This is the typical workflow used by groups developing stable cell lines for production of therapeutic recombinant proteins and biosimilars. In many cases, these proteins will be monoclonal antibodies (mAbs), but some companies also use them to produce enzymes for enzyme replacement therapies in rare diseases.
The importance of clonality
A key assessment in the development of any new stable production cell line is the 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 request assurance of clonality for the Master Cell Bank (MCB) as part of an IND process.
Additionally, it is very important to consider the quality and consistency of the protein therapeutic. This is important for batch to batch production, but also where companies are making biosimilars and they need to show equivalence.
Continued Protein Supply
Manufacturing the quantity of protein needed to supply therapeutics to patients in clinics, with a consistent quality, can only be achieved through stable cell line production. If the regulator decided to put a hold on the product or did not accept the clonality for a given cell line, then this could lead to shortages of the drug available to the market. This was discussed by Dr Audrey Jia in her video interviews.
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 which has nearly halved development times. More recently, novel single cell dispensing approaches, like the VIPS, have begun to replace traditional FACS as they can deliver higher cloning efficiencies, meaning potentially 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 which have led to more targeted integration for the Gene of Interest (GOI) rather than previous random integration. These approaches have included Zinc Finger nucleases, TALENS, CRISPR/Cas9 and transposases. This together with improved vectors and better transfection methods means that enrichment is becoming less important and far fewer clones need to be screened to find the high producers.
The need now is for a simple, dedicated portfolio of instruments for cell line development which do not require expensive specialists to run them, and which do not rely on specialist chips or consumables which could pose a supply risk. This portfolio will also provide data connectivity between respective instruments, providing a complete audit trail and assurance documentation for the production of cell lines for the client, and ultimately the regulator.
Single cell seeding
The requirements are to dispense single cells from a population into wells of microtiter plates, and then to prove that this has been achieved
This should be done at high efficiency so that most wells contain a single cell and they need to be viable after seeding so they grow into a colony over the next few days. For example, the high pressure of FACS (>20 psi) can destroy many of the cells on seeding.
Cell types used are predominantly CHO suspension cells which are expressing an antibody or recombinant protein of therapeutic importance.
Currently, methods for seeding single cells and selecting colonies include the following:
- Limiting Dilutions
- ClonePix picking of colonies from semi-solid media
- Newer single cell printers and opto-electronic approaches
Using our VIPS system provides far greater benefits and results compared with these other 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 hit picking
Regardless of the cell dispensing method used, it’s essential to show that the single cell successfully arrived in the well
From a regulatory perspective, the whole well image is fundamentally important as assurance of clonality.
With the advent of high quality automated imaging in the last 5 years, many customers now only perform a single round of cloning which significantly reduces their timelines for the whole cell line development process.
The numbers of plates screened can vary enormously between customers from a handful to several hundred per project. Part of this can be related to the nature of insertion (random versus targeted) and the efficiency of the seeding methods. After seeding, cells are allowed to settle to the bottom of the well or can be centrifuged.
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.
Earlier clone assessments
Aside from clonality, many customers also want to get an earlier handle on attributes for the best clones. For instance, they will look at growth rates and doubling time of clones, and also 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 outgrowth studies.
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 regulation
In recent years, regulators have indicated that assurance data provided by imagers should meet specific criteria and Solentim have automated this process, by way of generating the Clonality Report, to ensure no errors are made in the collation of this assurance data.
There are improvements to be made at several steps of the process. These contribute to an overall improvement through the combined effect of the complete Solentim portfolio. 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 further process. The VIPS can also carry out the Day 0 whole well image to confirm the clonality. This also removes the need for a second round of cloning, saving on average 4-6 weeks.
Then using the Cell Metric imager, the subsequent days of outgrowth imaging can be performed along with the further expansion studies of the best clones.
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.
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