A key workflow for the production of stable cell lines to facilitate the creation of therapeutic recombinant proteins and biosimilars
In many cases, the therapeutic proteins produced will be monoclonal antibodies (mAbs), but some companies also use these stable cell lines to produce enzymes for enzyme replacement therapies in rare diseases.
The importance of clonality
In the development of any new stable production cell line it is essential to provide assurance of clonality. Historically, this was assessed based on probability, but more recently, with the development of dedicated imaging, assurance can be provided by a high clarity picture of the single cell in the whole well shortly after seeding.
For any new biological drug, the FDA and EMA both recommend assurance of clonality for the Master Cell Bank (MCB) as part of an IND process.
Additionally, it is crucial to consider the quality and consistency of the protein therapeutic. This is important for batch to batch production, but also in demonstrating equivalence during the production of biosimilars.
Cell Line Development workflow evolution
Several issues have caused the workflow to evolve and change over the past five years. Firstly, there have been advancements in instrumentation. Second-generation dedicated imagers for clonality, such as Cell Metric, have been largely able to remove the need for a second round of cloning.
More recently, novel single cell dispensing approaches, like the VIPS, have begun to replace traditional FACS as they can deliver higher cloning efficiencies, potentially resulting in far fewer plates per project. Improvements in automated liquid handling have also meant more customers moving from 96 to 384 well plates.
Secondly, new cell engineering techniques have emerged, leading to more targeted integration for the Gene of Interest (GOI) rather than previous random integration. These approaches have included proprietary landing pads (e.g. Pfizer), Zinc Finger Nucleases, TALENS, CRISPR/Cas9 and transposases (see below).
Stable Transfection - improved vector design enriches for high producer clones
Up until recently, scientists would try and select clones which were indicative of high protein secretion (learn more). Some techniques try using a fluorescent label in a flow droplet to measure the secretion of a cell in transit, whereas other methods try to measure secretion in the form of immunoprecipitation or a halo in semi-solid media e.g. ClonePix. In reality, when these cells are isolated, the indicative level of protein expression does not hold true and these methods only truly served to separate non-producers from the population.
Most recently transposases, such as the ATUM Leap-In transposase technology have become commercially available for inserting a gene of interest at multiple target recognition sites in the genome, and typically at sites of open chromatin, for high resultant expression. These methods are compatible with forming stable cell lines for biotherapeutics and give rise to a transfected population where most clones are high producers. Using the ATUM Leap-In technology, for clonal distribution after transfection, typically over 60% of the clones are in the first quartile for expression level (see figure below). In practical terms, this means that scientists no longer need to try and pre-screen for high producers and far fewer plates of single cell clones are needed per project, saving time and money.
Benefits of using transposases to develop manufacturing cell lines
• Genomic integration in >90% of transfected cells = Rapid stable pool recovery
• Product from stable pools is comparable to clone derived protein = Earlier PD and tox manufacturing
• Multiple “single copy” integrations (2-60 copies per genome)
• Integration into transcriptionally active regions = High productivity
• Structural and functional integrity of the integrated transposon = Preserves heavy and light chain subunit ratios
• Exceptionally high degree of clonal genetic stability
Suitable for Next Generation Biologics
• Unlimited payload = Multiple ORFs can be co-expressed at optimal ratios
• Footprint free excision
Single cell seeding
From the stable transfection pools, single cells are dispensed into microtiter plate wells, then arrival confirmed, as per requirements.
Ideally, this should be done at high efficiency resulting in the majority of wells containing a single cell which is viable after seeding. These cells will then grow into colonies over the next few days. Cell types used are predominantly CHO suspension cells expressing an antibody or recombinant protein of therapeutic importance.
Current methods for seeding single cells and selecting colonies all have their drawbacks:
- Limiting Dilutions (inefficient)
- FACS (difficult to operate and high pressure leads to low outgrowth)
- ClonePix picking of colonies from semi-solid media (only removes non-producers)
- Newer single cell printers (need separate whole well imaging) and opto-electronic approaches (expensive to buy and run)
Using our VIPS system provides far greater benefits and results compared with other seeding and colony selection methods. VIPS will generate a resultant plate with high seeding efficiency in around 10 minutes, and due to the gentle nature of the dispensing method many of these wells will go on and successfully grow into colonies.
Clone screening, assurance and successful colony outgrowth
Regardless of the cell dispensing method used, it is essential to show the successful arrival of the single cell in the well.
From a regulatory perspective, whole well imaging is fundamentally important in providing assurance of clonality. With the advent of high-quality automated imaging in the last 5 years, many customers are now able to perform only one round of cloning, significantly reducing their timelines across the entire cell line development process.
Whole well imaging can be performed on both the VIPS or the Cell Metric. The optimal solution is to have the VIPS and Cell Metric connected via the Sync Server, producing plates with high seeding efficiency and outgrowth, along with the capacity for further outgrowth imaging to be performed as well as further expansion studies of the best clones.
The numbers of plates screened can vary enormously between customers from a handful to several hundred per project. After single cell seeding, cells can settle to the bottom of the well or can be centrifuged.
There are many important considerations for optimising the outgrowth from single cells. One of the key factors is growth supplements which can be added to the growth media. Solentim provide the InstiGRO supplements for both CHO and HEK cells which have been shown to significantly improve the outgrowth and cloning efficiency for the common commercial cells lines such as HD-BIOP3 (Horizon) and CHOZN (Millipore Sigma).
