Leveraging automation and a step-by-step approach are keys to success.
Cell lines determine the performance of bioprocesses and the quality of the biologic drug substances they produce. As such, selection of the optimum cell line for each specific biologic is essential. Given the number of potential factors that can influence cell-line quality, the increasing complexity of biologic drug substances and growing regulatory requirements, identifying the optimum cell line can be challenging.
Defining high-quality cell lines
A high-quality cell line will demonstrate high stability, scalability, and high titer, so that it can provide reproducible results—with all product quality attributes consistent with the desired profile—with high manufacturing efficiency, according to Ian Collins, senior director of process development at Catalent. “A high-quality cell line should be well-rounded among the critical chemistry, manufacture, and control (CMC) production cell line attributes,” agrees Jill Cai, vice-president of cell and protein sciences with WuXi Biologics.
Before scaling up to bioreactors, the productivity level for monoclonal antibodies (mAbs) should be 2–7 g/L (fed-batch culture in shake flask) or greater, observes Selexis CEO Igor Fisch. Once scaled up, the mAbs should be 5 to >10 g/L in the bioreactor. “This high level of productivity must also be maintained for more than 60 generations at the high-density and in the defined media conditions of large-scale manufacturing,” he says. In addition, the parent cell line must be characterized and maintain consistency between each cell-line development campaign. Similarly, production cell lines must be well characterized so that scale-up from shake flasks to large bioreactors does not require extensive optimization.
“Equally important,” stresses Fisch, “is the quality of the recombinant DNA expression and transfection systems. Incorporating recombinant DNA stably into a cell line’s genome in a manner that promotes high-level, stable gene expression while minimizing gene silencing is non-trivial. Therefore, both the cell line and the vectors systems must be optimal in order to achieve optimal results.”
These aspects are in general desirable for both new biomolecules and biosimilars, although cells expressing biosimilars should be able to produce the target proteins comparable to the cells expressing the original therapeutic proteins. Special attention in this case should be paid to critical quality attributes such as glycosylation, which can add a variable to clonal selection and process development and can lead to a scenario where titer may have to be sacrificed for comparability, Collins notes.
It is also desirable to leverage a cell-line development technology that has already been utilized in clinical trials or in approved products so that there is a precedent for regulatory filings, he adds.
Numerous influential factors
Many factors influence the selection of a quality cell line for a given biologic drug substance. First is the product itself, which can affect the choice parental cell line (e.g., Chinese hamster ovary [CHO] or other type). “The advent of more complex molecules rather than monospecific antibodies requires technology that can effectively express multiple genes, as well as titrate the genes as needed; this allows the correct product formation or the expression of other processing proteins to further ensure product quality,” Collins says.
The capacity of the cell line to cope with the secretory stresses associated with the high level of protein production, particularly with many of the newer, non-natural proteins such as bi-specifics or multi-specifics, is also important, adds Fisch. “It is within the secretory pathway the proteins are properly folded, secondary modifications like glycosylation are added, and protein molecules are paired, when necessary, such as in monoclonal antibodies. If the secretory system cannot handle the secretory throughput, the biotherapeutic proteins can become misfolded or have improper secondary modifications,” he explains.
This issue is particularly important for biosimilars, because the glycosylation patterns of biosimilars must meet biosimilarity specifications when compared to the innovator drugs, which can only be achieved when they are generated in cell lines with well-functioning and stable secretory machinery, according to Fisch.
The plasmid ratio and medium are also important, Cai observes. “A balanced plasmid ratio usually has dramatic impact on some formats of bispecific antibody cell line development to minimize impurities,” she explains. Factors such as cell growth and metabolism, as well as product quality attributes like glycosylation distribution, are closely related to the culture medium, and thus many cell-culture media components can impact cell line and product characteristics, she also notes.
Lastly, comments Fisch, optimized, well-defined, and maintained manufacturing conditions are absolutely critical to achieving the highest level of productivity of a high-quality product. “Feed streams, gas-exchange, metabolic bi-products, and temperature all play critical roles in the amount and quality of the biotherapeutic products. Only the highest quality manufacturing will result in the levels of productivity needed for clinical and commercial manufacturing,” he asserts.
Challenges with variability, complexity, and regulations
Selecting the optimum cell line must occur in the face of several challenges. For Fisch, the biggest hurdle to overcome is the variability in the expression and secretion requirements of different biotherapeutic proteins. “Each cell-line development campaign brings its own set of challenges that can affect cell-line productivity and product quality,” he says. “Recombinant biotherapeutic proteins can misfold and stress the secretory pathway, leading to protein degradation or cell death, multi-chain biotherapeutic proteins may not pair properly, or perhaps glycosylation is incomplete. The challenge is to have a cell-line development platform that is robust and flexible enough to allow for troubleshooting across a broad range of production issues,” Fisch concludes.
