Q&A: How to Maximise Cell Line Productivity and Efficiency

Firstly, a host cell line must be selected with the required authentication documentation in place. The transfection method must allow for optimal transfection efficiency and gene integration, for example, using Nucleofector® technology. The choice of vector is also a key consideration.

Here at Lonza, we have developed a suite of GSquad® vectors that use a simple two-step process to generate a vector containing up to four genes, designed to maximize productivity. An appropriate selection system should be used, either antibiotic selection such as puromycin or Zeocin®, or metabolic selection systems such as DHFR or GS. The speed at which cell line development can be achieved is also key. At Lonza, we have built upon many years of experience and expertise to design a program to achieve gene to IND in less than 11 months.

The importance of maintaining genetic stability is primarily to ensure the quality of the recombinant protein whilst maintaining high productivity and consistent growth. Mammalian cell lines can deliver good growth whilst being adapted to varying culture conditions and withstanding genetic manipulation. This also means there can be a tendency for these cell lines to undergo genetic changes, leading to instability.

Strategies to minimize the risk of instability include the vector design and expression system used. The choice of the promoter, number of integration sites, transfection efficiency, and sequence and codon optimization should be considered when preparing the vectors containing the genes of interest. Genetic engineering can be used to knock in/out specific genes associated with instability. Insulator elements can be utilized, such as MARs, S/MARS, cHS4 and UCOE chromatin-modifying elements, which insulate the expression cassette containing the gene of interest from surrounding genomic elements and genome positioning effects. 3’UTR enhancers can increase mRNA stability. The use of site-directed integrations (SSI) can also be beneficial in terms of improving stability.

Single-cell cloning is a technique used to generate monoclonal cell lines, which reduces the genetic heterogeneity of a cell line. This enables selection of a more stable cell line for manufacturing.

Maintaining a consistent culture environment may also be beneficial to reduce the chance of changes that may select for genetic variants, so a well-established and robust upstream process, including media and feeds, is key.

The selection of expression system technology can be key to maximizing cell line productivity. At the vector level, the incorporation of insulator elements and/or 3'UTR sequences can be utilized to optimize productivity as well as genetic engineering to knock in/out specific genes associated with productivity. At Lonza, the GS piggyBac® system, which uses an engineered hyperactive piggyBac® transposase enzyme to insert GS® expression vector cargos into the host cell genome with high efficiency, has been designed to preferentially target the gene of interest to stable regions of the genome associated with highly expressed genes. The GS piggyBac® system has been shown to increase productivity for clones expressing bispecific antibodies by 45% on average as well as increasing the cell recovery time post-transfection compared to random integration.

With increasing interest in the production of more complex therapeutic proteins such as bispecifics, solutions such as Lonza's bYlok® technology can be employed to increase the frequency of correct HC-LC pairing, thus increasing the yield of the required protein. Through the use of metabolomics, media and feeds can be designed to maximize protein expression based on analysis of the nutrient depletion and accumulation of waste products within a culture. The entire process can also be optimized using this approach.

Cell line screening and selection strategies can be designed to maximize the chance of selecting a high-producing cell line. Lonza uses the Beacon® optofluidic system to screen thousands of clones at the single-cell stage to identify those most suitable for progression.