Cells can then be imaged by the Cell Metric for assurance of clonality. The regulator has set out expectations for the imaging and stated that it should be carried out on a whole well basis and completed on Day 0 and then subsequent days of growth and division. The imager should also be used to validate your cloning method (FACS, LD, VIPS etc). This is only feasible if the imager can guarantee focus 100% for every well in the plate. The Cell Metric is uniquely able to do this by adjusting focus in each well for the curvature and distortion of each plate.
Validation methods can be designed for bright field and fluorescence modes. These experiments are essential to generate some statistical support to your cloning methods and the chances of a second cell being in a well which you believe to be clonal. It is generally not recommended or encouraged to use fluorescence labels in the real experiments and some of our customers have shown the Celltracker dyes do reduce outgrowth. Also, given that customers are developing therapeutics for patients, then extraneous fluorescence should be avoided. Imaging is carried out for Day 0 and subsequent days of growth and ultimately colony formation. This can be 10-21 days depending on cell types and protein being expressed.
A library of images is created for each well and it is possible to track back in time from the colony all the way back to Day 0 and confirm if it started from 1 cell or not. It can vary from customer to customer when they do this review of wells. Some customers do it immediately after colony formation and then hit pick the wells which formed a colony and started from a single cell. This could involve picking and re-arraying several hundred clones. Other customers re-array (or cherry-pick) all the wells that grew into fresh 24 DWP which are then shaken in fed-batch mode. Once they have found the best clones based on titre, they will take this smaller subset and review the original well images for assurance of clonal origin. For the clonal wells of choice, the Cell Metric can write a cherry-picking CSV file which can be exported to a third-party liquid handling robot for re-compiling into full 96 well plates of clones.
Finally, a report can be rapidly generated for the well history of the top clones. This documentation package is called the Clonality Report and is importantly something which customers can include as part of their IND submission to the regulator to support their clonality claims.
Clone attributes and earlier clone assessments
Aside from clonality, many customers also want to get an earlier assessment of attributes for the best clones. For instance, they will look at growth rates and doubling time of clones and will want to measure confluence to normalise with an early protein productivity assessment e.g. from Octet or ELISA. The Cell Metric is the ideal tool for these confluence measurements.
Once this subset of the top clones has been cherry-picked and are in shaking fed-batch culture, then more accurate assessments can be established for protein titre and Qp (Specific Productivity). Many customers carry out this expansion step using 24 DWP in shaking incubators. Solentim now also provide a supplement called InstiSHAKE which will enhance cell growth in shaking culture.
Once a subset of the clones has been cherry-picked and are in shaking fed-batch culture, then more accurate assessments can be established for protein titre and Qp (Specific Productivity). Many customers now do this using 24 DWP in shaking incubators. Again, the Cell Metric has an option for accurate viable cell counting (VCD) using Trypan Blue and these can be used in titre and Qp.
The Clonality Report - documentation for the Regulator
In recent years, regulators have indicated that assurance data provided by imagers should meet specific criteria. Solentim has automated this process by creating the Clonality Report, ensuring no errors are made in the collation of this assurance data.
Using a barcode system on the plates will eliminate errors in terms of clone history and data tracking. Finally, the Clonality Report generates a tamper-proof reliable report including relevant images for the end-customer, be that a client at a CRO or the regulator for an IND filing. These reports literally only take minutes to annotate and generate, and can be emailed in electronic format. From a user perspective, VIPS and Cell Metric share a common software interface for ease of use. Batch information only needs to be entered once and then is carried through automatically. VIPS data will also be added to the Clonality Report for even more complete tracking and assurance.
These reports, which can form part of the IND submission package, follow the valuable user-selected clonal wells from single cell to colony throughout the growth period with an image series (whole well and single cell image) in order to provide full time-and date-stamped data information for the well from single cell to colony. Annotation is possible of the single cell and any other features e.g. debris. The reports are generated in minutes in PDF or PowerPoint presentation formats which can be exported and shared with interested parties e.g. cell banking groups, or a CMO customer.
Remote data viewer software enables the user to analyse data and generate clonality reports from their office computer.
Summary - overall workflow efficiencies
In summary, there are improvements to be made at several steps of the process. Firstly, at the vector and transfection step, the ATUM transposases generate a stable pool population in which the majority of clones are already high producers with no requirements to enrich the population. At the single cell seeding stage, the VIPS delivers a plate with high seeding efficiency. This means that per project, you will have a lot less plates to image and process further. The VIPS can also carry out the Day 0 whole well image to confirm clonality. This also removes the need for a second round of cloning. Finally, using the Cell Metric imager, which automatically picks up the batch information from the VIPS, the subsequent days of outgrowth imaging can be performed along with the further expansion studies of the best clones.
Customers have already demonstrated that, by using this approach, they have been able to reduce cell line development project timelines from 6 months to <12 weeks (view our case studies to learn more).
The Clonality Report
“meets the FDA requirements of whole well image and where single cell feature is located; allows highlighting of other features which can then be described away”
Dr Audrey Jia, Former FDA CMC
Reviewer. IBC Asia, Shanghai, May 2017