The Biopharma Industry is Well Prepared for an Overhaul of the Lead-to-Clinic CMC Process for Antibodies

Abstract

The benefits of rapid drug development platforms were evident during the COVID-19 pandemic when investigational biological drugs were developed for emergency use on unprecedented timelines. Key speed-to-clinic approaches leveraged during the pandemic are being adopted more broadly to both accelerate development milestones and help start-up biotechs satisfy more aggressive investor outlooks. The use of well-controlled SBCs (stable bulk cultures; also referred to as “stable pools”) to accelerate the early development cycle is a scientifically sound and phase-appropriate chemistry, manufacturing, and controls (CMC) strategy that is gaining traction and regulatory acceptance. By deferring traditional single cell cloning (SCC) until a later point in development, a sponsor can utilize SBC-based platforms to generate reliable, reproducible, and representative preclinical and clinical materials months earlier relative to clone-based platforms. Wheeler Bio has developed an SBC-based mAb (monoclonal antibody) platform, Portable CMC™ that is open-source and easy to access. This article summarizes the main rationale behind SBCs and some key points to consider when evaluating a development pathway leveraging the speed of an SBC-based platform.

Next-Gen Biologics Require More Efficient Use of Capital

In 2022, the global market for biologics was valued more than U.S. $460 billion and is expected to grow at a 10.3% compound annual growth rate (CAGR) from 2023 to 2030.¹ The rising burden of cancer, genetic diseases, and autoimmune diseases, coupled with the approval of several disease-modifying therapies of these conditions, is driving the growth. Despite this, however, biological drug access remains constrained by high cost and payer complexities. Unsurprisingly, biologics represent only 2% of all U.S. prescriptions but 37% of net drug spending.² As biologics continue to flood the drug discovery pipeline, translation into widespread clinical impact will require more efficient uses of capital with minimal idle time, lower cost of goods, and improved patient access.

Outsourcing of CMC development continues to be an efficient use of capital for protein therapeutic–centered biotechnology companies, as evidenced by biopharmaceutical CDMO (contract development and manufacturing organization) industry growth.³ However, globally, start-ups are facing increased investor hesitancy and a reduction in both funding rate and magnitude, which is driving more conservative pipeline management. Investors are shifting their focus to proven sponsors that have drugs in the clinic and/or have hit clinical milestones for several drug candidates. This has led to poor alignment between the business needs of new start-up biotechs and their CDMO providers in this sector. Drug development sponsors need more cost-effective access to early development capabilities that span the translational space effectively to better bridge Seed and Series A rounds. It is incumbent upon this service sector to continuously improve processes and business models and adopt cutting-edge development strategies being devised and demonstrated by large pharma.

A Real-Time Paradigm Shift

The fastest timeline spanning Seed and Series A projects (i.e., from mAb lead candidate selection to the initiation of first-in-human (FiH) studies) is an important goal for all companies to help the patients they treat, maximize value creation, and advance their pipelines. Many large pharmaceutical companies have worked assiduously over the last 10 years to refine their CMC development strategies for more rapid clinical evaluation of lead candidates. The pandemic, especially in the United States with Operation Warp Speed funding, saw a combinatorial approach using multiple accelerative strategies to delivery extraordinary results.⁴

Among these strategies, application of SBC-based platforms was prevalent with companies developing SARS-CoV-2 neutralizing antibody products. More than six such products were granted emergency use authorization ((EUA: EUA 90, Nov. 9, 2020, EUA 91, EUA 94, EUA 100, EUA 104, EUA 111) within 11 months from the time the Public Health Emergency was declared by the Secretary of the Department of Human and Health Services (March 27, 2020) justifying authorizing the emergency use of certain drugs and biological products during the COVID-19 pandemic.⁴ In one company’s case, it took only 56 days to advance the lead antibody (April 15, 2021) to the initiation of clinical trials (June 10, 2021).⁵ Several more examples are provided below in the Sidebar: Leap-In Transposase^® Platform from ATUM.

