An Industry Perspective Approach and Control Strategy for Implementation of Ready-to-Use Cells in Bioassays: Survey Outcome And Recommendations

Abstract

The BioPhorum Development Group is an industry collaboration enabling the sharing of common practices for the development of biopharmaceuticals. Bioassays are an important part of an analytical control system. Utilization of ready-to-use cells can increase operational flexibility and improve efficiency by providing frozen cell banks uniform stock while removing challenges associated with maintaining cultured cells. The BioPhorum Development Group-Bioassay workstream conducted an intercompany benchmarking survey and group discussions around the use of ready-to-use cells for bioassays. The results of the collaboration provide alignment on nomenclature, production, qualification and implementation of ready-to-use cells to support the assay life cycle.

Method summary

Draft questions were composed by the authors based on prior knowledge of the issues associated with ready-to-use cell use. Questions were further refined through open discussion among the authors. Respondents answered the questions in a blinded fashion using Microsoft forms. One author compiled the results for open discussion among the BioPhorum members.

Keywords: :

• bioassay
• biologicals
• cell-based assay
• development
• ready-to-use cells

The definition of ’thaw-and-use’ or ’ready-to-use’ (RtU) cells, as defined by the BioPhorum Development Group (BPDG)-Bioassay workstream, includes cells that are thawed for use in a cell-based bioassay, often without further manipulation. This represents one of the major recent advances in bioassays for potency and characterization of biopharmaceuticals [1,2]. The BPDG-Bioassay workstream undertook a benchmarking exercise in 2022, conducting several surveys with up to 36 respondents on the use of RtU cells in GMP and non-GMP cell-based bioassays. The blinded responses came from a variety of global pharmaceutical companies of both small and large size as well as contract development manufacturing organizations (CDMOs) with diverse product portfolios and a wide range of business models. The survey results provide valuable insight into overall industry practices and direction. The responses and recommendations from discussions within the community are summarized in this article. The insights include general considerations for use of RtU cells in bioassays, cell banking logistics, implementation strategies (including qualification and release) and life cycle management of cell banks.

The concept behind the use of RtU cells is that each lot of RtU cells is cultured from a master or working cell bank, expanded and banked following a standard, optimized production procedure and then directly used after thawing in the bioassay. An RtU cell bank represents a uniform cell population that can mitigate against the impact of cellular changes (e.g., receptor expression) that may occur over time in a continual cell culture process [3]. The intent of this article is to outline common practices in the industry to establish, control and use RtU cells in potency assays from concept through to commercialization. The content of this article was derived through surveys and debate, representing a perspective on current industry practice for managing the introduction of RtU cells in GMP and non-GMP bioassay development. It should not be construed as the only way to utilize and manage RtU cells for use in GMP assays.

Results & discussion

Considerations for use of RtU cells in bioassays

In agreement with earlier publications focused on the use of RtU cells [1,2,4,5], the respondents have found that the use of RtU cells increases scheduling flexibility and can mitigate against changes in assay performance due to cellular changes that occur over time in culture [3]. In addition, RtU cell banks are less resource-intensive (reduced lab personnel, equipment, materials and footprint) to maintain than continuous cell culture. These advantages also simplify the lab-to-lab transfer of potency assays. One member company found that on a per cell line basis, the use of RtU cell banks saved approximately 14 h of work per week and an estimated €44,000 per year. For these reasons, RtU cells are used in potency assays by 80% of the respondent companies during some stage of assay development.

Once the strategic decision to employ RtU cells is made, there are several tactical considerations that the group would like to highlight. Although respondent companies use adherent and suspension cells equally, responses were made that adherent cells can be more challenging to establish as RtU because of the need for more labor-intensive culturing and banking procedures, and some mentioned that they are more likely to need a pre-assay recovery period after thaw. Time, space and equipment also need to be considered when deciding to pursue the use of RtU cells. The following questions should be considered before and during RtU work. Do you have the time (usually between 2 and 6 months) to establish culture conditions, passage limits and freezing conditions? Is a pre-assay recovery period required? If your clinical program is accelerated, the additional upfront work required to establish RtU cells in the potency assay may be prohibitive. Consider whether you or the contract research organization has the incubator and hood space required for the culture flasks to serve as the RtU cell source. In addition, is there sufficient space in liquid nitrogen or a mechanical freezer for storage of the bank or banks?

