Effects of the COVID-19 pandemic: new approaches for accelerated delivery of gene to first-in-human CMC data for recombinant proteins

ABSTRACT

The COVID-19 pandemic highlighted the urgent need for life-saving treatments, including vaccines, drugs, and therapeutic antibodies, delivered at unprecedented speed. During this period, recombinant antibody research and development cycle times were substantially shortened without compromising quality and safety, thanks to prior knowledge of Chemistry, Manufacturing and Controls (CMC) and integration of new acceleration concepts discussed below. Early product knowledge, selection of a parental cell line with appropriate characteristics, and the application of efficient approaches for generating manufacturing cell lines and manufacturing drug substance from non-clonal cells for preclinical and first-in-human studies are key elements for success. Prioritization of established manufacturing and analytical platforms, implementation of advanced analytical methods, consideration of new approaches for adventitious agent testing and viral clearance studies, and establishing stability claim with less real-time data are additional components that enable an accelerated successful gene to clinical-grade material development strategy.

KEYWORDS:

• Adventitious agents
• cell line development
• clonality
• developability product characterization
• manufacturing platform
• monoclonal antibodies
• stability studies

Introduction

By end of December 2022, the Johns Hopkins coronavirus resource center reported 658 million total cases and 6.7 million deaths since the initial COVID-19 outbreak occurred in Wuhan (China) in November 2019. Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the causative agent of COVID-19. Historically, coronavirus caused two previous outbreaks, SARS in 2002 and the Middle East respiratory syndrome (MERS) in 2012.¹ Whereas the first two outbreaks were quickly contained, SARS-CoV-2 spread globally due to its higher virulence and reproduction factor, and despite the substantial resources deployed to identify and develop therapeutics in record time. These data show that new viruses can emerge and may dramatically affect the global human population in a short period.

The COVID-19 pandemic highlighted the urgent need to make life-saving treatments in a timely manner. One approach was to repurpose approved drugs to treat COVID-19 relatively quickly.² The alternative approach was to develop targeted drugs such as vaccines based on mRNA or adenoviral viral vector, and monoclonal antibodies (mAbs) directed against SARS-COV-2 motifs.^3–9 Whereas new targeted drugs may show higher efficacy in principle, classical development pathways would not have been adequate, considering the speed at which the virus spread across the world. Thus, novel approaches to preclinical and clinical development and CMC were used to bring new drugs to patients at unprecedented speed.^10–15 As Kelley et al. noted, “Pandemic urgency led to novel development approaches that reduced the time to clinical trials by 75% or more without creating unacceptable patient or product-safety risks”.¹⁶

Here, we discuss recent advances in generating preclinical and clinical grade materials and CMC data to accelerate first-in-human trials for recombinant biotherapeutics, such as mAbs, Ig-fusion proteins and other protein-based moieties, intended to treat established diseases, diseases with critical unmet medical needs, or new emerging infectious diseases.¹⁷ Novel development approaches were used by companies to rapidly develop SARS-COV-2-targeted mAbs to treat COVID-19 disease in the context of an emergency use authorization (EUA) by the Food and Drug Administration (FDA) or conditional marketing authorization by the European Medicines Agency (EMA).^18–20 CMC levers enabling acceleration of development are discussed below.

Product knowledge and developability

Despite not directly affecting the speed at which a product moves through early stages of development, building strong product knowledge at the start of a development program is key to minimizing the risk of finding in later phases a product feature that could jeopardize its manufacturability and the overall program.

Developability assessments address not only the design of molecules, but also their suitability for manufacturing, storage, and administration. The developability assessment at the design stage of a new biological entity may lead to engineering out hot spots for degradation or undesired modifications to improve the manufacturability and the stability of a new product.^21,22 In recent years, computational approaches with the support of advanced analytical tools have been developed to predict, for example, the propensity of a molecule to undergo self-interaction, aggregation and formation of particles, chemical modifications, such as oxidation and deamidation, propensity to cleavage by proteases, conformational stability, sequence-based isoelectric point, and changes affecting the electrical charge.^23–32 These predictions could be further confirmed by carrying out forced degradation studies on the first grams of material produced that are traditionally used to identify product variants and degradation pathways and to support analytical method development.^33,34 In that context, forced degradation studies help assess the criticality of quality attributes through a comprehensive understanding of their severity of impact, their process- or storage-driven variability (occurrence) and the testing strategy/method capability (detectability).^35,36

Generating product knowledge early can also ensure that, in accordance with the quality by design concept and paradigm “the product makes the process”, a manufacturing platform and the associated panel of analytical methods fit with the product characteristics to improve speed and robustness.^21,37

Selection of a parental cell line with appropriate characteristics

Prior to generating a manufacturing cell line, the parental cell line should be chosen carefully. The choice of the parental cell line depends on manufacturing constraints and development strategy. For high demand products, Chinese hamster ovary-K1 (CHO-K1) or subsequent cell lines, such as CHO-K1SV have superior potentiality of expression compared to CHO-DG44 and CHO-S, but more extensive clone selection may be necessary, i.e., more clones may need to be screened to find ones with much higher levels of expression.^38–40 This inconvenience could be minimized through robotization and miniaturization of the initial screening processes post-transfection.⁴¹ Inversely, transfecting CHO-S cells delivers a higher proportion of clones with moderate levels of expression, slightly quicker growth rates and higher cell densities, but the likelihood of isolating a comparatively high expressing clone is limited. CHO-S parental cells could be the parental cell line of choice if priority is given to speed instead of performance.⁴² New generations of engineered CHO cell lines, such as the CHO double knocked-out for glutamine synthetase (CHOGSKO), may be considered because they combine the advantages of higher expression levels and lower risk of instability of expression through more stringent selective pressure.^43,44

