to optimize cell-line constructs they obtain from R&D for the production of the proteins destined for Phase I clinical trials.
“Our group transfects CHO cells using the expression vector containing the lead molecule characterized by the biological team. We grow up to thousands of clones and then seed 24-well deep-well plates. The focus is on defining the culture conditions that will best replicate those used in the bioreactor that will be used to produce material for the clinical trial,” said Dr. Majors.
“The protein is harvested from each of the clones after 14 days of growth. The proteins are purified in two steps: first, capture by binding protein A, and then size-exclusion chromatography for isolation of protein monomers. The purified proteins are then subjected to analytical analysis. A handful of clones that produce the best quantity and quality of product are processed further.”
The team uses a label-free method to monitor cell production of proteins during the 14-day culture period. A fiber optic tip coated with protein A is dipped into the culture media to capture and quantitate the protein in solution. The overall goal of the team’s effort is to establish a process to predict cell-line performance early in the development cycle to save time and money for the project.
At NovImmune, they are investigating possible extension of their portfolio of therapeutic antibodies by the introduction of a newly developed kappa-lambda body (κλ-body™). This fully human bispecific antibody format has typical functional and biochemical characteristics of a human IgG. The κλ-body features a common heavy chain but two different light chains (one κ and one λ), giving a different target specificity to each antibody arm.
The benefits of using a fully human format, without mutations or linkers, are numerous. They include PK profiles and Fc effector functions of therapeutic antibodies, robust productivity and stability during manufacturing and storage, and low risk of provoking immunogenicity.
The upstream processing group, headed by L. Di Grazia, further developed the novel tri-cistronic vector system for expression of the κλ-bodies in CHO cells, originally designed by the internal research group. CHO cell lines for two different κλ-body products were established, and the cell lines were observed to be genetically stable and maintain constant high-level productivity. The κλ-bodies were tested and shown to maintain biological function.
“Over the course of our work, we’ve grown these cells for over 50 generations without loss in productivity or κλ-body assembly efficiency,” said Di Grazia.
In order to manufacture batches for preclinical studies, the process has been successfully scaled up to the 100 L level using stirred bioreactors. The aim of the early cell-line screening approach is to minimize the impact of cell-line variability on downstream processing, notably with respect to the impact of varying concentrations of product-related impurities.
“To date we readily achieve multigram/liter titers for different κλ-body products using the platform USP,” noted Di Grazia. “These yields are equivalent to those achieved for our monospecific antibody controls, suggesting that κλ-body assembly is as efficient as that of therapeutic monoclonal antibodies.
“We routinely use a binding assay to verify that the purified products maintain the appropriate binding specificity as well as their ability to co-engage the two targets. This is a critical quality control assay for us. Our κλ-body products have also demonstrated favorable drug characteristics in vivo with PK properties equivalent to monospecific monoclonal antibodies.”
The κλ-body products developed to date by NovImmune have been shown to be readily amenable to a scalable platform manufacturing process. Di Grazia noted that the final product has the benefits of a monoclonal antibody plus the added functionality of multiple specificities: it is stable, has the expected half-life in circulation, a low potential for immunogenicity, and contains essential effector functions necessary for cutting-edge biotherapeutics.