Potency assays [6] are not platform methods because they must reflect the unique mechanism of action of each therapeutic. As a result, the respondent companies have experience with many different cell lines and have found some that are not amenable to RtU production (e.g., 4T1 cells, HCC2218 cells and HH cells) and some that may require more extensive development and optimization efforts (e.g., Jurkat cells and some adherent cells). The reasons for these difficulties can be complex and may include poor or variable viability or low receptor expression. However, evaluation of growth conditions, seeding densities, recovery time, fetal bovine serum (FBS) type and batch freezing conditions can be a good way to establish the groundwork for making successful RtU cell banks.

Generation & logistics of RtU cell banks

Establishing the RtU cell bank

One of the goals of the survey was to understand how member companies were implementing RtU cells in their potency assays. According to survey results, the use of RtU cells was evenly spread between assay development; chemistry, manufacturing and controls characterization; and GMP/quality control (QC) release methods (ratio 16:15:13). The survey revealed that not all cell lines are suitable for an RtU format; this could in part be due to cell modifications, receptor expression recovery and growth characteristics of the cell line, which can result in low viability, slow recovery and different growth rates upon thaw.

Both primary [7] and immortalized [1,2] cell lines can be used to establish an assay using RtU cells. Primary [7] cells are an example of RtU cells that are not passaged prior to use. In our experience, primary cells may require a short recovery period for consistent assay performance, and this should be assessed on a case-by-case basis. Compared with primary cells, immortalized cells are more commonly used in an RtU format (17 of 21 responses).

Details of the RtU bank configuration – namely, decisions regarding the desired size of the bank (number of vials), cell number/cell density and/or vial size – are informed by the cell-based assay format, operational needs and cell line-specific capabilities. The strategy for the generation of RtU banks could include multiple sequential and/or parallel RtU banks that do not necessarily need to be of the same size. The majority of the respondents (15 of 27) generate 50–200 RtU vials per batch. A total of 11 of 27 companies were able to generate RtU banks of more than 200 vials aided by the use of automated aliquoting of cells into vials and controlled-rate freezers, which is the typical mode of operation for commercial cell line suppliers.

There are several cell banking strategies used by the various companies to generate RtU cell banks. A total of 12 of 20 respondents reported preference for a three-tiered cell banking approach (generation of master cell bank [MCB], working cell bank [WCB] and subsequent RtU cell bank) compared with the two-tiered cell banking process (generation of MCB leading to RtU cell bank). The decision as to whether a two- or three-tiered cell banking system should be employed can be decided considering a number of factors, including the number of RtU cell banks that is likely to be made over the life cycle of the product. A two-tiered cell banking system is better suited when the number of RtU cell vials made in a campaign is high (>500 vials). By contrast, a three-tiered banking system may be better suited when the RtU banks are small and the testing demand is high. Two-tiered systems usually result in less storage requirements and simpler record keeping. However, it is important that the MCB is not depleted through the product life cycle.

RtU cell banks are critical reagents for bioassays. They can be generated in a non-GMP or GMP environment. Nonetheless, each batch is qualified for use in a GMP laboratory as part of the characterization required for critical reagents to ensure batch-to-batch consistency. A total of 13 of 15 respondents follow a protocol with preapproved criteria for the release of an RtU cell bank, with 11 issuing a certificate of analysis for each RtU cell bank batch that is used for QC release testing.