Despite not being mandatory, it is good practice to establish a cell bank system for the parental cell line as stipulated in ICHQ 5D: “The use of characterized parental cell banks is suggested but is not considered essential… ”.⁴⁵ A comprehensive testing strategy of a manufacturing master cell bank (MCB)/working cell banks (WCB) system is based on the risks to which the manufacturing cell line and the parental cell line have been exposed during their respective histories. Thus, establishing a fully characterized 2-tier serum-/trypsin-free cell bank system of the parental cell line, including testing for bovine and porcine viruses, will diminish the virus testing burden for qualifying the subsequent animal-derived component-free manufacturing cell line MCB/WCB system.^46,47 In addition, thanks to the genomic plasticity of parental cells, such as CHO and the inheritability of particular traits, cloning the parental cell line prior to establishing a cell bank system could improve the manufacturability of recombinant proteins by homogenizing the parental cell population with particular phenotypic and/or metabolic characteristics.^38,48–52

Right approach for generating a manufacturing cell line

Classically, the process for generating manufacturing mammalian cell lines for the expression of recombinant proteins, including mAbs, consists of five sequential activities once the genetic sequence of the product has been identified: 1) preparing or sourcing codon-optimized vectors incorporating the gene of interest and a selection system; 2) transfecting a parental cell line; 3) screening surviving pool of cells based on the level of expression and the activity of the protein; 4) cloning through one round of single-cell deposition followed by a cell imaging check to ensure the clonality of the cell line, screening and selecting lead clone candidates based on level of expression and critical quality attributes;^53–55 and finally 5) establishing research cell banks (RCB) of the lead clonal cell line candidates and selecting the final manufacturing cell line to generate the MCB as a compromise between the level of expression, the stability of expression and the quality profile of the recombinant protein expressed using a scale-down model mimicking the large-scale expected manufacturing process. Based on this traditional linear scenario shown in Figure 1, generating a clonal manufacturing cell line takes approximately 6–8 months.^13,56–58

Figure 1. Example of standard workflow from gene to final clone.

A flow chart describing the successive operations and timelines from transfection to selection of the final clone. The operations include transfection, cell pool generation, establishment of pre-research cell banks, cloning, establishment of research cell banks and evaluation of stability of expression.

In the context of accelerating CMC development, there are multiple approaches to reduce these timelines. For expressing recombinant proteins in CHO cells, the most common selection systems for transfection are the dihydrofolate reductase (DHFR) and glutamine synthetase (GS) systems.⁵⁹ Within the past decade, the GS-based methionine sulfoximine (MSX)-selection system has been increasingly used for the establishment of CHO-derived manufacturing cell lines. It is based on an incremental increase in MSX as the selecting agent, e.g., from 10 to 50 μM, that allows only cells that have the GS-containing vector inserted to grow and could be followed by a second round of 100–1000 μM for gene amplification.^60,61 The use of parental CHO cells with attenuated GS expression, or double gene knock-out for GS (GSKO), facilitates the generation of high producing clones in shorter timelines and improves the stability of expression. CHO-GSKO/GS attenuated cells showed a higher selection stringency than wild-type CHO-K1 and allowed generation of high producing cell pools and clones in the absence of MSX-based selective pressure, thus leading to time savings by implementing a single round of selection.^62–64

Another factor affecting cell line generation timelines is the mode of insertion of the gene(s) of interest into the host cells. Historically, transfection with a non-homologous recombination-based random integration (RI) was the common method for inserting foreign genes into a mammalian cell host to produce recombinant proteins.⁶⁵ Site-specific recombinase-mediated integration (SSI) is another method.^66–68 SSI has the advantage over random integration to reduce the selection and single-cell cloning stages from several weeks to a few days through negative selection (e.g., by supplementing cell culture medium with ganciclovir) to eliminate cells with unwanted recombination events, thus leading to the generation of more consistent and reliable clones.⁶⁹

The engineered Tc1/mariner transposons named Sleeping Beauty and Frog Prince, the insect-derived natural element Piggy-Bac and the Leap-In Transposase use transposon-based vectors and a cognate transposase enzyme.^70–72 Transposition stably inserts multiple independent copies of structurally intact transposons. The Leap-In Transposase system associates CHO-GSKO host cells and transposition, thus leading to more homogeneous stable pools with high gene copy number, higher productivity, and better comparability between pools and clones. Especially, the multiplicity of the insertion sites de-risks the use of cell pools instead of a clonal cell line for producing material for toxicological studies because of higher reliability of expression stability, productivity, and product quality profile.⁷³

While the traditional method of plating and screening stable cell pools usually takes 2.5 months, the SSI and transposon approaches may take as little as 1–2 weeks by skipping the labor intensive mini-pool screening, due to enhanced probability of genomic integration.⁷⁴ However, the SSI approach may limit the opportunity for the selection of high-expression clones through high throughput screening and may then be more appropriate for low-demand products.

Transduction is another alternative to transfection. The GPEx^Ⓡ transduction system uses replication-defective retroviral vectors derived from Moloney murine leukemia virus (MLV), pseudo-typed with vesicular stomatitis virus G protein to enlarge the types of permissible host cell lines, and stably insert multiple single copies of genes into mammalian cell lines.^75,76 The retrovectors are produced in HEK-293 cells expressing constitutively MLV gag, pro, and pol genes to sustain the replication of the defective retrovector. The HEK-293-derived retrovectors, which include the gene of interest, are purified and added to CHO cultures to infect the CHO cells where the RNA genome of the retrovector is transcribed into DNA, stably inserted into the CHO genome and conducts the expression of the desired recombinant protein. The absence of post-transduction selective pressure and a high homogeneity of cell pools due to the high efficiency of retroviral infection enables fast development of the manufacturing cell line and even faster if combined with GS doubled knock-out parental cell lines.^77,78 A similar system has been developed where the HEK-293 packaging cell line has been replaced by a CHO cell line, which limits the risks of virus infections favored by the use of adherent cells and serum during production of the retroviral vectors and a packaging cell line permissive to infection with human viruses.^79,80 In this system, the packaging CHO cells are co-transfected with a transfer plasmid together with the packaging plasmids to generate replication-competent retrovector. However, the transduced producing cells are submitted to selective pressure with an antibiotic prior to cloning, which limits the interest of this technology for accelerating the generation of manufacturing cell lines.