When applied more broadly, the use of SBC-based platforms could represent a paradigm shift for start-up biotech companies seeking to disrupt the cost/time cycle of early development. Despite the relative nascency of this timeline reduction strategy, SBCs are well described in scientific literature and are wholly compatible with regulatory guidance. The ICH Q5D specifies a requirement to prepare biological products from cells cultivated from cell banks of cell substrates. The term ‘cell substrates’ refers to cell lines derived from animal sources that possess the full potential for generation of the desired biotechnological/biological products for human in vivo or ex vivo use.⁶ Cell banks can be derived from well-controlled SBC cell banks and still be in keeping with the regulatory guidance.

Well-controlled SBCs (‘well-controlled’ distinction is made here, intentionally) are pools of recombinant cell lines serving as cell substrates (per ICH Q5D) that are derived from animal cells like Chinese hamster ovary (CHO) using specialized recombineering tools (described later). Traditionally, control of the cell substrate is assured through the SCC process. However, well-controlled CHO-based SBCs can be obtained with the proper tools, resulting in homogeneous cell pools characterized by high gene copy numbers and limited phenotypic diversity.

Although SCC is a long-established standard following transfection of parental CHO cell lines to minimize phenotypic diversity (while ensuring process consistency), SCC is not specified as an indication of clonality of transfected cell lines.^6,7 Rather, the determination of cDNA sequences of the predominant transcripts is acceptable as an indication of clonality.⁷ Well-controlled SBCs have limited phenotypic diversity and thus the predominant cDNA sequences are comprised of the target recombinant genes. Due to the relative ease of derivatizing a comparable clone with matching process and product attributes, the homogeneity of well-controlled SBCs de-risks the deferred SCC until after phase I. In other words, when the SBC comprises recombinant cell lines with a limited population diversity, the platform drug substance process doesn’t care whether the cell substrate is based on SBCs or on clones. This assures regulators that well-controlled SBCs possess the full potential for generation of the desired biological products, per ICH Q5D. As such, sponsors can be presented early on with an opportunity to save significant time, without risk to patient safety nor regulatory compliance (provided access to well-controlled SBCs).

SBC-based drug development platforms were hugely impactful during the COVID-19 pandemic response when multiple developers of SARS-CoV-2 neutralizing antibody products received EUA for their products using SBC-based cell substrates. In one example, it took only 56 days to advance the lead antibody (April 15, 2021) to the initiation of clinical trials (June 10, 2021).² Several more examples are provided below (see Sidebar: Leap-In Transposase^® Platform from ATUM).

For non-COVID products, the use of SBC-based cell substrates to generate toxicology and clinical materials would represent a paradigm shift for start-up biotechs seeking to disrupt the typical cost/time structure of early development. A recent (2022) industry survey revealed that 29% of large pharmas already use SBCs to produce toxicology supplies of non-COVID products and that another 39% are considering said SBC use within the next five years.⁸ Thirty percent of large pharmas are considering their use for clinical products in the next five years.⁸ Importantly, the majority of responders to this survey were not small risk-tolerant companies but rather a broad selection of development- and commercial-stage innovators, including AbbVie, Alexion, Bayer, Biogen, Boehringer-Ingelheim, CSL, Eisai, Gilead, GSK, ImmunoGen, Incyte, Janssen, Kyowa Kirin, Leo Pharma, Merck, Novo Nordisk, Pfizer, Regeneron, Roche, Samsung Bioepsis, Seagen, Takeda, UCB, and Vir Biotechnology.⁸
The survey results represent the view of medium-to-large companies that have the resources to be more conservative yet have acknowledged the value of their developmental approach. The results are consistent with the view that over half of the medium-to-large companies in our industry will be using SBCs in the next few years to add significant value to development programs.