There are a number of sources of cell lines for RtU banking, including in-house-developed cell lines, commercially available cell lines and licensed products (e.g., propagation cell line models of otherwise readily available commercial RtU vials). A total of 26 of 35 respondents generate their own RtU banks either in-house or via a CDMO, with a minority exclusively purchasing off-the-shelf RtU vials from commercial vendors. Challenges with outsourcing of RtU production to CDMOs have been highlighted in the survey. Examples include difficulty in upscaling, unsatisfactory cell bank quality or quantity and generation of consistent RtU lots. As these challenges are highly dependent on the appropriate RtU scale-up, freezing process development and knowledge transfer from the development lab, respondents recommended several control measures to improve these aspects. Recommendations include the provision of detailed handling instructions, with specified cell culture scale-up processes detailing cell banking reagents and freezing processes. Small pilot batches can be generated by the CDMO for client evaluation to confirm successful process transfer prior to preparation of a large RtU cell bank.

Logistics for how RtU cells are made & frozen

Cryopreservation of RtU cells follows general cell banking best practices, often already established for generation of cell banks for continuous culture cell-based assay format. In general, the aim is to freeze cells at a high concentration and at as low a passage number as possible. For RtU cells specifically, the scale-up process, cell freezing density, freezing media selection and/or proportions of individual components and actual cryopreservation process have been identified by survey participants as having the potential to impact the suitability of RtU implementation for a particular cell line. Creating an optimized cryopreservation protocol for the specific cell type of interest is therefore often a critical step in RtU cell-based assay development.

Cell culture scale-up & expansion

Generation of large RtU cell banks generally requires a significant investment in cell culture scale-up and an established process for culture and format of cell banks. This process may need to be redeveloped and further optimized during the life cycle of the asset.

Cryopreservation media ingredients

Selection of freezing medium containing the right cryoprotectants (type and concentration) and additives can significantly affect the post-thaw viability and suitability of the RtU cells intended for use in the bioassay. Selection of cryopreservation media ingredients can have an impact on the practical logistical considerations related to global transfer/import and export of RtU banks when using animal-derived materials. The use of in-house freezing media allows the option to fine-tune and optimize freezing media to achieve specific desired assay performance. Freezing medium is usually composed of a growth/culture medium containing FBS and a cryoprotectant such as DMSO. Optimization of the media components and concentrations (specifically FBS and DMSO) of the particular cell type or cell line is critical for optimal post-thaw recovery and suitability for RtU assay. A recovery time is cell line-dependent and may help to improve the reproducibility of the assay performance (e.g., signal to background). The DMSO concentration in freezing media typically ranges from 5 to 10%, with the concentration of FBS varying more widely (between 5 and 90%) (Figure 1A).

Figure 1. General experience of respondents when considering freezing media, equipment and transportation of RtU cells.

(A) Media used for freezing ready-to-use cell banks. (B) Process used to freeze ready-to-use cells. (C) Methods used to ship ready-to-use cell vials.

FBS: Fetal bovine serum; LN2: Liquid nitrogen.

In general, cell exposure time to DMSO should be limited (i.e., pre-assessment of sensitivity/maximum contact time with DMSO might be beneficial for optimal RtU cell banking). Only one of 23 respondents reported trying serum-free media, but not with success.

Additional considerations discussed by the survey participants were specific requirements for FBS, which is an animal product with various available options (heat-inactivated, dialyzed, gamma-irradiated, etc.) and various levels of characterization provided by the vendors. Certain cell types may require a specific type of FBS for optimal growth and performance in a bioassay.