Because CHO-derived cell lines often show a decrease in recombinant protein expression during long periods of culture due to genomic rearrangements, gene loss, transcriptional silencing or other epigenetic and proteomic changes, the last step that affects how long it takes to select the final manufacturing cell line is the evaluation of the stability of expression.^81–84 Traditionally, evaluating stability of expression is performed on the last 4–8 “pre-final-selection” lead candidate cell lines and the manufacturability of a cell line is acceptable if it exhibits a retention of not less than 70% of the volumetric productivity over 20–30 passages or 60–90 generations (Figure 1), which represents the number of population doubling level (PDL) from the RCB to the harvest of a large-scale bioreactor run operated in fed-batch or even longer in case of continuous biomanufacturing.⁸⁵

Cell line development strategies ensuring high probability of stable expression allow a shorter duration of the expression stability study used for selection of the final manufacturing cell line, without an excessive risk of finding at a later stage that a lack of long-term stability of expression jeopardizes the commercial production readiness.^{15,16,73,81, 86} To accelerate the final manufacturing cell line selection, the evaluation of stability of expression could be reduced to 30-PDL without selection pressure with support of a single-cell qPCR genetic stability test as a risk mitigating strategy.^15,87 By evaluating preliminary phenotypic and genetic stability together, the final manufacturing cell line can be selected as early as 2 weeks after clone fed-batch screening. The full evaluation of the cell line stability can subsequently be performed with the MCB in context of the evaluation of cell substrate stability as described in ICH Q5D.⁴⁵ Bolisetty et al. reported an effective selection of a final manufacturing cell line based on such a short-term stability assessment with acceptable quality profile.⁸⁴

In conclusion, where speed to clinic is a priority, use of selection systems combined with engineered host cells to make the selective pressure more stringent (e.g., GSKO CHO cells), high-efficiency gene integration (e.g., transduction, transposon) or site-specific integration strategies potentially associated with promotor engineering to further improve expression stability enable shorter lead time for the generation of more homogeneous and stable pools of cells, as well as for selection of the final manufacturing cell line.^88,89

Non-clonal cells for producing preclinical and first-in-human materials

Another way to accelerate CMC development is to parallelize activities by using materials derived from a stage of development that has not yet been fully completed, to perform another independent sequence of activity. This strategy can be considered provided that the use of a preliminary material will not bias the outcome of studies performed with that material.

Multiple regulatory guidelines discuss the topic of clonal derivation of mammalian production cell lines in the production of recombinant products for human use. ICH Q5D stipulates “For recombinant products, the cell substrate is the transfected cell containing the desired sequences which has been cloned from a single cell progenitor”.⁴⁵ Similarly, FDA’s points to consider in the manufacture and testing of mAb products for human use notes that the MCB should derive from a single cell and the EMA guideline on development, production, characterization, and specification for mAbs uses the term monoclonal cell line for characterizing the cell substrate.^90,91 These guidelines clearly require the use of a clonally derived cell line for establishing the MCB, which is the starting point of good manufacturing practices (GMP) and subsequent WCB established for preparing manufacturing seeds.⁹²

ICH S6(R1) on preclinical safety evaluation of biotechnology-derived pharmaceuticals notes “The product that is used in the definitive pharmacology and toxicology studies should be comparable to the product proposed for the initial clinical studies”.⁹³ As neither ICH S- nor ICH Q-guidelines indicate that the material for supplying the preclinical studies should derive from a clonal manufacturing cell line, this opens the door to using cell pools instead of clonal cells for generating preclinical materials.

In 2017, biopharmaceutical companies published six reports, described below, supporting the fact that materials issued from cell pools are not significantly different in quality from materials issued from the clones subsequently derived from cell pools.

Wright et al.⁵⁷ described an upstream platform capable of delivering equivalent quality material throughout the cell line generation process starting from subcloning the engineered CHO parental cell line to generate a more genotypically and phenotypically homogeneous starting host cell line. Then, using a standard random integration for generating recombinant cell lines, they compared the quality of three different molecules, an IgG₁, an IgG₄ and an aglycosylated IgG₁/IgG₄ hybrid at different stages of cell line generation: uncloned pools, pools of clones and lead clones. Using productivity (titer) and quality metrics (% high molecular weight impurities (HMW), impurities, charge variants and glycan distribution), they confirmed the comparability of materials produced at various time points of the cell line generation process.

Rajendra et al.⁷² used transposon technology to ensure a high level of homogeneity of pools to produce preclinical material. They compared four mAbs (three IgG₁ and one IgG₄) expressed in CHO cells for stability of expression, genetic stability, titer in bioreactors operated in fed-batch and product quality (HMW, charge variants, purity, glycan distribution, oxidized species, and peptide mapping by mass spectrometry) over 55 PDLs. The quality of materials produced at various culture ages was comparable one with the other and comparable to the control derived from a single-cell progenitor.

In addition, Hu et al.⁹⁴ compared titers and product quality (HMW, low molecular weight impurities (LMW) and charge variants) of cell pools and their corresponding top eight manufacturing cell lines expressing two mAbs, cultured up to 60 days in shake flasks and bioreactors operated in fed-batch. They concluded that: (1) pool and clones have comparable product expression stability and quality profiles for both mAbs and (2) parallelization of activities and use of cell pools instead of clonal cell lines could be applicable for the manufacture of preclinical material. That strategy allows savings of 4 months from gene to investigational new drug application (IND)/investigational medicinal product dossier submission (IMPD).

Fan et al.⁹⁵ proposed the use of mini-pools to supply preclinical studies and even first-in-human clinical trials. Using a CHO-GSKO parental cell line to ensure a stringent selection and site-specific integration technology, they showed the comparability of cell growth, productivity, and product quality (HMW, charge variants, glycan distribution, and sequence) of two mAbs produced from mini-pools and their corresponding clonal cell lines.

