Since the approval of the first monoclonal antibody (mAb) therapies in 1986,1 antibody-based drugs have become the predominant class of biopharmaceuticals. However, in recent years, novel antibody derivatives — multispecific antibodies, antibody-drug conjugates, antibody fragments, and others — have been investigated and offer substantial advantages over traditional mAbs.
Bispecific antibodies (bsAbs) are next-generation antibody therapies that have received significant attention owing to their increased specificity and efficacy relative to mAbs. They possess two distinct "arms" that bind to two distinct antigens (or different epitopes of the same antigen). Their ability to simultaneously bind to two targets and, thus, address two different disease pathways enables them to function as effective treatments for diseases with multiple mechanisms of action. For such diseases, manufacturers only need to produce a single biotherapeutic, and patients only need to take one bsAb rather than two mAbs.2
By the end of 2023, the U.S. Food and Drug Administration had approved nine bsAbs: seven for cancer, one for treatment for hemophilia A, and one for wet age-related macular degeneration.2 At the same time, over 100 bsAbs were in clinical development3 for chronic inflammatory, autoimmune, and neurodegenerative diseases; vascular (blood vessel-related), ocular (eye-related), and hematologic (blood-related) disorders; and infections, including COVID-19.2 According to Allied Market Research, the global bsAb market was valued at $5.5 billion in 2022 and is expanding at a compound annual growth rate of 34.8%, potentially reaching $109.4 billion by 2032.4
Despite this positive outlook, there are challenges associated with developing and manufacturing bsAbs, including issues relating to the quality, stability, and immunogenicity of bsAbs; difficulty maintaining the affinity of the parental mAbs; and complex manufacturability, particularly related to the production of bsAbs with correct heavy chain-light chain (HC–LC) pairing. The third issue arises because manufacturing bsAbs with two distinct chains often yields multiple final forms, only one of which is the desired product, while the other forms are impurities that are difficult to remove owing to their similarity to the preferred form.
Samsung Biologics has tackled these challenges head-on by introducing its S-DUAL™ bsAb platform. The commonly used knob-in-hole (KiH) design is combined with a novel asymmetric antibody structure created by introducing a CH3 (the third constant domain of the heavy chain) dimer in the antibody's fragment antigen-binding region, which delivers a 99% HC–LC pairing success rate (see Figure 1). This design also enables the seamless addition of complementarity-determining regions of interest without additional antibody engineering while maintaining high binding affinity and productivity.
Figure 1. The general structure of a bsAb developed with the Samsung S-DUAL™ platform
S-DUAL™ bsAbs have an immunoglobulin G (IgG)-like structure with an alternative effector function (IgG1/IgG4) and, thus, exhibit low immunogenicity while offering plug-and-play features for the variable domain. The KiH strategy ensures efficient HC–HC pairing, while an additional domain enables highly specific HC–LC pairing. Since the asymmetric structure makes it easy to distinguish, only by size, the desired product among the various impurities that bsAbs can create, it is possible to easily monitor only the desired product through size-exclusion high-performance liquid chromatography in the process development stage, thereby enabling process development that maximizes productivity and quality.
The initial results confirming the effectiveness of the S-DUAL™ bsAb platform used a process that had not been thoroughly optimized. As a result, an upstream process optimization initiative was launched to improve the productivity of the process and the quality of bsAbs, while shortening the development timeline (see Figure 2). An initial feasibility run was implemented to confirm the overall process performance. Different media and addition conditions for additive A were screened, followed by a design-of-experiment (DoE) study for process parameter optimization. Verification runs then demonstrated the robustness of the optimized process.
Figure 2. Upstream process optimization strategy for the S-DUAL™ bsAb platform
To expedite the timeline, pool cell lines producing S-DUAL™ bsAbs were cultured in a 2-L Biostat® B-DCU (Sartorius) bioreactor to monitor cell growth, metabolite, and productivity profiles. Based on this feasibility run, media and additive screening were conducted in Ambr15® (Sartorius) to determine media combinations and additives, which led to higher productivity and improved cell metabolism profiles.
Historically, various media combinations have been shown to increase productivity and quality profiles according to the cell line. In this case, the pool exhibited lactate accumulation, and it is challenging to change the lactate metabolic shift from the production to the consumption phase. Additive A was appropriately adjusted to modulate lactate consumption at a late phase of the cell culture (see Figure 3).
