Developing a cell line for production purpose is a very labor-intensive process. To increase the probability of obtaining a very high producer, a large number of cells need to be isolated at every step, which involves productivity variation from successive rounds of selection and amplification. In the past decades, lab automation and high- throughput technology have become an integral part of bioprocess development. Liquid and cell handling, both cell pool and cell clones, quantification of product titer, data acquisition, data processing and analysis, and archiving have all becoming automated. Many of the automated systems are based on culture plates, or wells, and resemble other liquid handling systems for high throughput chemical screening. The difference is that a incubation system, with temperature and atmospheric control for gas mixture and humidity, is necessary. Robotic arms are often used to move plates onto the working “stage” and allow multiple manipulations to be performed on multiple plates without human interference. In many cases, the system is installed inside a clean room or clean hood to minimize microbial contamination. The culture handling system is usually integrated with an assay system to assess product titer and cell growth. Multi-step assays and screening protocols can be performed by transferring culture fluid into automated assay systems. The results can be directly integrated into culture handling systems to further expand wells or plates selected for PerkenElmer - Basic Model
Tecan -- more automated with multi-plate capabilities and computer interface
Fig. 5.9: Examples of high throughput cell clone screening system.
product. Mutations causing protein sequence alteration may occur in one or more copies of product genes in all, or a subpopulation of, cells. Since not all copies of product genes in the cells are transcribed and translated at equal efficiency, not all mutations of product genes in a production cell population will be manifested to the same degree. With deep sequencing technology, one may be able to detect such mutations, even at minute level, in the consequent producing cell population.
• Production cell lines are tested for their ability to retain the product gene in the genome and produces the product
• The focus is production stability, not cell/genome stability
of stability is still labor intensive. The use of host cell lines, which have been adapted or modified to harbor all desirable growth characteristics, have greatly reduced the need of adaptation. There is an increasing movement toward miniaturizing cell culture while still simulating large-scale reactors, although this progress is still limited. The advance in genomics has brought about a fundamental change in the way we can study the process of cell line development and brightened the prospects that we can gain mechanistic insight into hyper-productivity. This knowledge may allow us to quickly select the “right” clone by examining the transcriptome or genome of the candidate cells. With more tools for genome engineering becoming available, it may also become feasible to impart on the cells favorable genome-wide modifications.
Concluding Remarks
further investigation. An integrated microscopic imaging system can provide the added capability of determining anything from clonal colony growth to colony morphology to ensuring that cells picked from each well are single-celled clones. Another type of automated system integrates cell cloning with product titer assessment by performing cell screening on agar plates. In this case, the secreted product molecules (mostly antibodies) are entrapped in agar that contains antibodies against the product. A halo ring of immunoprecipitation zone is formed around the colony. The size of the halo ring reflects the amount of product secreted. Image analysis is then used to extract the data for selecting high producing clones to pick.
• Basic liquid handling manipulations
• Distribution of liquid to 96 - 384 well plates • Sampling/removal of liquid
• Transfer from plate to plate
• Cherry picking – transfer from well to well
More complex models are capable of: • Cherry picking
• Multiple plate handing (i.e. movement of plates from a “hotel” to the pipetting stage)
• Multiple “steps” performed sequentially (e.g. DNA preparation protocols)
• Other “add-ons” like PCR machines, incubators, spectrophotometers, etc
Under best culture conditions, a hyperproducing industrial cell line derived from CHO or myeloma cells can secrete 50-100 pg protein/cell-day, a level which rivals professional secretors in vivo. Such remarkable cell lines are created through the combination of optimized genetic constructs, selection, amplification, cell clone screening, and the insight of picking the “right” clones. Although the specific productivity of the producing cells has increased by about one order of magnitude, the methodology of generating high producing cells has remained largely the same in the past three decades. The entire process is still empirical and very labor intensive. High-throughput cell handling and screening systems allow for the screening of a large number of potential high producing clones in the early stages of cell line development. However, subsequent steps of adaptation, growth characterization, and testing
Introduction . . . .147 Cell Mass and Composition . . . .148
Cell Mass and Size . . . . 148
Material Balance on Cell Growth . . . .149
Variation in Cell Volume . . . . 150 Amino Acid Composition . . . . 152 Intracellular Fluid . . . . 153
Growth of Mammalian Cells . . . .154 Quantitative Description of Cell Growth & Product Formation . . . .156
Stoichiometric Ratio and Yield Coefficient . . . . 158 Integral Cell Concentration . . . . 159
Kinetic Model of Cell Growth . . . .162
A Model Describing Growth and Production . . . . 162
Monod Model and its Derivatives . . . .164 Environment, Kinetics and Stoichiometry . . . .165 Concluding Remarks . . . .166