As cell-line optimization continues to push bioprocess productivity limits, several “loose end” issues persist. These include the use of design of experiment in clone selection, viral contamination, automation, and cell-level engineering.
Automation plays an increasingly significant role in cell-line development, particularly during clone screening and selection. When paired with the appropriate culture ware, liquid-handling systems provide consistency, fewer errors, and rework, and they free lab personnel from repetitive pipetting and sample manipulation.
At Informa’s upcoming “Cell Line Development and Engineering” conference, Jochen Schaub, Ph.D., associate director for cell line and cell culture development at Boehringer Ingelheim, will make the case for automation-driven cell-line screening with his company’s BI-HEX® platform, comprising BI-HEX expression system, cell-line development, and process development including a cell culture media platform.
As a platform, BI-HEX incorporates highly efficient vectors with novel genetic elements, serum-free operation throughout, FACS-based automated single-cell cloning, and high-throughput screening—which, according to the company, enables simultaneous analysis of more than 1,000 candidate clones. Boehringer Ingelheim employs imaging systems for automated cell counting.
“To cover the whole variety of today’s molecule formats, we apply different technologies for product titer determination such as homogeneous time resolved fluorescence or biolayer interferometry,” Dr. Schaub points out. Analyses are integrated within automated, robotic platforms to maximize sample throughput.
Greater insight into mammalian cell biology in the form of advanced systems biotechnology and knowledge of cell-line stability provides a scientific rationale for high-throughput cell-line development. “Despite the considerable scientific progress in these areas, the availability of robust and broadly applicable predictive tools in cell-line screening currently is still limited,” says Dr. Schaub. “Hence clone screening still relies on high-throughput workflows.”
Besides the benefits noted above, automation provides flexibility depending on specific project needs. “Examples are the optional incorporation of alternative single-cell cloning technologies or the early assessment of bioactivity measurements,” says Dr. Schaub.
In addition to continuous optimization of its cell-line development workflows to improve performance and to reduce timelines, Boehringer-Ingelheim has been focusing on early integration of process development ideas into cell-line development.
“Here, the aim is to perform screening in a representative cultivation environment through controlled, miniaturized bioreactor systems versus uncontrolled systems such as deep-well plates or tubes,” Dr. Schaub explains. Cell lines developed in this manner are selected not only for productivity and product quality, but also for performance during scaleup, process development, and for manufacturing.
“Through this combined approach, we recently obtained an increase in product titer by 85% in a 10-day fed batch process in clone selection without any further process development compared to a standard workflow.”
DOE Approach to Clone Selection
The interest over the last few years in parallel microbioreactor systems has arisen mostly from the manufacturing side, for scaledown and process modeling exercises. TAP Biosystems recently demonstrated that its ambr™ microbioreactor system serves cell-line development and optimization as well.
The ambr 48 system uses disposable bioreactors with 10–15 mL working volume. The system allows investigation of 48 unique combinations of cell-line and culture conditions. Since the system can vary temperature and stirring as well as cell, media, and feed conditions, customers have embraced ambr as a viable starting point for scaleup, TAP says. One 48 system customer is using the ambr to select the best antibody clones for production runs.
Benefits include a true stirred-tank bioreactor environment, parallel operation (12, 24, or 48 reactors), minimal operator interaction, compact design, rapid experiment turnaround, and reproducibility through full automation. “Once the system has started a run, the operator need only remove sample plates or tubes and replenish reagents or consumables,” says Barney Zoro, product manager.
One might ask, for cell-line development (vs. development or manufacturing applications), why not simply use microtiter plates, which are less expensive and provide greater parallelism? Zoro explains that multiwell plates have been used and continue to be, along with other technologies for early-stage selection of antibody-producing clones. “However, even when used in a shaken mode to increase gas transfer, they do not have any gas or pH control, which is a key characteristic of the production-scale bioreactor systems.”
A number of novel microtiter plate designs attempt to overcome these limitations, but these systems cannot provide the level of pH control, especially for cell culture, that faithfully reproduce bioreactor conditions. This, with lack of feeding capability, suggests poor correlation in productivity between clones selected in plates to performance in a full-scale bioreactor. Until microbioreactors from TAP, Dasgip, Pall, Invitrogen, and others came to the fore, the resources required to explore clone productivity in bioreactors prevented their use early in cell-line development.
ambr is typically employed at a stage when an initial clone screening has been performed either in plate- or image-based technologies such as ClonePix (Molecular Devices). Between 24 and 48 clones is about the right number to consider for a microbioreactor system. “Once the top-ranking clones have been selected from data obtained from the initial experiments, ambr can then be used for early-stage process development and media optimization to massively reduce the number of bench-scale bioreactor runs before transfer to pilot manufacturing,” Zoro says.