Scarcelli et al.⁹⁶ applied two different strategies for generating a manufacturing cell line dependent on the need of a particular project: (1) a random integration (RI) for high-demand projects where the level of expression is critical and (2) a site-specific integration (SSI) when speed to clinic is the main driver. Whereas the RI approach associates a CHO K1SV GS knockout, the SSI relies on a negative selection by addition of ganciclovir acting through removal of thymidine kinase gene from the landing pad and positive selection by addition of hygromycin and the presence of a hygromycin-resistant gene in the landing pad.⁹⁷ They compare the performance and product quality of cell pools and clones for three mAbs developed with the RI approach and six different mAbs issued from the SSI process. The SSI strategy generates highly stable expression across generational age as compared to the RI strategy and enables accelerated development strategies for programs where speed is critical. Whatever the strategy, similar process performance and product quality observed between process and materials based on pools or clones justify the use of cell pools instead of clones to support early product development and manufacture of material for preclinical studies. Similarly, Munro et al.⁹⁸ showed comparable stability of expression, fed-batch process performance and product quality (charge variants, HMW, LMW, glycan distribution, and other quality attributes by multi-attribute mass spectrometry (MAM), potency, and process-related impurities) of one glycosylated mAb and two aglycosylated mAbs produced from transfected stable cell pools or from clones. They concluded that “The successful implementation of this approach relies on an in-depth understanding of the expression system being used as well as the bioprocess and analytical platforms to generate and characterize the material irrespective of the cell substrate source”.

Since publication of this series of papers, additional data have accumulated to confirm the appropriateness of using non-clonal cells for accelerating process development and decoupling the production of material for preclinical studies from the final clone selection. In 2020, Bolisetty et al.⁸⁴ reported a parallelization of the evaluation of stability of expression, final clone selection, and manufacturing of preclinical material based on comprehensive observations that platform-derived pools of six top-producing clones mixed at the N-1 stage (one passage before production bioreactor) produce drug substance with very similar product quality profile to the final clone selected based on performance and stability of expression.

Stuible et al.⁵⁸ even proposed generation of material for preclinical studies through transient expression in CHO cells, which is possible due to significant improvement in the yield of transient systems observed in recent years.^99,100 The authors reported time-saving from 8 months to generate material from stable clones to 4 months for material issued from stable pools, or to 3 weeks when considering transient expression. For developing antibodies against SARS-CoV-2 in a timely manner, transient expression was used to support process development and even contemplated for supplying preclinical studies.^15,101,102

Effectively, in the specific context of FDA’s Coronavirus Treatment Acceleration Program, companies have used transient expression for selecting antibodies with appropriate characteristics, developing a suitable formulation, checking the suitability of analytical methods, and designing a manufacturing process. Production for preclinical and Phase 1 clinical studies started from RCBs established under GMP from stable pools generated using an SSI strategy or transduction to ensure high genetic homogeneity of cells and stability of expression.^12,14–16 The RCBs were tested in accordance with ICHQ5A and ICHQ5D to fulfill safety requirements.^45,46 In addition, product comparability between Phase 1 material and subsequent late-stage clinical material derived from clonal cell lines specifically selected for ensuring product comparability (the biased selection approach) was demonstrated retrospectively.^15,16 Xu et al.¹⁰¹ reported a global approach consisting of transient expression in CHO cells to support preclinical, IND-enabling toxicology research, and early CMC development, mini-pool materials to supply Phase 1 clinical trials, and a single-clone working cell bank for late-stage and pivotal clinical trials. Whereas there were clear differences in process performance between the transient expression and stable expression (mini-pools and single-cell clones), the authors showed comparable quality between cell types for production of three batches by using a large set of analytical methods covering the primary structure, higher-order structure, product- and process-related impurities, charge variants, glycan distribution, biological activity, Fc-driven biological functions, and forced degradation profiling.

The uncommon practice of using non-clonal cells and corresponding RCBs established under GMPs for the production of first-in-human materials has been accepted by regulatory bodies exclusively in the specific context of an emergency response to an urgent unmet medical need (i.e., COVID-19 pandemic). In that context, the absence of a demonstration of assurance of clonality of cells used for producing material intended for human use was offset by checking the genetic stability of end-of-production cells and/or augmenting the control strategy, e.g., monitoring of sequence variants by peptide mapping mass spectrometry in each batch of drug substance, monitoring of glycans affecting the mechanism of action, and/or tighter limit of in vitro cell age.^12,103

As an immortalized cell line, the CHO cell line is inherently unstable, and its history of development resulted in a genetically and phenotypically diverse family (e.g., CHO-K1, -K1SV, -DXB11, -S, -DG44).¹⁰⁴ This genome plasticity led some authors to describe CHO parental cells as a quasispecies and to reconsider the need for cloning manufacturing cell lines with adequate proof of clonality prior to cell bank establishment.^105,106 On one hand, this may come from a confusion between the semantic sense and the practical sense of clonality. Semantically, clonality means a population of cells that are genetically identical. Because of the high propensity of CHO cells to chromosomal rearrangements, i.e., about one major chromosomal rearrangement every 10 doublings, the semantic sense does not apply.^107–110 However, the practical sense refers to a population of cells with a high probability of being clonally derived from a single-cell progenitor through a manipulation termed “cloning”. This is the regulatory sense to clonality.¹⁰³ On the other hand, the industry view is that the clonal state of a manufacturing cell line is only one factor that could affect product quality consistency, whereas emphasis should be placed on an augmented control strategy for confirming product consistency.¹⁰⁴ This aspect was supported by the observation that single-cell-derived subclones of clonal manufacturing cell lines generated by random integration in CHO-K1 displayed a variable range in titer, cell-specific productivity, expression stability, growth, and product quality attributes, thus showing that cell heterogeneity exists in a cell population even when derived from a single-cell progenitor.⁵²

In contrast, FDA’s position on proof of clonality of the population of cells that compose an MCB, which is the starting point of GMP and manufacture of biotechnological products for human use as per ICH Q11, has four key points: (1) a nonclonally-derived cell bank may induce more variability in process performance and cellular phenotype-dependent quality attributes (e.g., glycans), and may result in drift, shift, or unforeseen selective pressure following a process change; (2) the industry view to augment the control strategy is not aligned with the aim of health authorities to promote the quality-by-design concepts; (3) it may be difficult to differentiate non-clonality from other possible root causes in case of a drift identified by the quality system, including the continued process verification; and (4) a non-clonally derived cell bank system may generate variability in product quality due to a lack of consistency in replenishing a working cell bank or during the life cycle of a product.^37,55,92,111 Welch & Arden, FDA’s representatives who signed a paper on clonality, concluded “Nevertheless, a demonstration that even clonally derived cell lines possess tremendous heterogeneity (or clonal variation) and that nonclonally derived pools can in some cases produce drug substance with critical quality attributes (CQAs) matching those of drug substance produced by a clonally derived line fails to address key unresolved questions”.⁵⁵