Vial aliquoting using automated systems is generally recommended for RtU cell banks because of their size and specific requirement for consistency, though a majority of the survey respondents still used manual aliquoting. One advantage of automation is that larger RtU cell banks reduce the requirement to frequently qualify multiple small RtU cell banks. One justification for investing in automated equipment includes resource savings and simplification in the process of making RtU cells. Outsourcing or partnering with CROs or utilizing existing lab automation equipment, such as liquid handlers, is possible, but the latter must be assessed for aseptic conditions. A total of 50% of survey respondents utilized a controlled-rate freezer (Figure 1B). The use of a controlled-rate freezer allows the operator to customize the rate of temperature change before, during and after the freeze transition phase. This helps to preserve optimal cell viability while also offering an operational advantage (e.g., larger cell bank size) by reducing the time of a typical cryopreservation process. The remaining 50% of respondents used a manual process of placing cryogenic vials in an isopropanol freezing container, where a slowed rate of freezing is achieved over a longer period of time. It is acknowledged that there is a limited vial capacity per freezer container, which may influence the potential size of the RtU bank upon use of the manual process.

Storage

RtU cells are commonly stored in liquid nitrogen vapor phase or a cryogenic ultra-low temperature freezer at temperatures (-135°C to -196°C) that suspend cellular metabolism and preserve cells for long-term use.

Inventory

Upon qualification of the RtU cell bank all respondents treated the RtU cells as a critical reagent. Storage and inventory management of the RtU cells, should follow the site-specific Standard Operating Procedure. Generally, RtU cell banks are stored in two or more separate areas at the testing site and/or in a central critical reagent management facility. Separation of RtU cell banks into multiple storage locations will not only minimize exposure during access but will also protect a portion of the inventory in the event of freezer failure. Smaller local RtU stocks are typically kept for day-to-day operational support (i.e., at QC labs, CDMOs). Life cycle and inventory management considerations are discussed later in the article.

Shipping

Shipment of RtU cell banks is routinely performed from the production site to storage locations and from storage locations to bioassay testing laboratories. Shipping biological materials requires attention to the type of material transported, adherence to regulatory requirements, packaging materials, proper assembly, labeling and engagement of reliable carriers. Best practices associated with shipping, permitting, transport of dangerous goods and methods of maintaining cold chain storage are documented in the literature [8]. The selection of FBS sources is an important consideration for shipping RtU cell banks. Typically, FBS should be sourced from countries with certified zero incidence of transmissible bovine diseases, including spongiform encephalopathy.

The transportation of RtU cell banks, without impacting their function, is a critical factor when selecting the appropriate shipping conditions. Measures should be taken to ensure that cryogenic temperatures are maintained for the entire duration of the transportation. For shipments of RtU cells, the selection of the proposed shipping condition was influenced by the cell line, where the majority of survey respondents utilized dry ice or liquid nitrogen shippers (Figure 1C). Further considerations behind the selection of the shipment method included criticality of the material, feasibility/cost/availability of shippers, carrier limitations and domestic versus international shipment. Replenishment of coolant by the carrier to mitigate any unexpected delays during the shipment process was a widely utilized practice among survey respondents.

Release strategy including assay performance

Cells are a critical component of accurate and robust cell-based bioassays. Dilution schemes, reagent concentrations, incubation times and temperatures and other assay parameters are all optimized around cellular components, for which consistency is critical. As described earlier, steps and options associated with generating a cell bank have the potential to influence performance in the cell-based assay and should therefore be carefully defined. Consequently, cell bank qualification for RtU cells is critical and ensures consistent and expected responses and characteristics following the banking process.

After generation of RtU cell banks, the assay developer needs to decide the appropriate release assays and release criteria for the cell banks (Figure 2A). For phase II to commercial, there was agreement among respondents that viability, sterility, mycoplasma and assay performance are the main release parameters for MCBs, WCBs and RtU cell banks. Additionally, screening for adventitious viruses is sometimes performed for the release of MCBs (nine of 20 respondents), whereas only a few respondents confirmed doing this test in WCBs and RtU cell banks (three and two of 20 respondents, respectively). For RtU cell banks, some additional tests on cell density and cell dispensing volume in the cryovial may be considered. Several companies test sterility only in MCBs or WCBs and not in RtU cell banks (decrease from 13 to eight responses). Of 21 respondents, one-third test cell identity for MCBs or WCBs, whereas only one does so for RtU cell banks. For RtU cell banks, assay performance (e.g., system suitability test) is the most popular release assay (16 of 21 responses) followed by viability (14 of 21 responses) and mycoplasma (11 of 21 responses).

