September 2, 2022 PAO-06-022-CL-12
Innovators of INDs (investigational new drugs) often face uncertainties in forecasted dosages, titers, process yields, and manufacturing costs. Coupled with the current biotech bear market, the unpredictability of biology, the increasing complexity of therapeutics, and the stringency of regulators, innovators will continue to face extraordinary challenges while moving toward their first-in-human (FIH) studies. Emerging biopharma companies, where the majority of innovators are located, are further challenged when outsourcing to a network of providers and suppliers whose complex processes are not integrated or harmonized. These innovators take on huge risks with every single outsourcing decision made. More trust and transparency are needed in the pharma services sector to ensure that the innovators’ pipelines advance as fast as possible to deliver clinical impact. It is time for new, disruptive approaches in the outsourcing space and novel business models to give innovators a better chance of success. To this end, Wheeler Bio has developed a deployable solution called Portable CMC™.
Portable CMC™ is a state-of-the-art, open-source, bundled development platform that integrates with CROs’ antibody discovery processes to accelerate clinical impact. With Portable CMC™, Wheeler Bio is helping innovators reduce manufacturing risks by giving them unfettered access to critical tools and a validation-ready method for the preparation of drug substance. Their platform delivers manufacturing readiness and features three open-source, digitalized processes: Lead Selection, Clone Selection, and CDMO Selection. This article will focus on Clone Selection. The open-source nature of the platform stems from the freedom that customers have when selecting the best outsourcing provider for clinical manufacturing. Customers have the choice of retaining Wheeler Bio for GMP manufacturing or taking their Portable CMC™ process to another CDMO — with no strings attached- but assurances of scalability, quality, and speed.
Since only 0.1% of preclinical drug candidates reach the market, and many failures are due to lack of efficacy and safety (immunogenicity), stability and aggregation issues can greatly increase cost and delay timelines. This is the main driver for why Wheeler is moving critical tools further upstream into the late-discovery space — to give innovators access to scalable manufacturing technologies, like Chinese hamster ovary (CHO) cells and safe expression constructs that produce genetically stable recombinants.
Per Section 1.2 ICH Q5D Derivation and characterization of cell substrates used for production of biotechnological/biological products, the cell substrate is the cornerstone of CMC, so its quality control is an imperative from the very beginning: “Historically, some quality concerns for cell-derived biological products have originated from the presence of adventitious contaminants or from the properties of the cells used to prepare the product. Recombinant DNA (rDNA) – derived products also carry quality concerns regarding the expression construct contained in the cell substrate. Thus, it is well established that the properties of the cell substrate and events linked to the cell substrate can affect resultant product quality and safety and, further, that effective quality control of these products requires appropriate controls on all aspects of handling the cell substrate.”
For these reasons, Wheeler Bio uses proven cell lines, expression constructs, and gene delivery methods, as well as cutting-edge workflows from Solentim and Sartorius for ensuring clonality and manufacturability screening.
Taking a step back to clarify concerns with traditional clone selection for biotherapeutic manufacturing, we look at the issues in more detail. Early on in the production of biologic products, quality concerns have involved the presence of adventitious contaminants or the properties of the cell lines used to generate the product, which led to the establishment of the ICH Q5D Guideline Derivation and Characterisation of Cell Substrates Used for Production of Biotechnological Biological Products. This guidance establishes standards for cell line development (CLD), including the crucial early step of generating an optimal cell substrate — a cell line with desired characteristics that will be utilized in the generation of a robust stable cell line for biotherapeutic production. Since cell substrate selection can strongly influence the safety and purity of the biological product, it is critical to establish an ideal substrate from the outset and to develop analytical methods based on that substrate. Generating a cell substrate later and outside cGMP quality design principles may create the need for additional documentation, testing, and production steps later in development.
Downstream of host cell selection, the processes used in conventional CLD often present significant bottlenecks in biomanufacturing. The process begins with the transfection of host cells with recombinant plasmids encoding the therapeutic protein, followed by selection of a pool of stable transfected cells that express the protein of interest. Subsequently, large numbers of individual clones are isolated and screened, with lead clones often being selected with a focus on high protein titer. Each selected clone must then be confirmed, validated, and characterized, after which validated clones are expanded and scaled up. There are a number of inefficient steps in this workflow, particularly relating to the clonal screening to separate high-producing protein secretors from low producers, which involves screening thousands of clones over months and requires excessive resources.