Better Business Alignment Needed Between Sponsors and CDMOs

Start-up biotechs need better ways to bridge the translation from preclinical to clinical development. Since outsourcing is compulsory for the sake of capital management, the onus is squarely on the pharma services sector to ensure that the most current, capital-efficient CMC development platforms are well designed and accessible to start-ups. As such, the CDMO sector needs to align with a broader, post-pandemic SBC-based time-saving strategy. Start-ups do not only need CDMOs to offer efficient platforms; they also need access to their supporting data sets to facilitate risk assessment and regulatory filings. Vir Biotechnology recently described a scenario of saving 5–6 months off the early development cycle using an accelerated platform featuring SBCs.⁹ At an illustrative “burn rate” of $250,000 per month, this company would save significant capital and help keep its other pipeline molecules advancing. Another example of an accelerated platform featuring SBCs is Wheeler Bio’s Portable CMC™ platform. This platform leverages large data sets garnered from both SBCs and derivative clones using Leap-In Transposase^® (ATUM) technology to enable well-controlled SBCs, high titers, process robustness, scalability, and speed-to-clinic.

Points to Consider for SBCs

It is essential that speed-to-clinic by virtue of SBCs does not compromise the safety, identity, strength, purity, or quality (SISPQ) of investigational biologic drugs. To minimize additional delays and costs during development programs, preclinical materials must be representative of clinical materials. Therefore, to ensure that SBCs provide a reliable cell substrate, the underlying tools and technologies supporting their generation need to be well tested and validated. Several of these have been discussed in the literature and are available “off the shelf” today through vendors and pharma service providers like Wheeler Bio in Oklahoma City, OK.

The CLD Process
The CLD process is a series of time-consuming steps requiring extensive infrastructure, expertise, and recombineering tools. The process enables the developer to interrogate thousands of single cells before identifying a good producer clone with desirable attributes (process and product), from which a clonally derived cell substrate is then established, tested, banked, and stored. Historically, the CLD process can take up to six months or even a full year to complete depending on the complexity of the target proteins.

The CLD process begins with the transfection of a well-characterized parental cell line with expression plasmids encoding the desired recombinant protein product. Recombinant cells are selected by virtue of plasmid-bearing selectable markers, such as glutamine synthetase (GS) or dihydrofolate reductase (DHFR) genes (to enable methionine sulfoximine (MSX) in a GS knockout background or methotrexate (MTX) selection in thymidine-lacking medium, respectively). Gene delivery to the host cell genome, depending on the recombineering tools being used, is mediated by one of several types of recombination mechanisms, expanded on below.

Following selection to generate a pool of recombinant cells, single recombinant cells (clones) are isolated through a series of dilution, single-cell sorting, imaging, and analysis steps. New technologies, such as the Solentim Ecosystem (VIPS™, Cell Metric^®, ICON™, STUDIUS™), are available to improve the automation, throughput, consistency, repeatability, and success rates of the CLD process. This equipment ecosystem is integrated at ATUM and Wheeler Bio.

Advancements in CLD recombineering tools have been key enablers allowing the derivation of robust cell substrates exhibiting enhanced titers, consistent process performance, and consistent product quality. There are three types of CLD tools that have made it possible to establish SBCs from the lengthier CLD process, although the second two mentioned below are more reliable than the first. SBCs can be banked and used later to resume the full CLD process (i.e., “deferred cloning”) without having to repeat the initial transfection and selection steps of CLD. When integrated with a robust upstream, downstream, and analytical platform for drug substance manufacture designed based on SBCs, the predictability or control of the SBCs aids the robustness and control potential of the entire integrated platform.

Random Integration (RI)
The majority of CLD processes still involve RI for inserting mAb expression plasmids into the genome of CHO cells. RI can be used to generate SBCs; however, there are several recombinational liabilities causing truncations of expression cassettes, random sequence scrambling, and concatamerization that massively limit their robustness and hence utility as a source of well-controlled SBCs enabling use of a deferred cloning strategy.

