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Multichain recombinant proteins are a growing class of biotherapeutics used to treat cancer and other indications. These proteins, which are typically assembled from three or more constituent polypeptide chains, can pose unique challenges during development and manufacturing processes, owing to their increased structural complexity. State-of-the-art mammalian expression systems, including innovative vector technology can potentially overcome hurdles in titer, chain pairing, and product quality, and help accelerate these biologics towards the market.
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Multichain biotherapeutics: bispecific antibodies and Fc-fusion proteins as promising therapeutic modalities
Multichain biotherapeutics continue to be a promising therapeutic modality. “These therapeutic recombinant proteins are typically assembled from at least three distinct polypeptide chains,” says Rachel Da Rosa, Product Manager, Licensing and Early Access Services at Lonza. “They can include engineered fusion proteins that integrate multiple functional domains into a single therapeutic, and multispecific antibodies capable of binding to two or more molecular targets, amongst other formats.” For example, Fc-fusion proteins contain the Fc domain of IgG linked to a peptide or protein of interest which can help enhance the half-life, solubility, and stability of the fused protein. Bispecific antibodies (bsAbs) can have a multitude of formats and structures, but they all have the distinguishing feature of being able to bind to multiple targets simultaneously.
Since the first bsAbs were described in the 1960s, Da Rosa says, “Most bsAbs in clinical trials are aimed at treating solid tumors and blood cancers, while a smaller number aim to treat non-oncology indications like immune-mediated diseases.” In 1998, the first Fc-fusion protein approved, etanercept, was aimed towards treating rheumatoid arthritis and in 2009, the first bsAb approved was catumaxomab to treat solid malignancies but was later withdrawn for commercial reasons. Now, there are at least 16 bsAbs launched across key markets and hundreds more in clinical development.
Multichain therapeutics such as bispecific antibodies have many potential advantages over more traditional modalities.
“Bispecific antibodies can achieve their therapeutic impact on solid tumours via mechanisms not accessible to standard format monoclonal antibodies. For example, one arm of a bispecific T cell engager can be designed to bind a tumour-associated antigen while the other engages a T cell via CD3, ultimately leading to lysis of the tumour cell.”
However, these therapeutics can present a unique set of challenges during the biomanufacturing process.
Challenges of manufacturing multichain biotherapeutics
Optimizing host cells, expression vectors, and titer
Two of the most critical components in the synthesis of any biotherapeutic protein are the host cell line, such as the commonly used CHO cells, and the DNA expression vector. “We routinely expect recombinant CHO host cell lines to synthesize the individual constituent protein chains of multichain biotherapeutics at high levels, and to efficiently assemble them together into the correct format for secretion,” says O’Callaghan. “But along the intricate intracellular biosynthetic pathway that runs from gene to fully assembled and secreted protein, there are myriad opportunities for that process to go wrong, or to operate at sub-optimal efficiency.” This is why he suggests optimizing all stages of the bioprocess to maximize yields of high quality biotherapeutics.
Historically, it’s been harder to get titer yields for multichain biotherapeutics to match those of single proteins. However, advances in vector design and bioprocess steps have boosted titers for these complex molecules. “At Lonza, the typical titers we obtain for bsAbs and Fc fusion proteins are broadly similar to what we achieve for typical monoclonal antibodies, a result which represents the significant amount of work we have invested to ensure we can consistently deliver on customer expectations for these multichain format molecules,” says O’Callaghan.
Chain pairing in multichain biotherapeutics
While titer is important, a unique challenge in developing multichain biotherapeutics is chain pairing, or making sure that the different pieces are assembled properly. “Drug developers have become much better at designing these molecules as we’ve gained more experience with them over time,” says O’Callaghan. “We’ve understood better how to spatially bring together the different parts of the molecule so that they engage the appropriate targets, achieve the desired efficacy, and assemble at high efficiency.”