Figure 2. List of most common criteria used in the industry to assess (A) the quality of RtU cell banks, (B) along with the criteria used for cell bank release.

CV: Coefficient of variation; EC₅₀: 50% effective concentration; MCB: Master cell bank; RtU: Ready-to-use; WCB: Working cell bank.

System suitability criteria offer a reliable and readily available quantitative measure to monitor assay performance, and method performance trending of system suitability parameters provides a valuable data set to ensure suitability and consistency within and across RtU cell banks [6]. Although most respondents indicated that meeting system suitability criteria would suffice to demonstrate method performance and release of an RtU cell bank, additional criteria may be considered; for example, method performance parameters (slope, asymptotes, dynamic range, etc.) can be established for RtU cell bank qualification. When possible, decisions around acceptance criteria ranges should be data-driven and justified based on historical performance during assay development, validation, robustness studies and routine use [9].

As discussed previously, numerous steps in the cell banking process, particularly cell dispensing and freezing, can influence bank consistency. Depending on the size of the bank, enough vials should be tested to ensure consistency across the cell bank. The number of vials used for RtU cell bank qualification varied among the different companies. Depending on the RtU bank size, a minimum of three vials seems a common practice used to test and release an RtU cell bank. For banks greater than 200 vials, it may be appropriate to test additional vials for release. The use of a controlled-rate freezer allows the operator to customize the rate of temperature change before, during and after the freeze transition phase. This helps to preserve optimal cell viability while also offering an operational advantage (e.g., larger cell bank size) by reducing the time of a typical cryopreservation process.

Generally, expiration dates are not assigned to RtU cell banks when stored under ultra-low temperatures, which are expected to remain usable following long periods of storage under these conditions. Nearly half of the respondents (six of 13) reported successful use of RtU cells 3 or more years following generation of the bank, and one respondent reported use of RtU cells 15 years after cell banking.

Life cycle management of RtU cell banks for analytical testing

Introduction & management of RtU cells

The introduction and management of RtU cells in a cell-based bioassay are important and should be considered carefully. The process of introducing RtU cells is tied to the stage of assay and product development, with early introduction being the most simplistic. Introducing RtU cells once the product has advanced to commercial is the most challenging. Some guidance regarding these aspects can be gleaned from ICH Q14 [10]. In general, to successfully support a qualified or validated assay, RtU cells would need to behave in such a way that the bioassay is reproducible and accurate over the life cycle of the method. System suitability measures for RtU cells are expected to be similar to the system suitability criteria for propagated cells [6].

Traceability & documentation

To ensure lot-to-lot consistency, there needs to be proper documentation of how the RtU cell banks are sourced, generated, stored and tested for release into the assays. An inventory management system to track RtU cell bank inventory and critical reagent documentation is important for sample testing support. RtU inventory management is particularly critical in late-stage and commercial programs. An increase in demand for the therapeutic product is directly tied to an increase in batches to be tested and therefore the number of RtU vials used. Here the RtU inventory burn rate must be balanced with the time needed to generate and release a new RtU bank when the trigger point for the new batch is reached. Preemptive steps to maintain RtU inventory will mitigate program stalls and ensure the sponsor is prepared for increases in testing demand.