Targeted Integration (TI)
TI is a precise form of genetic recombination also referred to as site-specific recombination. Several large pharmas have developed cell lines using targeted integration of mAb expression vectors.^10,11 TI can be facilitated by site-specific recombinases and specialized expression vectors containing recombinational sequences, but resulting copy numbers are low. A few relevant tools include CRISPR-associated protein 9 (CRISPR-Cas9), zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). ZFN- and TALENS-based genome modification methods are costly and time-consuming, extending CLD programs out by several years to allow time to develop and validate genomic target sites. CRISPR-Cas9 is the simplest, most versatile, and most precise method of genetic manipulation; however, there are limitations on commercial licensure.

Semi-Target Integration (STI)
STI is another precise form of genetic recombination and a very robust and accessible modality for SBC generation. STI is mediated by transposase enzymes and specialized transposon expression cassettes, such as the Leap-In Transposase^®.¹² Eleven of the top 20 pharmas, including Janssen, Novartis, AbbVie, Bristol-Myers Squibb, Merck, and Boehringer-Ingelheim, have utilized the Leap-In system, as have several CDMOs including Wheeler Bio, Rentschler, and Just-Evotec. Leap-In is well documented as supporting the generation of consistently high-expression SBCs with homogeneous phenotypes.

Integrated Platform
The intrinsic advantage of well-controlled SBCs is the limited diversity in phenotype. This facilitates the ability to isolate a derivative clone that responds in the same way to bioprocess controls. Because the SBC is homogeneous, the screening effort to identify a comparable clone is significantly reduced. It should be noted that, in some studies, transgene sequence variants can exist in SBCs as determined via next-generation sequencing. Therefore, further data should be gathered on late-stage processes to gain more insight into genetic drift in SBC-based cultures. However, SCBs would not be unique in exhibiting genetic drift. The CHO genome is well known for its plasticity. It has been proposed that there may be less sequence diversity in SBCs than in clones, particularly if generated using TI or STI, which significantly limits the diversity of clones in the population and concomitant risk.¹³ In general, the focus for speed-to-clinic should rest on phenotype integrity (i.e., batch-to-batch product quality and process performance) rather than on genotype integrity, as the tools exist to later ensure clonality and quality.¹⁴ Indeed, STI minimizes cell-to-cell variability, makes the pool and clones more similar, and reduces the effort in finding the ideal clone.

Overall, given the current tools and technologies available and their associated case studies in the public domain, expression of recombinant therapeutic proteins in SBCs for use as toxicology supplies and early clinical development materials is a practical means of dramatically speeding up new drug development without compromising quality, efficacy, or safety.

Well-controlled SBCs can be generated using the early steps of the CLD process and have been well described in scientific literature for nearly a decade. The recombineering tools utilized are important points to consider when contemplating SBCs, since not all tools are useful for enabling well-controlled SBCs.

Old Scientific Precedent for SBCs

SBCs emerged long before the pandemic in the literature, which provided a scientifically valid platform enabling development on unprecedented timelines. Large pharma companies, including Merck,¹⁴ Amgen,¹³ Boehringer Ingelheim,¹⁵ Bristol-Myers Squibb,¹⁶ Pfizer,¹⁷ Genentech,¹⁸ and Biogen,¹⁹ and global CDMOs, including WuXi Biologics²⁰ and Lonza,^21,22 have all examined the utility of SBCs for the generation of toxicology and early-phase clinical materials based on innovative academic research conducted over many years.^12,23,24 This foundational science provided evidence of process consistency, product quality, and phenotype comparability between SBCs and derivatized clones (i.e., recombinant protein expression levels, cell culture properties, generational stability, bioprocess performance, and product quality attributes). Additionally, research directed to the investigation and characterization of genomic instability of CHO clonal populations has resulted in the suggestion that the very notion of a clonal cell substrate, in the context of ICH Q5H, could be considered moot.²⁴