In contrast to monoclonal antibodies, which have identical heavy chains (HCs) and light chains (LCs), multichain biotherapeutics can have three or four unique antibody or antibody-like polypeptide chains, so it is important to make sure that all the constituent parts assemble together into precisely the right format. If that doesn’t happen, or not at sufficiently high levels, the efficacy and safety of the material can be severely compromised. A failure to ensure correct assembly will prevent a therapeutic candidate progressing through the development pipeline, requiring lengthy and costly rework. One way to ensure that the different heavy chains are correctly assembled together is to use the “knob-in-hole” (KiH) strategy which uses introduced mutations in the heavy chains to promote heterodimerization. O’Callaghan notes that while KiH is the gold standard for classic IgG-like bsAbs, for asymmetric molecules with two different HCs and two different LCs, further protein engineering can help ensure that the final product is correctly assembled. O’Callaghan advises parallel screening of several protein engineering technologies to find the best strategy on a per molecule basis.
Beyond titer and chain pairing, other measures of product quality should be addressed including aggregation, glycan profiles, and IgG purity. “For some molecules you can certainly observe a complex interplay between titer and product quality, where a correct balance has to be found so that high titers are not achieved at the expense of product quality,” says O’Callaghan.
Importance of expression system selection and vector design
The selection of a high-performing expression system and design of the expression vector are foundational steps that can impact expression levels, product quality and scalability. Da Rosa emphasises that integrating a well-established, GMP-compatible expression system early in development can help to de-risk the drug development process.
“By starting discovery and development with a proven platform like Lonza’s GS Gene Expression System®, drug developers can build early confidence in product quality and future scalability,” says Da Rosa. “This approach can help mitigate the need for potentially costly cell line and process changes down the line and ensure compatibility with CDMO processes during later stage development”. She adds, “With a strong regulatory track record and demonstrated scalability, such systems can not only help to minimize technical and commercial risk, but may also strengthen investor confidence.” To date, Lonza’s expression systems have powered the expression of over 100 commercial biotherapeutics.
In choosing and designing an expression vector, drug developers should consider how gene order, gene copy number, and promoter choice affect titer and product quality early in the development process. O’Callaghan says, “Currently, we find the balance between titer and quality by empirically testing different expression vector designs in parallel. In the future, we may potentially be able to use in silico predictive tools to significantly reduce the upfront vector design and screening work.” He adds that the ideal clone would be one that has a high titer, produces correctly assembled molecules with negligible impurities, and does it stably over long term subculture to support commercial scale manufacturing.
Lonza has developed an optimized range of expression tools to boost expression of the diverse protein formats coming from drug discovery groups. For example, the GS Gene Expression System® is an integrated set of components to express recombinant therapeutic proteins and consists of two host cells lines, GS Xceed® CHOK1SV GS-KO® and GS Effex®; the GSquad® Pro vector system to contain the gene of interest; and the GS piggyBac® transposon technology, to insert the gene of interest into the host cell line’s genome. For drug developers at an earlier stage, like candidate screening and selection, the GS Discovery® Transient Expression System enables them to use the same expression tools from discovery to commercial stage.
New synthetic gene promoter boosts multichain biotherapeutic titers
To further improve expression vector technology for multichain biotherapeutics, Lonza has developed a novel gene promoter, available as part of the updated GSquad® Pro vector system. This new promoter, LHP-1, is a synthetic promoter developed by Lonza’s in-house R&D team. “In all our testing to date, the LHP-1 promoter is outperforming the legacy mCMV promoter in our previous system,” says O’Callaghan.
Real-world anonymized customer data from a difficult-to-express bispecific protein finds that the LHP-1 promoter was able to boost titers, on average across clones, by over 40%, increasing the titer up to ~5 g/L (Figure 1).
In another internal study, the LHP-1 promoter enabled higher titers for a Fab-scFV protein, and when considering product quality, the increased titer did not have a negative impact on chain pairing (Figure 2).
Source: Lonza internal data.
Improvements in vector technology over the years have helped the industry overcome challenges with chain pairing and expression stability leading to more high-quality multichain biotherapeutics. “Optimizing the entire bioprocess is rooted in expression vector design,” O’Callaghan says. “Beginning the development process with a strong foundation ensures that we can deliver economically viable manufacturing yields that increase patient access to these critical drugs.”
