In a recent webinar for The Medicine Maker, I outlined practical, next-generation strategies for improving bispecific antibody expression in CHO cell lines, including an antibiotic-free platform that uses only the glutamine synthetase selection marker.
Bispecific and multi-specific antibodies are becoming more effective therapeutics because they can be engineered to simultaneously bind two or more distinct antigens or epitopes. However, the design and manufacture of these complex molecules is challenging for many companies because it requires careful consideration when selecting a scalable process to ensure delivery of high levels of purity with high mass output.
The mission of Just-Evotec Biologics is to design and apply innovative technologies to enable global access to biotherapeutics. In line with this mission, we have designed a proven approach that uses innovative expression systems and manufacturing workflows to boost yields of bispecific and multi-specific antibodies
Our platform covers the entire range of bioprocessing development:
The J.MDTM: Molecular Design performs an initial assessment of molecule developability using artificial intelligence and machine learning approaches, and applies molecular engineering mitigations when needed. The process includes biophysical characterization.
J.MDTM Molecular Design performs an initial assessment of molecule developability using artificial intelligence and machine learning approaches, and applies molecular engineering mitigations when needed. The process includes biophysical characterization.
J.CHO® CHO-K1 Expression System gives clients access to host cell lines, media and expression systems and vectors in our optimized cell and development workflow. This helps to generate high-expressing clonal cell lines.
JP3® Process & Product Design is a fully integrated and automated continuous process manufacturing platform, supported by in-house process analytical and formulation capabilities, and J.POD®, a GMP continuous manufacturing facility. The facility enables clinical and commercial supply to be generated, while minimizing scaling risks from fed-batch processes.
The Advantages of a Perfusion Process
Figure 1 shows a schematic of our perfusion process. The process supports healthier cells by continuously supplying fresh nutrients, removing metabolic waste and reducing product retention time in the bioreactor. This allows us to enhance the productivity of the cell lines and product quality of the antibodies. Some advantages include:
The perfusion process gives control of critical quality attributes, making it easier to match previously produced fed-batch materials. The unit operation sequence completely aligns with fed-batch processes and sophisticated automation allows surge vessel capacity to be used in the event of a process disruption. The flow rate is kept very low and we always keep the bioreactors running. Therefore, for late-stage products, we can extend the run up to 25 days for a fully end-to-end continuous process.
Our continuous perfusion system yields significantly higher productivity compared to a standard fed-batch culture process. We have data showing that a 25-day perfusion cell culture process yields almost a 10-fold increase in productivity compared to standard fed-batch culture.
We have produced a wide variety of antibody products using our process, including IgGs, fusion proteins and bispecific antibodies. This process can also be customized according to the needs of the clients. Recently, we produced more than 6 kg of bispecific mass output from a single 500 L, 15-day run.
Inside the expression system
The J.CHO Expression System uses proprietary CHO-K1 glutamine synthetase (GS)-knockout host cell lines adapted to grow in serum-free media and can deliver high-specific productivities and high-density growth in bioreactors. We also have transposon-based expression vectors with strong promoter sequences that allow stable integration and high expression of genes of interest. These are optimized for our upstream perfusion platform process and scalable directly from 3 L up to 1000 L bioreactors for clinical or commercial production.
We generated our in-house CHO GS knockout cell line in 2020 and have been using it successfully for several molecules, including bispecific antibodies.
This schematic in Figure 2 shows the essential elements of our expression vector. The inverted terminal repeat (ITR) sequences allowed the transposase to insert these sequences containing the antibody chains and a selection marker into the host genome. We create stable cell lines from the start that ensure a high level of expression in pools and clones – and allow us to further characterize the cell line better.
In our cell line development workflow, shown in Figure 3, we start off with high-throughput transfection where we transfect our expression vector containing the molecule of interest, or co-transfect with a combination of plasmids containing different molecules to make up the bispecific or other complex molecules. We use a high-throughput transfection method, which means that up to 25 transfections can be performed at the same time. These will then be put into selection in glutamine-free media, or with antibiotic selection to generate stable pools.
Once the cells recover from selection, the stable pools are put into a 10-day fed batch production assay. We perform high-throughput analytical assays on the stable pools to select pools for further screening in bench-scale bioreactors. From here, a stable pool will be selected for single-cell cloning.
Clones are expanded and bulked from 96-well to 24-well deep-well plates, and automation is used during cell passaging and bulking throughout the entire process. The clones are then put into a 10-day fed batch production assay and high-throughput analytical assays are performed to identify top clones for screening in bioreactors.
Optimizing bispecific expression
To optimize bispecific expression, it is important to begin at the earliest stage of cell line development by adjusting the configuration of the plasmids and the relative ratios used during transfection. In this case, the first light-chain and heavy-chain pair is encoded on a plasmid that also carries the GS selection marker. The second light-chain and heavy-chain pair is encoded on a separate plasmid that carries the puromycin selection marker.
Both plasmids are co-transfected at a range of ratios. This step is critical for achieving balanced chain expression, which supports correct heavy- and light-chain pairing and reduces the formation of misassembled or partial species. Because we can perform up to 25 transfections in a single high-throughput run, we can include replicates and test multiple plasmid ratios and vector configurations in parallel within the same experiment.
We generate stable pools from each vector ratio and produce material using a 10-day fed-batch assay. We then apply our high-throughput analytical methods, such as non-reduced capillary electrophoresis and mass spectrometry, to assess the purity and quality of the molecules produced. These analyses are carried out both at the stable-pool stage and later during clone screening. By comparing the data across vector ratios, we can rapidly determine which vector configuration performs best.