Added to this challenge is the strong focus by regulatory authorities on monoclonality, according to Collins. “Whichever method is used for clonal selection should be well validated and ideally utilized in programs that have gained regulatory approval,” he states.
The sharp increase in the complexity of biomolecules is also increasing the difficulties associated with optimum cell-line selection, adds Cai. “Quickly developing and delivering high-quality cell lines is getting more and more challenging for bispecific antibodies and fusion proteins. It is difficult to rapidly screen more cell types clones to enable the selection of scalable cell lines capable of generating better cell productivity and product quality that are stable over extended cell-line generations and the long production cycle from vial thaw to harvest at large manufacturing scales,” she explains.
Two specific challenges, Cai continues, include clippings for bispecific antibody and fusion proteins, which may require many constructs screening and extensive mass analysis or peptide mapping to detect the problem early on. Another challenge for bispecific antibody cell line development is the percentage of the desired product vs. byproduct impurities, which requires early downstream involvement to determine the types of impurities that are easy to remove, and consequently influences clone selection.
An experienced team is essential
The importance of an experienced team in cell-line development should not be underestimated, according to Fisch. “While there can be straightforward cell-line development campaigns with mAbs, there are also numerous examples of difficult-to-express mAbs. In addition, the newer bi-specifics and multi-specifics, which are non-natural proteins, can really challenge a production cell line’s capacity to produce and secrete,” he states. A team that has seen and solved many of the issues associated with difficult-to-express proteins has the know-how to identify issues early and a history of solutions to address them, Fisch asserts. Having an experienced team with access to the right technologies can save months on any cell-line development campaign, he adds.
Bioinformatics can help
Bioinformatics tools can also be invaluable. “The ability to sequence the host cell-line genome, and more recently the transcriptome, along with the development of the bioinformatics to analyze that data has allowed for significant advances in cell-line development,” Fisch says. Bioinformatics analysis may help in getting genetic/epigenetic information that could offer insights into issues with protein production or cell stability in advance, agrees Cai.
“Transcriptomics and proteomics can provide a more comprehensive and quantitative understanding of metabolic, signaling, secretory, and other pathways to determine whether a cell line is good for production,” Cai comments. “Additionally, Cai notes that bioinformatics methods can help rationally design a host cell through determination of ideal genome sites for target integration and pathway engineering based on RNA sequencing results,” she notes.
For instance, Fisch observes that secretory requirements can vary from protein to protein, and the genomic and transcriptomic analyses of the parent manufacturing cell lines helps to determine where there might be deficiencies or inappropriate expression of cellular proteins affecting a given biotherapeutic’s production.
Detailed genomic data can also be used to assess cell lines for transgene integrity at junctions within the genome, transgene copy number and correct DNA sequence, number and locations of genomic integration loci, and possible adverse effects from transgene integration, according to Fisch. “[These] data, along with the right bioinformatics, can allow for direct assessment of the clonality of the biotherapeutic production cell line and provide traceability, validation, and origin of cells used to generate the production cell line—a vast improvement over the historical, indirect ways of determining clonality that makes it considerably easier to monitor drift and possible contaminations in production cell lines,” he remarks.
Moving toward multiparameter screening
The best instruments for selecting high-quality cell lines ideally enable the simultaneous screening of productivity (i.e., protein amount) and product quality (e.g., aggregation and cleavage), according to Cai. “Unfortunately,” she says, “currently available instruments mainly focus on productivity-based screening. Flow cytometry does, however, provide a relatively easy and economical way of screening high-producing cell lines.”
New instruments based on microfluidic and nanofluidic technologies such as Cyto-Mine (Sphere Fluidics) and Beacon (Berkeley Lights), which allow the screening of cell-line productivity during the single-cell cloning step, are emerging, according to Cai, but such instruments are generally more expensive than flow cytometers and their reliability, time savings, and acceptance by regulatory agencies have not been widely demonstrated.
Other new instruments designed for single-cell deposition are also available such as single-cell printers and verified in-situ plate seeding. Cai notes that these instruments are easy to use and may one day replace flow cytometry for single-cell deposition.
Catalent has focused on the Beacon platform, which the company believes to be an emerging leader in the area of clonal isolation and selection. “The Beacon platform is an automated system that is able to select the highest expressing clones faster than traditional methods and provides sufficient data to allow for a single round of cloning, which can reduce timelines for early cell-line development,” Collins states.