Scheduled review of RtU banks

Once RtU cells have been implemented for a validated method, there needs to be a periodic performance review of the RtU cells. The performance review should trend SST parameters for the method, as is typical for assays using cultured cells. With regard to RtU cell bank requalification, nine of 17 respondents reported using a data-driven strategy based on control trending or method performance data from ongoing or recent release and stability testing to justify continued use of a cell bank. Four respondents reported using experimental determination of RtU cell performance to requalify RtU banks at the end of shelf life. The rest of the respondents reported mixed strategies using both historical data and experimental determination or making business-driven decisions based on what the cells are used for (e.g., release assay or nonclinical). Using a data-driven approach, the majority of relevant functional parameters can be monitored against established control limits, which will enable early detection of drifts in RtU cell performance or the method as a whole. Using cultured cells as the backup strategy will enable the sponsor to generate, qualify and release a new RtU bank and can support testing without pauses or further delays to the system.

Assessing performance differences between RtU & cultured cells

Establishing and comparing RtU cells with cultured cells are sometimes needed for supply reasons or to allow both cell sources to be used in the method interchangeably. Comparisons between propagated and RtU cell sources can highlight some performance differences between the two approaches. Overall, the respondents collectively determined that small changes in any of the parameters are acceptable as long as the assay is still meeting the required performance criteria and is therefore suitable for the intended purpose. Since performance differences have been observed between RtU cells and cultured cells, we asked respondents to identify specifically what performance differences they deem acceptable (Figure 2B).

Across respondents, nine identified a change in the absolute EC₅₀ between RtU cells and cultured cells as acceptable. However, within that acceptance criterion, five respondents indicated that they would not accept a difference greater than twofold, whereas five would accept a difference of between twofold and tenfold. None would accept a difference greater than tenfold. Additionally, 11 respondents identified a change in the A/D ratio (upper asymptote divided by lower asymptote ratio) as acceptable, and 11 respondents also found changes in the signal background to be acceptable, with the majority accepting a 10–80% lower A/D ratio of RtU cells compared with propagated cells (Figure 2B). However, the degree of change in this criterion depends on the initial assay window. Interestingly, about an equal number of respondents would accept a smaller or larger background change (lower asymptote), with a slight preference toward a decrease in lower asymptote value. Five of the respondents found a change in the regression slope acceptable (B-parameter), as long as the assay still meets SST criteria and thus is still suitable for its intended use. Six of ten respondents would accept a 0.1 to 0.5 difference in the B-parameter and three of ten respondents would only accept a <0.1 change in the B-parameter between RtU and propagated cells. Cell viability is an important parameter that is usually part of the assay acceptance criteria. When questioned about the viability of RtU cell banks, respondents stated that they would accept some reduction in viability compared with the typical viability of propagated cells. Only one of 13 respondents would accept viability below 80%, with the remainder (12 of 13) accepting viability between 85 and 95%.

The percent coefficient of variation (CV) between sample replicates can be an important acceptance criterion in assay performance. When there was a difference in %CV between RtU and cultured cells, the majority of respondents (11 of 13) required the %CV to be less than 20%, with two respondents allowing a %CV of between 20 and 25%. No respondent would accept an RtU %CV above 25%. All of the aforementioned differences have been noted by respondents when comparing RtU cell sources with propagated cell sources; however, unless these differences have an impact on the method performance, they are not considered significant (Figure 2B).

To augment the results of the survey, we requested assay data from the BPDG-Bioassay workstream community comparing the performance of RtU and propagated cells. We received four responses from groups that had run side-by-side comparisons for qualified RtU and propagated cells. Figure 3 shows the comparison of these results as a bar graph. For the four different experimental mechanisms of action run with RtU and propagated cells, there is remarkably consistent performance. The only significant difference was the lower, upper and A/D window in the system suitability criteria. Despite this slight difference, the two sources of cells are considered equivalent and could be used interchangeably in the same method and use the same assay acceptance criteria.

Figure 3. Comparison of system suitability parameters observed when using propagated and ready-to-use cells in the same cell-based assay.

(A–D) Each graph represents a different mechanism of action. The number of individual assays performed for each assay was between 1 and 10 individual experiments.

CV: Coefficient of variation; EC₅₀: 50% effective concentration; RtU: Ready-to-use.