In conclusion, as of today, the use of a non-clonally-derived master cell bank to produce materials for human studies could be acceptable solely in a specific emergency context, or in case of a product fulfilling an unmet high medical need and where a program exhibits a high benefit-to-risk ratio provided that: 1) the RCB has been established in compliance with GMP and tested for adventitious agents in accordance with appropriate ICH guidelines, and 2) the residual uncertainty of the impact of non-clonality is counterbalanced by testing for genetic stability of aged cells and implementing an augmented control strategy, such as batch-to-batch amino acid sequence variant analysis.

There is now accumulated evidence on the limited risk of genetic instability, non-reproducibility, and non-comparability between non-clonally derived preclinical and clonally derived Phase 1 materials if an appropriate cell line development strategy has been applied. Therefore, there are some precautions to take before implementing a non-clonal strategy for the production of preclinical material. One could consider cell engineering methods, such as transduction, DNA transposon integration or SSI, to deliver stable pools with high levels of cell homogeneity and a lower risk of significant genetic diversity and instability of expression. Alternatively, transfecting engineered parental cells with a GS double knockout allows higher stability of expression than wild-type parental cells. In addition, selecting the final manufacturing cell line by screening clones derived from the cell pool used for producing preclinical material may further minimize the risk of non-comparability between preclinical and Phase 1 materials.⁹⁴

New technologies for transient expression make that approach an appropriate way to very quickly generate enough material for the development of a manufacturing process, analytics, and a suitable formulation. However, there are still limited published reports to really appreciate the probability of non-comparability when using transient expression for generating preclinical material and stable clonal cells for the production of Phase 1 material. It may be prudent to further accumulate data prior to considering transient expression as a viable strategy for the manufacture of preclinical material.

Maximized use of manufacturing and analytical platforms

To reach first-in-human clinical studies faster, companies developing anti-SARS-CoV-2 antibodies maximized the use of manufacturing and analytical technology platforms, and prior knowledge.^12,84 Moreover, as noted by Brian Kelley, “delivering these mAbs in record time was only made possible by the industry convergence on mAb platform processes”¹⁶ and “The industry’s COVID-19 experience demonstrated that mAb process and formulation development has matured to a point where a ‘one-shot’ development is a viable strategy for all products developed with a strong platform and production knowledge base”.¹⁶

Practically, the process design stage can be reduced to a performance check of a manufacturing platform and suitability check of analytical platform technologies, which are analytical methods used for the same class of products and product sample matrices without modification of their procedures. Process development may then be limited to slight adjustments where needed only.^{14,15,101,112,113}

In-process control strategy, specifications and strategies for justification could also be set up based on shared learning across the biotechnology industry with appropriate extended ranges and the expectation that they will be reviewed and potentially narrowed after additional manufacturing experience.^{16,21,114–116}

One additional benefit in using manufacturing platforms is the potential timesaving for raw material and consumable supplies. A lead time of more than 6 months to obtain raw materials and consumables after the decision to initiate a batch has been taken is quite common. Manufacturing delays due to the constraint of supplies could then be lifted through continuous improvement of the manufacturing platform by implementing dual-sourcing of key raw materials, resins, and membranes and/or at the expense of larger ready-to-use inventories.

Implementing advanced analytical methods

In addition to using platform analytical technologies as discussed above, other approaches have been considered for a quicker development of mAbs in the context of the COVID-19 pandemic. To expedite the chromatographic analysis of casirivimab and imdevimab, ultra-short columns, retention modeling, and automated method development have been successfully used for characterizing these antibodies.¹¹⁷ As an example, the development and verification of four complementary analytical methods required only 2 days of experimental work and a comprehensive characterization of the mAb cocktail by four different profiling techniques was achieved within a one-hour turnaround time.

When accelerating antibody development, attribute information is continuously built into multiple integrated risk assessments that inform and suitably shape specifications, and therefore, also define the limits of CQAs. In complement or even alternatively to rapid methods, MAM can be used to directly and simultaneously characterize and monitor numerous CQAs, thus saving time.¹¹⁸ MAM enables identity testing based on primary sequence verification, detection, and quantitation of post-translational modifications and impurities, including new species identification.¹¹⁹ This ability to determine CQAs of therapeutic glycoproteins makes MAM simultaneously and directly a more informative and productive workflow than conventional chromatographic and electrophoretic assays.¹²⁰ MAM drastically contributes to the acceleration of CMC data delivery, including support to process development, product characterization, stability data, and quality controls.^121–124 In addition, thorough monitoring of critical quality attributes would minimize the need for a cell-based assay and allow use of simple-binding assays for product release.

Considering new approaches for adventitious agent testing and viral clearance studies

The prevention of viral contamination risks is based on three pillars: prevent, detect, and remove.¹²⁵ Testing the cell banks and unprocessed bulk materials for adventitious agents, and demonstration of the capability that a process can clear viruses should be carried out in accordance with the current ICH guideline Q5A(R1) and completed before moving to first-in-human studies.⁴⁶

Cell bank testing and delivery of viral clearance data are usually on the critical path to Phase 1 application. Whereas sterility and mycoplasma testing of the cell bank system takes only a few weeks, testing of cell banks for viruses using in vitro and in vivo assays may take 1.5 to 2.5 months. Use of cell lines and vector constructs with substantial prior knowledge on their safety profile, the maturity of new broad molecular methods and the application of revision 2 of ICH Q5A would reduce the testing time of a new cell bank system.