Additionally, conventional CLD is not grounded in alignment between drug discovery and manufacturing. Typically, a discovery company will use whatever host and expression system they can access. A considerable amount of time may need to be dedicated to tech transferring these systems to the manufacturer, as well as addressing associated regulatory concerns. Wheeler Bio is partnering to align discovery CROs and manufacturers to ensure that customers move into scalable systems as soon as the lead molecules are identified.
Wheeler Bio’s Portable CMCTM Clone Selection service — an innovative CLD service — begins the physical workflow with the synthesis of a recombinant transposon containing the gene-of-interest (GOI) flanked by transposase recognition elements, inverted-terminal repeats (ITRs). The transposase enzyme recognizes the inverted repeats, cuts out the piece of DNA between them, and places it at various preferred sites within the target genome.
Following sequence verification of the transposon construct, host cells are transfected with a proprietary mixture of the new DNA construct and an exogenously supplied mRNA reagent encoding the cognate transposase enzyme. Once inside the cell, the mRNA gets translated to a functional transposase enzyme, which binds the ITRs on the transposon, forming a mobile transposon. Transposons are highly mobile and readily integrated at accessible locations in the chromatin. Transposon-mediated gene delivery is associated with enhanced protein expression levels and potentially improved cell line stability, due to the underpinning molecular genetics of transposition, which leads to stable pools that are potentially highly representative of the prospective final clones. This is one aspect that distinguishes the Wheeler Bio strategy from conventional CLD processes, which often use a random integration approach for gene delivery, leading to significant heterogeneity across the clonal pool and increasing the need to screen many more individual clones. In contrast, with the Wheeler Bio approach, the customer will gain access to representative material much earlier in the life cycle. It takes only a few weeks to complete the vector design and engineering, one month to generate the pools, and another month to perform stable clone selection. The next phase is validation and ranking of the clones, which is achieved using a high-throughput screening approach using the Sartorius Ambr® 15 Microbioreactor System. The best candidates are then subject to stability testing. An added advantage to the Portable CMC™ platform is that, in parallel to stability assessment, the customer has the option to accelerate process development using the Sartorius Ambr® 250 Bioreactor System, thus building confidence in clone selection for manufacturing scale-up and potentially reducing time to clinic.
The entire cell line development process — from receiving the customer’s sequence through stability assessment — takes approximately eight months, at the end of which the customer receives a comprehensive report on their clones (potentially including the optional time-saving Ambr® 250 assessment) and a development cell bank (DCB). Between the in-licensed technologies and the extensive experience of the team, Wheeler Bio is at the forefront of the industry in terms of titers, stability, and cell substrate manufacturability alignment, helping to make Wheeler Bio a compelling option that is similarly enabled to incumbent mega-CDMOs.
Indeed, Wheeler Bio’s CLD leadership team has over 40 years of experience in platform engineering and cell line development for mAbs, bispecifics, antibody fragments, fusion proteins, and enzymes in the areas of protein expression, vector optimization, host-cell engineering, stable cell cloning, stability assessment, and product quality.
To take a deeper dive into Wheeler Bio’s differentiators, the following sections expand on key aspects highlighted above.
Discovery overlap is enabled by Wheeler Bio’s key differentiator; a unique partnering model (Portable CMC™ Collaboration Agreements) with discovery CROs, which can optionally put Wheeler Bio’s people, processes, and equipment inside of the CRO, giving customers unprecedented access to manufacturing capabilities. Alternatively, the customer will have the option to adopt Portable CMC™ lite, where the same services are provided through physical resources located at Wheeler Bio’s hub in Oklahoma City. By accessing CMC bedrock tools like CHO expression technology and turn-key analytical methods, customers can more quickly evaluate the developability and manufacturability of biotherapeutics and more readily advance their lead molecules. In doing so, Wheeler Bio’s novel approach helps innovators progress more smoothly into clinical development and reach their FIH studies faster.