Considerations for introducing RtU cells at different stages of asset development

The stage at which RtU cells are introduced in an assay can be important in understanding the ease with which the introduction can be accomplished. With regard to introducing RtU cells into a method, most respondents (15 of 31) recommended introducing RtU cells before method qualification, whereas ten indicated that RtU cells are introduced after method qualification but before method validation. Another four respondents indicated introducing RtU cells after method validation, and two respondents introduced RtU cells after commercialization. Based on these responses, a decision tree was established to understand the potential obstacles encountered when introducing RtU cells during assay design, during early- or late-stage development or once the product was commercialized [10]. Figure 4 depicts a decision tree outlining the processes and considerations for introducing RtU cells during the different stages of assay development. As can be seen in the flow diagram, and as discussed by BPDG-Bioassay workstream members, the degree of complexity associated with introducing the RtU cells increases as a product progresses through development, with the most complex switch occurring once the product has reached commercialization, when a comparison study between propagated and RtU cells is recommended. At earlier stages of development, comparison of assay performance may be sufficient to introduce RtU cells.

Figure 4. Complexity and risk when introducing ready-to-use cells at different stages of drug development and recommended risk mitigation.

MoA: Mechanism of action; RtU: Ready-to-use.

Strategies for switching from cultured cells to RtU cells for a validated method

Respondents were surveyed regarding what strategy they used to add or switch from cultured cells to RtU cells. For a smooth transition, it is generally preferred that parallel qualification/validation using both cultured cells and RtU cells is performed if the RtU cells are available. However, when the method was validated prior to the availability of the RtU cells, the major strategy the respondents used to enable the switch to RtU cells was equivalence testing and statistical analysis (12 of 22 responses). Alternatively, other respondents indicated that method revalidation was used to enable the change (five of 22 responses). Two respondents also indicated that they used control chart trending to facilitate the change to RtU cells. For these latter respondents, they indicated that a need to transition to RtU cells after method validation has not yet occurred for them, as both cultured cells and RtU cells are used during early phases to first demonstrate equivalence followed by method validation and GMP implementation using the RtU cells [9,11].

The approaches taken by the respondent companies are aligned with the change assessments outlined in ICH Q14 [10]. Namely, the introduction of RtU cells as a replacement of or as an addition to cultured cells, which does not impact assay performance, can usually be accomplished with a simple bridging study (e.g., representative stability and or stress samples). This approach was used by the aforementioned 12 respondents. If performance characteristics of the assay are impacted by the introduction of RtU cells, then the revalidation approach, taken by five respondents, is more appropriate. Guidance and examples of analytical change evaluations and associated bridging strategies can be found in Table 2 of the draft ICH Q14.

Conclusion

This article provides valuable insight into overall common industry practices employed for the production, introduction, qualification and maintenance of RtU cells used in cell-based potency assays [6]. RtU cell banks have received strong endorsement from development and QC laboratories over the last few years as a means to improve both assay performance and flexibility in the cell-based bioassay testing environment. Through questionnaires and discussion, we found a number of common practices that aid the use of RtU cells. Among these was the timing of RtU cell use introduction in bioassays. One common practice was to introduce RtU cells as early as possible to gain maximum experience with making and using the cells before validation. Another key finding of the survey was the concept of using a two-tiered cell banking system for the manufacturing of RtU cells, where RtU cell banks would be derived from a similar MCB/WCB model. Although RtU cell banks do not need to be prepared in a GMP facility, they are considered a critical reagent. As such, the BPDG-Bioassay workstream consortium members recommend treating them as a critical reagent with appropriate criteria and testing before use. Another critical finding from the survey was the acceptance of small performance differences between propagated and RtU cells. The general consensus was that small performance differences were noted and deemed to be acceptable as long as the assay still performed essentially the same when using propagated and RtU cells.