In accordance with ICH Q5A(R1), testing for adventitious agents of a cell bank system is performed using infectivity assays (e.g., S⁺L⁻ focus assay, XC plaque assay), transmission electron microscopy (TEM), and reverse-transcriptase (RT) assays for the detection of retroviruses, and numerous assays for detecting endogenous viruses, including: (1) in vitro assays using indicator cells; (2) in vivo assays in guinea pigs, mice, suckling mice, and embryonated hen’s eggs; (3) mouse/hamster/rat antibody production assays (MAP/HAP/RAP) for rodent cell lines; and (4) virus-specific PCR-based assays based on specific process risks.

In accordance with ICH Q5A(R2), which was published for public consultation in September 2022, in vivo assays could be replaced by broad molecular methods (e.g., next-generation sequencing (NGS) and MAP/HAP/RAP tests by targeted rapid molecular assays in the context of the 3 R (replace, reduce, refine) testing approaches.^126–129 In addition, the replacement of in vitro assays for the detection of non-endogenous viruses by broad molecular methods may also be considered on a case-by-case basis upon acceptance by health agencies. Once ICH Q5A(R2) comes into force, replacing the current methods by unbiased NGS and other targeted nucleic acid technology (NAT)-based methods may save more than 1 month on the testing and release of cell banks.¹³⁰

Generic modular claims may reduce the time to complete viral clearance studies, which usually take between 4 and 6 months.¹³¹ Generic modular claims, which should be based on in-house prior viral clearance data sets supplemented with literature data describing clear mechanism of clearances, would support the performance of viral clearance studies made simpler by testing single confirmatory runs in lieu of duplicate testing, replacing infectivity assays by qPCR assays for virus removal steps, as well as reducing the testing to known endogenous viruses (e.g., MLV for CHO cells) for chromatography steps and viral inactivation steps (e.g., low pH hold) where applicable and to small virus model (e.g., minute virus of mice) for nanofiltration steps.¹³² Additionally, using a qPCR assay in place of TEM, which takes usually 8–12 weeks, for the determination of the number of virus-like particles in the cell culture supernatant for cell lines known to be contaminated with endogenous retroviruses (e.g., CHO) could further reduce testing time to several weeks. Combining these approaches may reduce the overall turnaround time of viral clearance studies by half.

In the context of accelerated programs and in accordance with the Medicines Adaptive Pathways to Patients Initiative, the use of manufacturing platforms covering both upstream (e.g., cell line, media, and process ranges for products expressed in cells known to contain endogenous replication-incompetent retroviruses, such as CHO cells) and downstream (e.g., capture and purification steps, viral inactivation and removal technologies, operational ranges) processes and appropriate accumulated prior knowledge could support not performing product-specific viral clearance studies prior to first-in-human studies. However, a thorough analysis of accumulated prior knowledge on the capability of the platform process to remove/inactivate viruses should be included into the dossier and is subject to pre-approval by competent authorities. In the context of the development of anti-SARS-CoV-2 mAbs, replacing new clearance data by accumulated prior knowledge saved 4–6 months in a development program.¹²

Stability claim with less real-time data

Acceleration of development timelines is also constrained by the availability of real-time, real-conditions stability data to justify the shelf-life of the product. In accordance with ICH Q1E, “the proposed retest period/shelf-life can be up to twice but not more than 12 months beyond the period covered by long-termdata”.¹³³ There are scientific approaches to generate stability understanding under expedited programs.²¹ For chemical entities, stability claims could be leveraged from accelerated stability studies and modeling.¹³⁴ For biologics, stability claims for first-in-human products may be derived from real-time, real-conditions, early development, and preclinical stability data, supported by forced degradation studies, thermal stress studies, and modeling.^16,21 The ability to predict biological product stability, instead of extrapolating a shelf-life from real-time, real-conditions studies, is now emerging with advanced kinetic modeling tools. Long-term stability profiles could be deduced from the analysis of stability profiles of biotechnological products submitted to thermal stress at different temperatures over a few weeks.^135,136 This approach requires an appropriate justification that the risks in extending a stability profile are mitigated by sufficient prior knowledge, and that commitments to report deviations from the expected stability trends and out-of-specification results are provided.¹³⁷ In addition, these new tools could help in screening and selecting early suitable formulations and limit the risk of discovering too late a major product stability issue that may compromise the development of the product.

Discussion

The emergence of deadly pandemics and outbreaks has greatly affected the speed at which a biologic can move from the design stage to its first use in humans. Whereas it may take about 17 months for a biotechnological product to move from its initial design to the submission of an IND/IMPD when applying a traditional sequential development approach (Figure 2), multiple companies were able to reduce those timelines to less than 6 months when developing anti-SARS-CoV-2 mAbs and approvals were granted in record times under specific regulatory pathways, such as the EUA in the US and fast-track development support and approval in Europe.^16,20,138 These record times were made possible thanks to a comprehensive combination of acceleration strategies based on multiple levers described in Table 1.

Figure 2. Traditional and accelerated development timelines.

Two Gantt charts describing the sequence and critical path of CMC activities in accordance with the traditional or accelerated approach.

Table 1. Acceleration options to CMC development (from gene to First-in-Human) (text in bold relates to acceleration options for traditional programs whereas text in italic reflects additional pathways potentially applicable to accelerated programs only).

Stage	Lever
Parental cell line	Select an appropriate parental cell line depending on the development strategy: high or low demand product
Cell line generation	Prioritize a gene integration approach that maximizes the homogeneity of the post-insertion cells and the stability of expression Select the final clone candidate based on abbreviated stability data but supported by a genetic stability test (e.g., single-cell qPCR) Establish non-clonal RCBs in accordance with GMPs
Product and process development	Use in silico predictive tools for ensuring an adequate product design and manufacturability Prioritize the use of upstream, downstream, and analytical platforms Check that product design fits with the manufacturing and testing platforms Use transient expression for early process/analytical development activities
Preclinical manufacturing	Produce preclinical material from a non-clonal RCB
Phase 1 manufacturing	Minimize (no) process changes between preclinical and Phase 1 manufacturing Produce Phase 1 material from a non-clonal RCB and commit with authorities (1) to establish a clonal MCB immediately afterward with a clone selection process aiming at ensuring high level of comparability, (2) to assess the comparability of materials issued from the RCB and the MCB, and (3) to manufacture for Phase 2/3 using a clonal cell bank system Mitigate risk of genetic instability through an augmented control strategy (e.g., peptide mapping – mass spectrometry) Implement wide-ranges specifications and commit with regulatory authorities to revise the control strategy based on accumulated experience Support stability claim with available clinical stability data, preclinical stability data, forced degradation studies and stability modeling and commit with authorities on continuous delivery of real-time, real-conditions stability data, and information in case of out-of-specification Implement conditional release of drug substance
Prevention of viral risks	Implement broad molecular methods in lieu of in vitro and in vivo testing for adventitious agents Reduce product-specific viral clearance data based on modular claims Replace product-specific viral clearance data with accumulated platform data and prior knowledge