Wheeler Bio will initially be working with the CHOSOURCE™ expression platform for cell line development, which we have in-licensed from Horizon Discovery. This validated cell line is widely used in biologics drug development, with over 100 licenses worldwide since 2016 and 20 confirmed regulatory filings globally. In addition to this proven track record, CHOSOURCETM offers a high degree of adaptability; it has been successfully used with various vector systems and multiple media and production processes to generate a wide variety of biologic formats (including monoclonal antibodies, Fc-fusions, bispecifics, etc.). CHOSOURCETM has the added advantage that it comes with a full cell line history package to support regulatory submissions when the time rises.
One of the ways that Wheeler Bio is compressing the cell line development timeline is through the use of transposon-based gene delivery, a cutting-edge approach that enables the assessment of product quality and manufacturability from transfectant pools. The initial pools that are generated early in the selection process are representative of the stable clones produced later on, so there isn’t a need to go through a laborious clone isolation step before getting a representative data set.
Application of this technology thus allows for targeting of many expression cassettes — between 40–60 copies — to different locations within the genome. With that number of copies, the resulting clones are more stable. In addition, the pools that are generated are more representative, because there are so many copies incorporated into the genome.
The beneficial aspect of transposon technology, which generates the pools that are representative of final clones, is the “cut-and-paste” basis of the transposase mechanism. Unlike random integration, where there is wide variety in the actual fragment of the expression vector that is incorporated into each clone’s genome, the fragment of DNA introduced by transposon integration is identical in each case. Hence, the system significantly reduces the clone-to-clone variability and increases the efficiency of final clone selection.
There are other advantages over traditional vector integration approaches as well. In addition to the large number of copies, the sites of integration of those copies are typically areas of the genome that are highly actively transcribed, which results in higher expression levels. Furthermore, since the transposase enzyme is necessary for translocation of the transposon, and it is encoded by short-lived mRNA at the time of transfection, GOI integration into the genome is more stable. This higher stability of numerous copies of the GOI also means that titers don’t drop off, even if a few copies are lost. There also isn’t a limit to the size of the DNA fragment with the transposon system like there is with viral integration systems, which enables the inclusion of more genetic material encoding the gene(s) of interest.
Although the use of transposons is a relatively new technology for biologic drug production, it has been used and validated for recombinant protein expression for several years. On the basis of current data, it is a robust approach that produces stable cell lines while saving significant development time and is being embraced by the manufacturing community.
For CDMOs like Wheeler Bio, using this innovative transposon technology can produce significant benefits, because fewer clones or pools need to be screened to identify a lead molecule, making it both faster and cheaper for customers. In the end, our approach to clone selection delivers on the three key technical demands: speed, titers, and stability, ultimately resulting in better economics and a better chance of success for the customer.
The cornerstone of Wheeler Bio’s Portable CMC™ platform is a next-generation cell line development capability that features the Solentim Ecosystem comprising the VIPS™, Cell Metric®, ICON™, and STUDIUS™ technologies.
Wheeler Bio will be among the first labs in the world to integrate the entire Solentim Ecosystem — a comprehensive system from cell seeding to clone selection to clonality report generation — into its workflow. The first instrument in the ecosystem is the VIPS™ double-lock technology. This is a high-efficiency and high-viability single-cell seeder that photographs each nanodroplet as is it placed into a well to confirm clonality. The cloning efficiency is therefore much higher with this instrument than it would be with backspace technology or manually with limiting dilution. In addition, with 70–80% of wells having a single clone in them, less incubator space, handling, and consumables are required.
The Cell Metric® instrument is the second imaging part of the technology. It takes daily pictures of each well so that the growth of each clone can be tracked over two weeks or until there is sufficient material to assay for titer. The ICON™ instrument — the newest component of the ecosystem — is a multi-assay platform designed to monitor titer and cell viability through the clone scale-up stages.
The entire Solentim Ecosystem is driven by the STUDIUS™ software package, which collates all of the clone selection data produced by the different instruments into a verified data package that shows every step of the process from single cell isolation to healthy clone productivity to enable and track decision making. Importantly for Wheeler Bio, the Solentim Ecosystem output is suitable for clients to submit to regulatory authorities. The fact that the Cell Metric® instrument has been widely used in the industry for several years and is thus well-validated is also invaluable. The Solentim Ecosystem package will enable us to ensure to our clients that they have a highly productive clonal cell line.