New Clinical Precedent for SBCs

A significant amount of clinical data was collected across the pandemic that effectively correlates the foundational science with clinical drug safety and efficacy. In its Guidance for Industry: Development of Monoclonal Antibody Products Targeting SARS-CoV-2, Including Addressing the Impact of Emerging Variants, During the COVID-19 Public Health Emergency published in February 2021, the FDA supported the use of a stable cell pool in lieu of a clonally derived cell line to generate early clinical batches for early-phase development.²⁵ Dr. Maria-Teresa Gutierrez-Lugo, an FDA product quality review chief in the Office of Biotechnology Products, recently shared similar views on SBCs.⁴ Notably, Gutierrez-Lugo emphasized caution in assessing the appropriateness of certain CMC regulatory strategies in the context of their associated risk and benefit, the intended purpose of the product (treatment vs. prophylaxis), the uncertainties present, the mitigation strategies, and the available knowledge of the disease and technology being applied.

Leap-In Transposase^® Platform from ATUM

ATUM’s Leap-In Transposase^® enables robust, seamlessly implemented, and rapid generation of robust high-titer SBCs within weeks of transfection. Genome integration occurs via an enzymatic cut-and-paste mechanism into transcriptionally active chromatin, with the integrity of the integrated transposon cassettes retained intact and stable.

More specifically, the transposase enzyme recognizes inverted repeats that flank the transposon that contains the gene of interest (e.g., mAb), removes that entire piece of DNA from its current location (e.g., plasmid), and inserts it at a new site harboring a short nucleotide recognition sequence within the target genome. This technology allows targeting of many expression cassettes — titratable and can exceed 60 copies — to different locations within the genome. The resulting clones are both genetically and phenotypically stable, and the generated SBCs are representative of the final clones due to the homogeneity of clonal phenotypes within the SBC.

With predictable growth, titer, and product quality across scales and compared to final clones, these representative bulk cultures can be readily used for early upstream and downstream process and analytical development, tox lot generation, and potentially phase I manufacturing.

Leap-In Transposase^® was launched in 2018 and has been used in over 150 projects via ATUM’s service business and has been deployed to over 40 active licenses with many large pharmas and emerging biotech companies. With 22 regulatory filings across three regulatory jurisdictions in the past three years, including for phase II/III trials; this is the leading and industry-standard transposase platform. Likewise, the platform is very well suited for large, complex, and routine molecules, including traditional proteins, mAbs, and fusion proteins, as well as the various multi-specific antibody formats bispecifics, and it can be applied within a wide range of host cell types, both CHO and non-CHO in origin.

Notably, Leap-In Transposase^® was used to enable accelerated CGMP manufacture of multiple COVID-19 mAbs.^13–15 In one case, selection was completed in approximately three to four days and afforded SBCs providing titers of 2.5–5 g/L.²⁶ Material was shipped to the client’s CDMO within 38 days of ATUM receiving the sequence of the mAb of interest. At the CDMO, via an intensified fed-batch process, a titer of >12 g/L was obtained, and an IND was approved. In another program, a stable pool with a titer of 6.0 g/L was first used to produce a COVID-19 mAb for preclinical and safety testing at the 200-L scale and then to manufacture phase I material at the 2000-L scale within 4.5 months.¹⁴ GMP manufacture of another COVID-19 mAb was achieved within three months using an SBC generated using Leap-In Transposase^®.¹⁵

Portable CMC™ Platform

Over the past few years, investors have become increasingly risk averse and are asking sponsors to deliver more data with the same capital. Sponsors are now expected to produce initial clinical data from early rounds of funding (Seed and Series A), which traditionally would have been reserved for later rounds.^27,28 In the face of rising R&D costs and speed-to-clinic pressures by investors, the relationship between start-ups and service providers is critical.

Portable CMC™ is a mAb development platform that is purpose-built for early development but scalable for commercial supply given the underlying technology stack, process robustness, and proven control strategies. Portable CMC™ efficiently translates from discovery to scalable manufacturing (gene-to-GMP) while reducing CMC risk across the entire development lifecycle by virtue of a Quality-by-Design (QbD) platform approach. Additionally, the platform is open source, which enables unincumbered technology transfers and regulatory support.