The best selection tools depend upon the cell-line development platform, adds Fisch. “In some platforms with lower transfection efficiencies, identifying high-expressing cell clones depends upon screening tens of thousands of clones, which is achievable using flow cytometry and high-throughput screening systems. When transfection efficiencies are high, identifying high-expressing and stable cell clones requires far less screening,” he explains. Selexis uses systems such as the Beacon optofluidic platform and ClonePix to provide faster, more efficient, multiparameter screening approaches for clone selection.
Catalent is leveraging automation to create high-quality cell lines and timelines. The company combines its proprietary GPEx cell-line development technology with the Beacon platform to generate highly stable, high-titer clonal cell lines for even difficult-to-express proteins, while the ambr15 system (Sartorius AG) allows for early evaluation of the individual clones in a bioreactor environment, according to Collins.
Considering each step
WuXi Biologics carefully considers each and every step involved in cell-line development using its WuXia platform to ensure the generation of high-quality cell lines. The strategy, according to Cai, typically starts with the molecule of interest and a codon optimization step to optimize the sequence of the gene(s) of interest. Proprietary vectors are then specifically engineered to enhance the expression level of the target genes. The company uses a tandem pool development and clone-screening approach to maximize the chances of deriving high-producing and high-quality cell lines. “We work closely with our analytical and process development teams to ensure the developability, monoclonality, and scalability of our cell lines and to achieve the key product quality attributes that our clients and partners desire,” Cai observes.
Taking a modular approach
With the Selexis SUREtechnology Platform, Selexis scientists apply a modular approach to generate high-quality cell lines that meet the production needs of each recombinant therapeutic protein. At the foundation of each cell-line development program, according to Fisch, are Selexis SGEs (Selexis Genetic Elements), unique epigenetic DNA-based elements that control the dynamic organization of chromatin in all mammalian cells and allow for higher and more stable expression of recombinant proteins.
A panel of tens to hundreds of high-expressing clones are assessed for production levels, product quality, and cell viability. If any of these parameters are suboptimal, Selexis has a suite of solutions for addressing them. With this approach, Selexis is able to express almost every protein, including difficult-to-express proteins that have failed in other systems, Fisch says.
Selecting cell lines for CAR-T therapies
As opposed to a synthetic drug, chimeric antigen receptor (CAR)-T cells are living therapies that are sensitive to manipulation and difficult to manufacture with consistent quality. As a result, the process of preparing them so they are safe and effective is multifaceted and complicated, according to Kris Simonyi, global marketing manager with Bio-Rad Laboratories. “One must confirm that all replication-competent viruses have been removed before injecting CAR-T cells into the patient, that the transgene has been inserted in the right location in the genome with the right dosage, and that the cell line is free from microbial contamination,” he explains.
As a result, a high-quality CAR-T-cell line should not contain any replication-competent viruses or microbial contaminants, but should contain one to four copies of the CAR gene (more than four copies exposes patients to the toxic effects of T cells, which include organ dysfunction and death).
One of the biggest challenges in selecting high-quality CAR-T cells, Simonyi notes, is that even if a transfection is technically successful, it does not mean it will provide a clinical benefit. “Transgenes integrate into random sites and may have detrimental effects on a patient,” he says. For example, a CAR gene could integrate into and activate an oncogene and lead to cancer. It could also integrate into a non-coding region of DNA and never be expressed.
To detect low levels of CAR genes and microbial contaminants and precisely quantify CAR genes, Bio-Rad’s Droplet Digital PCR (ddPCR) technology is of increasing interest. The technology selectively amplifies targets of interest including genetic alterations and nucleic acid contaminants, making them easy to identify, according to Simonyi.
Samples are partitioned into 20,000 nanoliter-sized droplets and a polymerase chain reaction (PCR) takes place in each droplet. A fluorescent probe binds to the target DNA, making it fluoresce when it gets amplified. The nucleic acid fragments that do not contain the target sequence do not get amplified and do not fluoresce. By counting the number of fluorescent droplets versus the total number of droplets, the concentration of the target DNA strand can be determined.
Custom probes can be used to select for cells containing the CAR gene and bacterial contamination. Because the system performs absolute quantification without the need for a standard curve, it can also measure transfection efficiency and precisely quantify the transgene copy number in CAR-T cells. “Our ddPCR technology can be 100- to 1000-fold more sensitive than quantitative PCR, enabling it to detect down to one CAR gene copy per cell,” Simonyi comments.
It can also detect and precisely quantifying various types of DNA and RNA alterations that can factor into the quality of genetically engineered T cells, including minimal levels of nucleic acids of interest, such as genetic material from replication-competent viruses and other contaminating microbes. In addition, following next-generation sequencing to map transgene integrations, Bio-Rad’s ddPCR technology can be used to track the cells through the manufacturing process, according to Simonyi.