Novel Expression Methodology
Genome-editing technologies now permit precise positioning of deletions, modifications, and transgenes within living cells. These ideas have led Zsolt Keresztessy, Ph.D., senior research fellow at Proxencell, to a method employing sequence-specific meganucleases and TAL effector nucleases to generate stable monoclonal cell lines expressing membrane-bound antigens, FCGR receptors, and monoclonal antibodies. TAL effector nucleases are novel sequence-specific nucleases, formed by fusing a transcription activator-like (TAL) effector DNA binding domain to the catalytic head of an endonuclease.
As a core facility for the University of Debrecen in Hungary, Proxencell provides protein expression services using optimized synthetic genes and expression organisms that include bacteria, yeast, and both insect and mammalian cells for small- and large-scale protein production.
Dr. Keresztessy explains that specific genome editing technologies are still in the initial evolutionary phase—true especially for TAL effector nucleases. “That means, in addition to requiring substantial optimization work, investigators must also innovate in the adaptation of commercially available systems from, for example, Cellectis Bioresearch or Life Technologies.”
Uncovering effective ways to transfer and express sequence-specific nucleases (e.g., plasmid DNA, mRNA, or proteins) into your target cells or cell lines, together with accessory sequences including like templates for homologous recombination or genome editing reporter constructs, is critical.
“As a result, we were forced to develop new technologies for assessing genome modifications at early stages of TAL transfections, strategies and tools for detecting and enriching knockout cells, and new approaches for mapping TAL specificity in vivo in automated and high-throughput assays.”
Dr. Keresztessy and colleagues have enjoyed several successes in designing bioassay-worthy cells through these strategies. More relevant here are production cells. When the goal is overexpressing a protein—for example, a monoclonal antibody—for large-scale production, a variety of choices exist from commercially available reagent kits. Dr. Keresztessy’s system of choice is the cGPS CHO-Sa CEMAX system from Cellectis.
“However, we needed to construct our own version of the integration matrix vector provided by the vendor to efficiently express our mutant therapeutic antibodies, which we aimed to use as cellular assay controls in our research applications. Into the vector, we cloned the synthetic genes of the light and heavy chains of a human IgG framework, with the desired mutations, linked via an in-house designed and optimized IRES sequence.”
A total of 20 unique restriction endonuclease sites were engineered into genes to allow subcloning of variable regions for future applications.
Dr. Keresztessy sees great potential in sequence-specific nuclease-assisted genome editing for cell-line development. Applications include basic research for functional studies using reporters with gene tagging, promoter and enhancer modifications, and gene disruptions. The general technique could be employed to produce more potent reporter effector cell lines incorporating molecular sensory systems for the specific detection of macrophage activation, apoptosis, necrosis, differentiation, and others for cell-based assays.
“In my opinion one of the main limitations of the technology is efficiency variations for various cell types,” Dr. Keresztessy says. “The cost of specific genome modifications may also be limiting for academia and research institutes.”
Virus Testing Strategies
Contamination is one of the most dreaded events in cell culture, regardless of the scale or purpose. For biologics, contaminating organisms include bacteria and viruses. The latter are of particular concern because they arise naturally from starting materials of animal origin, or from mammalian expression systems themselves. Alison Armstrong, senior director for development services at Bioreliance (a Sigma-Aldrich subsidiary), will speak about contamination testing and avoidance with an emphasis on viruses.
Armstrong says that biosafety testing for biologics manufacturing and development is a tightly regulated, multitiered exercise that ensures the quality of raw materials and subsequently, at various junctures, to assess the ability of a process to remove or inactivate viruses. The trend away from animal-derived components (ADCs) is based on the fact that media and feed ingredients originating from mammalian sources can be serious sources of contamination.
“One viral contaminant in particular, minute virus of mouse (MVM), was thought to arise from media components manufactured under poor rodent control,” she says. “But MVM is not the only agent to consider. There’s a fairly significant number of potential contaminating viruses ranging from bovine-based agents, reoviruses, and hemhorragic disease viruses.”
One issue in the fight against MVM and other viruses is the interplay of infectivity assays and molecular-based amplification like PCR. Infectivity assays require a suitable cell medium and can take several days, while PCR is much faster. However, infectivity assays have emerged as the gold standard for demonstrating the absence of potential contaminants, while PCR remains an effective screen that might trigger more extensive testing.
“Molecular-based tests target specific sequences on specific viruses,” Armstrong explains, and as such are subject to the same issues of cross-contamination and false positive readings as any other PCR assay. “Infectivity assays actually confirm that the virus is present.”