The rapid deployment of multiple mAbs has been a remarkable accomplishment and many lives have been saved by preventing COVID-19 from progressing to a life-threatening condition. The marketed mAbs generated substantial sales prior to their withdrawal due to the emergence and predominance of genetic variants (Table 2). The Omicron variant became the dominant strain within 2 months after its emergence in November 2021, which caused resistance to mAb-based therapies to treat COVID-19, such as casirivumab + imdevimab, sotrovimab, bamlanivimab + etesevimab, and bebtelovimab.^148–152

Table 2. Approved COVID-19 monoclonal antibodies.

INN (tradename)	Code	Company	Regulatory status	Clinical Trial ID	Sales (2021/2022)	References
Casirivimab + imdevimab (REGEN-COV)	REGN10933 + REGN10987	Regeneron + Genentech	EUA granted but no longer authorized due to lack of efficacy against new subvariants, approved in Japan	NCT04452318	M$ 6190/1832	^Citation7,Citation14,Citation139
Bamlanivimab + etesevimab	LYCoV555 (LY3819253) + LYCoV016 (LY3832479)	AbCellera/Eli Lilly	EUA for bamlanivimab/etesevimab combination therapy granted but no longer authorized due to lack of efficacy against new subvariants	NCT04501978	M$ 2240/2024	^Citation140
Sotrovimab (Xevudy)	VIR-7831/GSK4182136	Vir Biotechnology & GlaxoSmithKline	EUA granted but no longer authorized due to lack of efficacy against new subvariants, approved in Australia, UK, EU	NCT04545060	M$ 1317/2846	^Citation13,Citation16,Citation141,Citation142
Tixagevimab + cilgavimab (Evusheld)	AZD7442 (AZD8895 + AZD1061)	AstraZeneca	EUA granted, approved in Korea and EU	NCT04625972	M$ 855/2184	^Citation143
Bebtelovimab	LYCoV1404	Eli Lilly	EUA granted but no longer authorized (Dec. 1^st, 2022) due to lack of efficacy against new subvariants	NCT04634409	M$ 720/0	^Citation144
Regdanvimab (Regkirona)	CT-P59	Celltrion	Approved in Korea and EU	NCT04602000	M$ 0/10	^Citation145
Amubarvimab + romlusevimab	-	Brii Biosciences, Tsinghua University and the Third People’s Hospital of Shenzhen	Approved in China	-	-	^Citation146
-	JS016	Shanghai Junshi Biosciences, China National Institutes for Food and Drug Control	Approved in China	NCT04479644	-	^Citation101,Citation147

The rapid acquisition of resistance to approved mAb-based treatments discouraged some companies from further development of their investigational molecules. However, others realized the potential of neutralizing anti-SARS-CoV-2 mAbs targeting conserved epitopes for treating vulnerable populations, preventing evolution of the virus in immunocompromised patients, and treating patients infected by new variants, or considered development of new mAbs against pathogens in small outbreaks and localized epidemics, rather than under pandemic settings.^153–158 Thus, the need for the rapid generation of new treatments with adapted specific targeting remains.

The acceleration options applicable to the development of mAbs against SARS-CoV-2 were proposed to, discussed with, and accepted by the regulatory authorities in the context of quickly developing solutions for a new disease that expanded world-wide at an exceptional speed, whereby the product quality should not be compromised as supported by a benefit/risk assessment.^159–161

The approaches to accelerated CMC, preclinical and clinical development, and the regulatory pathways for early access to anti-SARS-CoV-2 were not substantially different in essence to how key challenges could be solved, scientific elements developed, and regulatory tools used to address shortened development timelines in the context of expedited programs, such as PRIME, Breakthrough Therapy, and accelerated/conditional approval. These regulatory pathways were developed prior to the COVID-19 pandemic and their aim is to allow patients early access to new drugs fulfilling an unmet medical need when the product showed sufficient preclinical and clinical evidence of its benefit without compromising the assurance of a safe product with an appropriate and reproducible quality. For CMC, innovative approaches and/or deferring the delivery of specific datasets (e.g., completion of process performance qualification) can compensate for the reduced time to complete the CMC package prior to submission in case of an accelerated/conditional approval.^{21,137,162–164} Flexibility shown by regulatory authorities was essential during the COVID-19 pandemic because of the high unmet medical need.

The question is then “Which acceleration options could become new development standards or be used outside of the context of fulfilling urgent and high unmet medical needs?”

Regardless of whether the program is accelerated or not, product knowledge is critical at the start of a new program. Sources of product knowledge are multiple, starting from in-house prior knowledge for the same class of product, literature, structure–function relationship studies and forced degradation studies in combination with the use of advanced analytical tools for product charatcerization. Product knowledge allows the determination of the suitability of process and analytical platforms to deliver a product with the desired quality or, for new modalities, to design an appropriate process.