Figure 3. Historical data on productivity and lactate control for media and additive screening
A DoE study was then performed to optimize critical process parameters. As one of the DoE approaches, response surface methodology (RSM) was applied in the DoE design platform using JMP® statistical software, which is specifically used in process optimization. In DoE RSM, a central composite design was chosen to identify the effect of various process parameters, with four factors and two levels that historically showed improvements in productivity and quality. Factors that were considered included cell conditions, pH and gas levels, metabolite profiles, osmolality, titer, and quality/purity.
The determined set of physical experiments was then performed in Ambr15® bioreactors to increase both the productivity of the process and the quality of the bsAbs using a single clone derived from the pool cell lines and the best media combination established in the previous step. Predicted and actual titer high-performance liquid chromatography (PA–HPLC) results are shown in Figure 4. The correlation was strong.
Figure 4. Statistical analysis of productivity and quality for upstream critical process parameters
Process verification at the 2-L bioreactor scale was then conducted to confirm that the established upstream development process, including harvest, provided the expected results. Following process development, the operational differences between the feasibility run and the verification run were the cell line (pool to single clone), media combination, the addition of Additive A, and process parameters. It was confirmed that the advanced upstream development efforts for the S-DUAL™ bsAb platform successfully improved lactate metabolism, productivity, and quality (see Figure 5).
The productivity of the verification run was more than 3.4 times higher than that of the feasibility run, with the final average titer approximately 8.0 g/L, as determined by HPLC (Waters Alliance™ 2695 with 2489 UV/Vis detector) using a 2.1 × 30 mm column (Thermo Fisher Scientific POROS™ A20). Lactate metabolism showed more dramatic improvement. In the feasibility run before process development, the final lactate concentration was maintained at 3 g/L or more because the lactate metabolic shift had not occurred; in contrast, the final lactate concentration of the verification run was maintained at 0.1 g/L or less as a result of a complete shift to the lactate consumption phase following the addition of Additive A.
Figure 5. Overall results for S-DUAL™ bsAb upstream platform development
The optimization of the upstream S-DUAL™ bsAb platform process is reflective of Samsung Biologics' commitment to considering commercialization needs from the start of development, particularly the maximization of bsAb titer and quality. Extensive analytics performed during the early stages help identify potential problem areas and mitigate risks. Process optimization efforts, such as the one described here, are routinely performed and often increase titers by between 3 and 5 g/L while achieving purities above 95%, further minimizing risks as projects advance from cell line development to process development and manufacturing. Meanwhile, starting at the 2 L scale and progressing to 100 L before moving to 1,000 L de-risks scale-up. If issues are detected during any run, full testing is conducted to mitigate problems before advancing to the next scale. Equally important, the equipment used for small and large batches is consistent, ensuring that results obtained at smaller scales accurately reflect the results expected during commercial production.
BsAbs are unique, next-generation antibodies with dual specificity, creating considerable opportunities for therapeutic applications, such as redirecting T cells to tumor cells, blocking two different signaling pathways simultaneously, dual targeting different disease mediators, and delivering payloads to targeted sites. However, as bsAbs are much more complex molecules than traditional mAbs, they present unique manufacturing challenges, including increased incidence of mispairing, the manufacture of undesired fragments, and increased levels of aggregates. The S-DUAL™ bispecific antibody platform by Samsung Biologics addresses these complexities.
Having a development and manufacturing partner with experience and proven expertise in working with and providing strategies for such advanced molecules is fundamental, since creating high-producing and high-yielding processes for this class of products is a complex endeavor. Samsung Biologics embraces its responsibility to overcome these challenges, driving the development of innovative bsAb therapies that offer therapeutic options to patients with currently unmet needs.
By conducting all processes and testing in-house at a central location, Samsung Biologics provides confidence across all concerns associated with each client's molecule and a plan to effectively address each concern when moving to the next level of production. In addition, as a fully integrated drug development and manufacturing partner providing integrated services from cell line development to commercial manufacturing, Samsung Biologics provides customers with the advantage of reduced timelines from DNA to Investigational New Drug application (IND) and Biologics License Application (BLA). Furthermore, with locations in South Korea and the United States, Samsung Biologics is ideally positioned to support clients worldwide.
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