Finally, the BPDG-Bioassay workstream recommends the following process to qualify a new RtU cell bank as a critical reagent before use: 1) sterility and mycoplasma testing; 2) assessing cell count and viability either immediately or 24 h after thaw depending on the time course of the bioassay; and 3) confirming uniformity of the RtU cell bank by testing, at a minimum, one vial from the beginning, middle and end of the freezing process. The use of RtU cells in assay development is increasing, and we therefore think the guidance on practical implementation of RtU cells contained in this article is timely and useful for those who have not yet either undertaken assay development with RtU cells or switched to using RtU cells.

Future perspective

RtU cells are becoming center stage in the development and implementation of bioassays in the GMP testing space. RtU cells have already become an important opportunity for many companies when considering the development of a new bioassay. The convenience of performing assays with RtU cells that have a long shelf life will transform the stability and release testing of biopharmaceuticals. The assay will transition from a carefully planned and timed event, due to the nature of propagated cell growth, to one that can be performed with little notice or planning. Furthermore, the intersection of RtU cells and full automation will deliver both increased flexibility (no cell culture) and increased throughput (automation), with the potential to reduce assay variability and increase assay reliability. In the future, bioassay analysts will benefit from an increased skill set, integrating automation and biology in stability and release testing.

Executive summary

Generation & logistics of ready-to-use cell banks

• Ready-to-use (RtU) cell banks are considered critical reagents. They can be generated either in-house or commercially. The banks can be established using either primary or immortalized cell lines.

Considerations for use of RtU cells in bioassays

• Using RtU cells increases assay scheduling flexibility and is less resource-intensive than using continuous cell cultures. It also simplifies the logistics of internal or external potency assay transfers.

Generation & logistics of RtU cell banks

• Across the companies surveyed, the use of RtU cells was evenly spread between assay development; chemistry, manufacturing and controls (CMC); and GMP/quality control release methods.

Logistics for how RtU cells are made & frozen

• Establishing a successful RtU cell bank that delivers consistent results involves the use of an optimized cryopreservation protocol, up to and including a set cell freezing density, freezing media and supplements and the actual vialing and cryopreservation process.

Storage

• RtU cells are commonly stored in liquid nitrogen vapor phase or a cryogenic ultra-low temperature freezer at temperatures (-135°C to -196°C) that suspend cellular metabolism and preserve cells for long-term use.

Inventory

• For assay use, RtU cell banks undergo a critical reagent qualification process with subsequent storage and inventory management. Transport of RtU banks between production and testing facilities must be performed with cold chain management to ensure preservation of cell viability and function while adhering to regulatory requirements and shipping regulations.

Assessing performance differences between RtU & cultured cells

• The most common parameters assessed for releasing an RtU cell bank include viability, sterility and assay performance. Additional parameters may include identity, EC₅₀, doubling time and relevant biomarker expression. Establishment of system suitability criteria is data-driven and specific to each method, where historical data play a large part in the setting of the method performance parameters. These performance parameters are also used to confirm cell bank consistency across the vialing process as well as during long-term storage, requalification at the end of shelf life and generation of new cell banks.

Life cycle management of RtU cell banks for analytical testing

• The use of RtU cell banks can be introduced during various stages of assay development. This includes once the product has advanced to the commercial phase, when introduction of RtU banks can be supported by adhering to International Conference on Harmonisation guidelines.

Author contributions

All authors contributed equally to writing the survey questions, the discussions that led to the interpretation of responses and writing and reviewing the manuscript. In addition, JR White sought, analyzed and interpreted the data submitted for Figure 3.

Financial disclosure

The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with an interest in or conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

Acknowledgments

The authors wish to acknowledge H-S Lam (GlaxoSmithKline), L Mackenzie (GlaxoSmithKline), L Bonnet (Fresenius Kabi SwissBioSim), A Viaud (Fresenius Kabi SwissBioSim) and T Milhiet (UCB) for ready-to-use and propagated cell data in Figure 3 and other members of the BioPhorum Development Group-Bioassay workstream for the useful discussions that contributed to this article.