Once individual clones have been isolated with the Solentim Ecosystem, second-generation Ambr® 15 Microbioreactor Systems from Sartorius will be used to identify high titer robust clones. The Wheeler Ambr® system enables parallel assessment of up to 48 samples (or 24 clones in duplicate) and includes integrated controls and integrated metabolite analysis for fast-tracked implementation of quality-by-design principles. The microbioreactor design also aligns with production-scale manufacturing solutions for seamless scale-up. With this system it is not only possible to select better clones more quickly, but processes can be more readily scaled as well.
The customer will also have the option to take lead clones coming out of the Ambr® 15 Microbioreactor System and perform early process development in the larger-volume Ambr® 250 Bioreactor System, thus validating clone scale-up and promoting lead clone identification. This optional service is performed in parallel to the clone selection stability assessment, helping to reduce the potential time to the clinic for the customer’s molecule by an average of six weeks.
The ability to readily scale processes from the Ambr® 15 microbioreactors (and optional Ambr® 250 bioreactors) enables Wheeler Bio to integrate drug substance processes development with cell line development. From the early stage of generating cell lines, we will already be looking at how they will move through manufacturability and into production. The integrated Ambr® systems are great screening tools to ensure that the clones that we generate are going to be good for manufacturing later.
In addition to offering the Portable CMC™ platform for non-GMP activities, Wheeler Bio is building GMP capacity in their headquarters in Oklahoma City. While construction of the company’s 35,000-ft2 GMP facility, which is slated to complete in Q2 2023, the Portable CMC™ platform will create opportunities for Wheeler Bio to interact with customers and build credibility in the industry. In the short term, Wheeler Bio is seeking to collaborate with other CDMOs, providing cell line development services and then transferring the lead clones to a partner CDMO.
As mentioned previously, Portable CMC™ is an open-source platform, and the customer is welcome to take Wheeler Bio–generated cell lines to their CDMO of choice. Once such a decision is made, Wheeler Bio will support the customer’s request and fully engage in the transfer. However, the ultimate goal is to give those customers the option to keep their projects in-house at Wheeler Bio.
Having described Wheeler Bio’s Portable CMC™ in the sections above, Wheeler Bio’s first CRO partnership is with the Waltham, Massachusetts–based CRO, Alloy Therapeutics. Alloy is a premier discovery company with several offerings, including monoclonal antibody (mAb) discovery services using proprietary ATX-Gx™ (humanized) mice. Wheeler Bio and Alloy are collaborating to validate Portable CMC™ and to establish a set of key initial questions that will garner critical information needed to facilitate the seamless transition from research to manufacturing.
Wheeler Bio does not plan to stop at the new technology that we are incorporating today — our model is to look at the next innovation and see what we can bring in to offer value to our customers and offer improvements to our processes. We are actively attending conferences, learning about new aspects, talking to reps, and investigating what new technology might be on the horizon.
Wheeler Bio’s clone selection service will be launched in July, 2022 from our 3,500-ft2 cell-line development lab at the Oklahoma University Research Park and a Boston-based R&D center inside Alloy Therapeutics in Waltham, Massachusetts, for vector development and small-scale protein production.
Lead clone selection services will be introduced in September 2022 and CDMO tech-transfer services will roll out early in 2023. The new production facility that Wheeler Bio is building will feature two closed-processing drug substance lines, cell-banking capabilities, and a process development lab with extensive analytical capabilities and AI-powered workflows.
Dr. Hamilton has over 20 years of industrial and academic research experience delivering on key technical milestones in both small biotech and large pharma environments. He has an extensive track record of leading successful platform and product development projects resulting in numerous regulatory filings and patent awards. Across the span of his career, he co-authored over 25 research articles, including two first author manuscripts in the journal Science. His technical expertise is in protein production, glycobiology and host cell engineering, and his areas of interest are in innovation and technology development around optimized workflows and new product development. He completed his postdoctoral research at Dartmouth Medical School in Hanover, NH. Steve earned a Ph.D. in biochemistry and molecular biology from the University of Leeds, UK, and a B.Sc. in biochemistry from Heriot-Watt University in Edinburgh, UK.