Where speed-to-clinic is a priority, using process and analytical technology platforms is essential for accelerating the early stages of development through the replacement of process and analytical development studies by implementing process and analytical platforms and checking their appropriateness to deliver a product with the desired quality. This greatly minimizes time and effort prior to producing materials to fulfill preclinical and Phase 1 product needs. It implies that companies should allocate enough resources to continuously improve in-house platforms, ensuring a sufficient level of performance considering that a platform-derived process and its associated control strategy could be the basis for producing material from early development phases up to launch. This is a right-first-time strategy of development based on, as Kelley et al. wrote, “battle-tested”, proven, robust, and reliable manufacturing technologies.¹⁶ Such an approach could now be considered by companies, but it necessitates acceptance of uncertainty and a “recalibration of the tolerance for business risk related to development and manufacturing”.¹⁶

The use of transient expression for production of the first grams of material for process, formulation and analytical development, preliminary toxicological studies, and efficacy evaluation in animal models, and non-clonal cells for generation of material for pivotal preclinical studies, if this is combined with adequate gene insertion technologies ensuring high level of cell homogeneity and limited risk of genetic instability, can become common place. The risk of non-comparability between preclinical and Phase 1 materials could be further limited through the concept of biased selection of the final manufacturing cell line from the pool of cells used to produce preclinical material. However, the firm position of the regulatory authorities on the need to initiate the production of material for human use from a clonal master cell bank is clear, and, up to now, that approach may not be considered valid for non-expedited programs.⁵⁵

Broad molecular methods, such as NGS, are now sufficiently mature to be considered adequate methods for replacing in vitro and in vivo assays in adventitious virus testing.¹⁶⁵ ICH Q5A(R2) will introduce four advances since the first revision: (1) new classes of biotechnology products; (2) additional validation approaches for virus clearance; (3) virus clearance validation and risk mitigation strategies for continuous biomanufacturing; and (4) new adventitious virus testing methods. Once in force, ICH Q5A(R2) will allow the reduction of testing times when qualifying a cell bank system, as well as quantifying how much virus may be present in the unprocessed bulk. Replacing product- and process-specific viral clearance studies by prior knowledge was acceptable in the context of the development of anti-SARS-CoV-2 mAbs. For more “traditional” products, the use of in-house prior knowledge in lieu of product- and process-specific viral clearance studies might also be applicable on a case-by-case basis, provided the process steps and the mechanism underlying viral clearance are well-understood, there is no interaction between virus and product, and there are no substantial changes between the product-specific process and the platform process from which in-house prior knowledge has been built.¹²⁶

Finally, a right-first-time strategy further streamlines product development by avoiding product comparability studies between preclinical and Phase 1 materials, and by using stability data of representative preclinical materials and modeling to support shelf-life claim for Phase 1 material despite limited real-time, real-conditions data.

These accelerated options and the mitigations described above minimize the risk in carrying out multiple parallel, instead of sequential, activities, which has a major impact on the reduction of development timelines. However, as shown in Figure 2, when combined, these options move the critical path from Phase 1 manufacturing to preclinical manufacturing and the generation of pivotal preclinical data. Thus, to materialize the gain on the overall timeline from gene to Phase 1 IND/IMPD through a right-first-time approach, it is essential to streamline the preclinical studies for authorizing reliable, safe, and effective products by, for example, determining the minimum data package required to move a compound into clinical development safety, using fewer animal studies and more alternatives, and considering bioengineering tools as applied for COVID-19 vaccines.^10,166,167

In addition, the pharmaceutical quality system (PQS) should allow regulatory flexibility to accommodate accelerated options. For example, the following seven options may be considered: (1) using non-clonal manufacturing cell line for the manufacture of material for toxicological studies; (2) using drug substance instead of drug product for toxicological studies by preformulating the drug substance and parallelizing quality controls, and product shipment; (3) accelerating the transformation of drug substance to drug product and shipment of drug product to clinical sites by applying a conditional release approach for Phase 1 supply; (4) shortening viral clearance studies through modular claims based on prior knowledge; (5) introducing broad molecular techniques for virus testing in alignment with ICH Q5A(R2); (6) filing with broader product specification acceptance criteria based on prior knowledge built on the same class of product; and (7) reducing the amount of real-time, real-condition stability data for Phase 1 material leveraged by prior stability data on toxicological material if preformulated, accelerated stability data and modeling techniques to support final conclusions.

In conclusion, COVID-19 provided an excellent use case to enact innovative strategies for the rapid development of anti-SARS-Cov-2 mAbs. These innovative strategies, except the use of non-clonal cells for producing Phase 1 material, could also be applicable to any traditional development program. They would enable the reduction of the time to clinic to about half of the standard development timelines (Figure 2) and could substantially lower CMC development costs. However, their implementation would necessitate a change in the level of risks companies are ready to accept because of parallelization of activities and an extension of innovation to other activities (e.g., preclinical development, quality assurance, regulatory functions), which then become critical to the development path. Additional value-driven acceleration innovative strategies in biopharmaceuticals development based on the COVID-19 experience could also be applied to late-stage development, thus reducing the time to Biologics License Application or Marketing Authorisation Application submission.^16,168

Abbreviations

CHO	=	Chinese hamster ovary
CMC	=	Chemistry, Manufacturing and Controls
CQA	=	Critical quality attribute
DHFR	=	Dihydrofolate reductase
EMA	=	European Medicines Agency
EUA	=	Emergency Use Authorization
FDA	=	US Food and Drug Administration
GMP	=	Good manufacturing practices
GS	=	Glutamine Synthetase
HEK	=	Human embryonic kidney
HMW	=	High molecular weight
ICH	=	International conference for harmonization of technical requirements for registration of pharmaceuticals for human use
IMPD	=	Investigational medicinal product dossier
IND	=	Investigational new drug
LMW	=	Low molecular weight
mAb(s)	=	Monoclonal antibody(ies)
MAM	=	Multi-attribute mass spectrometry
MAP/HAP/RAP	=	Mouse/hamster/rat antibody production
MCB	=	Master cell bank
MLV	=	Murine Leukemia Virus
MSX	=	Methionine sulfoximine
NAT	=	Nucleic acid technology
NGS	=	Next generation sequencing
PDL	=	Population doubling level
PQS	=	Pharmaceutical quality system
qPCR	=	Quantitative polymerase chain reaction
RCB	=	Research cell bank
RI	=	Random integration
RT	=	Reverse transcriptase
SSI	=	Site-specific integration
TEM	=	Transmission electron microscopy
WCB	=	Working cell bank

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Additional information

Funding

The authors reported there is no funding associated with the work featured in this article.