1. Methodological validation of monoclonal origin
One round of limiting dilution or single-cell plating equipment with monoclonal imaging equipment. Different from probabilistic calculations and statistics, this method can provide image data of monoclonal origin, but at the same time it faces certain challenges. It is necessary to consider whether the cells are adhesional, whether they sink to the bottom, and the position of the cells after the fall. The hole position alignment of the imaging equipment, the bottom focus of the microplate, the light source, the lens shift, the resolution, the edge effect, etc., all affect the accuracy and validity of the image.
Validation of the monoclonal origin methodology can be carried out around the following aspects:
1. Verification of natural cell sedimentation process.
Brightfield imaging was used to investigate whether the cells of the platform process could settle during the plateau sedimentation time, and the effect of sedimentation at different time points.
2. Centrifugal sedimentation process verification.
Brightfield imaging was used to investigate whether the cells of the platform process could settle under the conditions of platform centrifugation, and the effect of sedimentation at different time points after centrifugation.
3. Cell identification verification.
Brightfield and fluorescence imaging were used to investigate the ability of imaging equipment to identify monoclonals.
4. Cross-validation of monoclonal plating equipment projects.
Brightfield and fluorescence imaging were used to investigate whether there was cell line crossover in monoclonal plating equipment between different projects.
It is necessary to verify the monoclonal origin of single-cell plating equipment and imaging equipment with single-cell plating equipment and imaging equipment. The resolution ability and sorting efficiency of single-cell plating equipment for single cells were investigated, and cell identification and cross-validation of single-clonal imaging equipment were also carried out through the validated cell sedimentation method. At the same time, the confidence of single-cell plating and imaging equipment as a single-cell sorting method for monoclonal origin was validated and improved.
Genentech used a single-cell sorting method combining single-cell printing with plate imaging to verify the efficiency of its sorting and confidence in its monoclonal origin. As can be seen from the sorting images of the single-cell plating device, there are 959% single-cell wells, 26% empty cell wells, 15% of multi-cell wells, due to low image contrast, cells falling into the ROI (Region of Interest) outer ring area, or the software failed to identify adherent cells, resulting in false positive wells.
Cells sorted by mixed populations of green and red fluorescence were photographed and tracked using an imaging device, which was able to identify 879% of the single-cell wells, after 14 days of culture, 9 wells were red-green mixed cell populations, 03% false-positive monoclonal rate. Therefore, systematically verifying the monoclonal origin of the single-cell platform sorting method can effectively improve the reliability of the experiment and provide sufficient data for the verification of the monoclonal origin, which plays an important role in providing good cell line monoclonality in the whole drug development process.
In 2019, FDA reviewers made some observations on the monoclonal origin of cell lines: Monoclonal origin verification is to reduce the heterogeneity of cell banks, maintain consistency and predictability in the production process, and ensure that the yield and quality of the final product are within the preset range. If there are major defects and risks of monoclonal origin that affect the quality of products, it is necessary to carry out research on the control strategy of monoclonal origin as part of the overall control strategy. Verification of monoclonal origin is mainly to evaluate the safety and efficacy of the final product, and the view that monoclonal origin is not equal to the genetic homogeneity of the cell line. It is further pointed out that monoclonal origin plays a very important role in the quality and safety of products.
2. The difference between the concepts of probability and assurance in monoclonal origin
probability: Calculate the probability of a monoclonal mathematically (e.g., Poisson distribution). assurance: The information required to assess the origin of a monoclonality, including probability calculations, supplementary documents and data.
In general, the following monoclonal origin cases are acceptable: high probability, low probability + high assurance, low probability + little assurance + adequate control evaluation strategy.
3. Methods of analysis supplemented by evidence of monoclonal origin assurance
Unique genetic features (plasmid integration sites, plasmid rearrangements, etc.) are unique and generally unaffected by CHO cell plasticity and can serve as evidence of monoclonal origin. There are typically several methods for the analysis of plasmid integration sites:
01. Fluorescence in situ hybridization technology (FISH): Observe the integration site at the chromosome level, without obtaining the integration site sequence, and directly visually test the position and uniformity of DNA on the chromosome. Moreover, in situ hybridization is not affected by intra-site variation and inter-site copy number, and has become an important means for repetitive sequence and polygenic family mapping. However, it is susceptible to chromosomal rearrangements and has high sensitivity for low-copy sites.
02. Southern blot: KB-level DNA detection level, the bands produced by it are unique to each integration site, and there is no need to obtain the integration site sequence, and it is not easy to be affected by chromosome rearrangement. However, the workload is large, the resolution is not high, and it will be affected by the rearrangement of the attachment sequence of the integration site.
03. Junction PCR: BP-level DNA detection level, the critical sequence of the gene integration site is obtained by NGS or traditional methods (Inverse PCR, SPLINKERETTE-PCR or LAM-PCR), and then the critical sequence is amplified by junction PCR to determine the existence of unique integration site. This method is not susceptible to mutations and rearrangements, but requires subcloning and obtaining the sequence of the integration site.
04. Next-generation sequencing technology (NGS): This method can be directly used for the identification of the monoclonal origin of the integration site, which does not necessarily require a large number of subcloning, but the operation and analysis are cumbersome and time-consuming, and some sites may be missed.
Fourth, the control strategy (CS) of monoclonal origin
The control strategy for non-monoclonal cell lines depends on the product and its application. Some of the conventional control strategies include, but are not limited to, additional detection methods such as LC-MS to detect sequence variability, glycosylation levels, etc.;Comprehensive systematic evaluation beyond the limited number of cell passages in vitro;Add more critical process parameters;Trend statistics of process control;Risk assessment of changes to important raw materials;Strict control and comprehensive identification of newly established cell banks, etc.
5. Summary
Monoclonal origin is part of the overall control strategy in the biopharmaceutical manufacturing process and plays a crucial role in the safety and efficacy of the product. Validation of monoclonal origin is designed to reduce the heterogeneity of cell banks, ensure consistency and predictability in the production process, and ensure that the yield and quality of the final product are within preset ranges. It is recommended to do a good job of monoclonality from the cell line screening stage as soon as possible to reduce the risk of subsequent drug development.
References:
1. achieving greater efficiency and higher confidence in single-cell cloning by combining cell printing and plate imaging technologies. biotechnology progress, (doi:10.1002/btpr.2698
2. considering “clonality”: a regulatory perspective on the importance of the clonal derivation of mammalian cell banks in biopharmaceutical development.