A case study
A bispecific antibody was engineered from two different molecules using the knob-into-hole format and expressed using our J.CHO high expression system. I will refer to them as Molecule A and Molecule B.
We performed a dual selection method using GS selection and puromycin to generate stable pools. High throughput 24-well plate fed-batch cultures of these pools demonstrated expression levels ranging from 1 to 3 g/L.
Figure 4 shows that when the ratios of the GS plasmid are varied, there is a difference in titer. To further optimize expression and molecule assembly, we also swapped the molecules on expression vectors. Molecule A (shown in blue) was on the GS vector, but we placed this on a puromycin vector and also swapped Molecule B. This led to a significant increase in titer. Strategies like this can help to balance chain ratios and show that empirical testing of the vector configurations can significantly benefit the yield of bispecific antibodies.
One potential drawback to this dual selection approach where more than one expression vector is transfected is that an additional antibiotic selection is needed, which adds to the cell line development timeline. It can also impart a higher risk to the process. A solution is to design an expression system for multiple chains that only uses the GS selection marker. In that way, no additional antibiotic selection will be needed.
We can do this using mRNA trans splicing. This is a naturally occurring splicing process that takes two different pre-mRNAs to create a mature chimeric mRNA. It occurs when a separate pre mRNA is spliced with a target pre-mRNA – and is typically used to alter functions of genes or to correct mutations in gene therapy.
In our vector system, the GS gene is split into two parts. The first half of the GS gene goes onto one plasmid containing all the elements needed for trans splicing. The second half of the GS goes on the other plasmid. After co-transfection of these plasmids, mRNA synthesis occurs, followed by trans splicing of the pre-mRNAs to form a mature mRNA. In this case, the two halves of the GS are trans spliced together to form a fully functioning GS at the mRNA level.
When the cells are put into selection in glutamine-free media, only cells that have fully trans-spliced GS to form fully functioning GS will survive. A PCR strategy is also used to determine successful trans-splicing. In this case, the forward primer sits on one half of the GS and the reverse primer sits on the second half, so a PCR product should only be detected on a cDNA, which is generated from the mRNA and not on genomic DNA.
We expressed a regular monoclonal antibody with two chains to demonstrate proof of concept of this strategy. To test the split GS method, we put the heavy chain on one plasmid containing the first half of GS and the light chain on the second plasmid containing the second half of the GS. For this to successfully occur, the two halves of the GS will have to come together during successful trans-splicing.
In Figure 5, the agarose gel shows a clear PCR product in the control. In our experimental replicates, a PCR product is detected only when using RNA, indicating that trans-splicing successfully occurred. The cells also recovered well during selection and reached full recovery within 14 days, producing good titers. This demonstrates that the split GS method can support monoclonal antibody expression.
We then applied the approach to a bispecific molecule, varying the ratios of the two plasmids and swapping which molecule (A or B) was placed on each plasmid. Again, the cells recovered after selection. Notably, the resulting samples displayed a range of titers depending on both the plasmid ratio used and the arrangement of the molecules on each vector.
Comparing the split GS method with dual selection
Using the split GS approach, molecules A and B are placed on separate plasmids that each contain half of the GS gene. With the dual-selection method, molecule A is linked to the GS marker and molecule B to the puromycin marker. We also tested swapping the positions of molecules A and B in the dual-selection system.
Across the different configurations, we observed a range of titers. Data from size-exclusion chromatography showed that all samples had good purity, with the main peak above 85 percent, although there were some differences depending on the configuration, suggesting that how we express these molecules using different vector systems can influence purity.
Similarly, the non-reduced CE assay showed small differences among the different vector configurations. The assay, performed under non-reducing conditions, is used to assess the purity and integrity of the intact bispecific antibody by separating it based on size and charge, and it can reveal related impurities or degradation products. Altogether, these high-throughput analytical methods help us optimize vector design and generate high-quality bispecific antibodies.
In conclusion
I have discussed how Just-Evotec Biologics’ continuous perfusion platform process yields significantly higher productivity. I've also described how vector design strategies can help boost titer and the purity of bispecific antibodies.
Split GS is a new expression system that we have developed that can stably express complex molecules using just the GS selection marker, thereby eliminating the need for further antibiotic selection. Finally, I showed that empirically optimizing bispecific antibody expression and product quality by varying the plasmid ratios and vector configurations during transfection is necessary to obtain the best yield.
Additional Insight from Yiting Lim
What other host cell lines does Just-Evotec Biologics offer?
We offer a variety of other host cell lines for optimal expression of antibodies. We have an inducible host cell line, which is very effective for difficult-to-express antibodies and has been used for bispecifics. We also use another GS knockout cell line that we license from another vendor and we're developing new host cell lines for a variety of purposes.
What is your approach to producing a difficult to express antibody?
Oftentimes, we can start by using J.MDTM Molecular Design to optimize the sequences – not so much for expression at this stage but for developability, which can significantly impact the ability to produce the antibody.
We also evaluate different codon optimizations of the DNA sequences because this can improve expression, and we can try expressing it in our inducible host cell line. With our JP3® process, we can optimize process parameters in the bioreactors to support better cell growth. For our in-house CHO K1 host cell line, we typically achieve very high cell densities, so optimizing that growth process in the bioreactors can also help with increasing yields.
Click here to access the webinar: A Simpler, Smarter CHO Strategy for High-Quality Bispecifics
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