Portable CMC™ comprises three service modules with 10 work packages designed to advance four lead mAb candidates to a clinical lead while delivering ample materials and module 3 inputs. Asynchronous programming of the work packages enables rapid, SBC-based toxicology and clinical material generation, as desired by the client. Traditional SCC programming exists as well.

When using the SBC strategy, a post-phase I process change from SBC-based cell substrate to an SCC-based cell substrate is relatively easy. Given the deep characterization of the platform and supporting data sets, it is relatively painless to derive a narrow set of top clones from SBCs and know how to run/optimize the platform process to achieve the same product quality as that obtained from the pre-change SBC. This ensures that pre-change preclinical and clinical data are relevant to post-change clinical data.

SBCs: Towards Maximum Acceleration

More than ever before, developers are looking for more speed and agility in the face of rising R&D costs and escalating investor concerns over CMC risks. However, such timeline benefits must not risk the quality or safety of investigational biologic drugs.

New tools, technologies, and development strategies are available today to accelerate the translational process between discovery and the initiation of clinical trials. To have broad impact, these approaches need be readily accessible to start-ups, allowing industry-wide adoption and impact on the drug development process.

We argue here that well-controlled SBCs, enabled by STI and TI, allow for significant improvements in speed and agility without compromising product quality nor safety. By incorporating SBCs into biomanufacturing platforms, CDMOs can help democratize access to this important accelerative strategy while fostering a larger impact on the industry.

When contemplating the adoption of this approach for speed-to-clinic, start-ups will now be able to leverage considerable scientific and clinical evidence, as well as assurance of regulatory buy-in. SCC was established by the industry as a standard for INDs in the late 1990s. This standard, however, did not arise in response to any specific safety or quality incident (but rather out of an abundance of caution). SBC-based cell lines still satisfy the original regulatory requirements for cell lines/cell substrates contemplated in PTC 1993, 1997, and ICH Q5D.^4,5

We also noted here that SBC-related efficiencies stem not only from deferring SCC but also from the relative ease of finding the ‘one clone’ later due to the homogeneity of the cellular phenotypes within the SBC.

We also highlighted the common wisdom that CHO genomes naturally vary significantly and often as function of generational age. As a result, from a genotype perspective, there may not be a significant difference between a “clonal CHO cell” derived from RI and a highly homogeneous, well-controlled SBC (i.e., one generated from transposons). The stability of the CHO genome has been challenged in the literature to the extent that the practicality of demonstrating clonality is questionable.³ Nevertheless, the FDA guidance (from the 1990s) has had a lasting impact on risk perception of cell substrates, which has led to a limited exploration of alternative cell substrates – until now. Studies specifically focused on comparing the expression performance and product quality of non-clonal SBCs to those of clonal cell lines provide increasing evidence towards addressing concerns about the heterogeneity of these cultures and have shown that SBCs remain sufficiently stable for the uses described herein. It has further been shown that the SBCs consistently produce high-quality recombinant protein products at small to large scale for many different molecule types while reducing timelines to toxicology studies and early-stage clinical trial materials by a factor of two or more.

As with any approach used for drug development and manufacture, the risks and benefits must be appropriately balanced. The use of STI (such as is achieved using the Leap-In Transposase^® technology) coupled with a standard cell culture process (such as Portable CMC™) helps mitigate potential risks, particularly those related to product comparability and cell bank characterization. This speed-to-clinic option can now be evaluated by start-ups armed with a significant body of scientific literature, clinical safety, and regulatory track record.

Acknowledgements- The authors would like to thank several individuals who provided thoughtful comments and useful discussions; Lorenz Hasler, Jean Bender, Howard Levine, Marc Helouin, Yvonne Lungershausen, Stewart McNaull, Brian Berquist, and David Alvaro.

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