Assuring Monoclonality with Direct Measurements: Why Imaging Prevails
September 28, 2017
By Steve Wiltgen, PhD, BioSpace.com Contributor
The market for therapeutic monoclonal antibodies (mAbs) has grown rapidly in recent years due to the significant clinical advantages they provide to customers and the economic benefits they offer pharma/biotech companies when compared to small molecule development. This rapid growth has heralded much promise for new therapies, but many challenges remain.
Although the development of a therapeutic monoclonal antibody takes less time than small molecule drug discovery (10-15 years on average), manufacturing costs are much higher. Maintaining quality production is also challenging due to the biological nature of the product, which often introduces variabilities into the process. These biological fluctuations are not always well understood or characterized, leading to substantial rework in order to maintain the same quality product.
Combine these challenges with one of the most regulated markets in the world, and you have a situation where even modest improvements in process efficiency and quality can have millions of dollars’ worth of impact.
Challenges Facing Early Stage Development of Clonal Cell Lines
As with any product life cycle, bringing a therapeutic monoclonal antibody to market begins at the R&D stage. And just like any other product, the largest proportion of time spent on developing a monoclonal antibody is associated with R&D activities. Not surprisingly then, this stage is also where most products fail, making it an attractive area for workflow improvements.
Because monoclonal antibodies, by definition, are originally derived from a single cell, the isolation of individual cells is critical to the success of any R&D efforts surrounding novel mAb discovery. However, traditional methods for isolating single cells such as limiting dilution and flow cytometry suffer from several major drawbacks.
First, these methods are very inefficient at isolating viable single cells. Typical clonal outgrowth efficiencies for both techniques are <25%. Second, limiting dilution and flow cytometry also rely on indirect measurements to isolate single cells. Limiting dilution relies on the Poisson Distribution to determine the likelihood of a single cell in a well, whereas flow cytometry relies on the reflection/refraction of light as it passes through a cell.
These indirect measurements make certain assumptions to infer the presence of a single cell. For example, flow cytometry makes the logical assumption that two cells should reflect more light than one cell. However two, small cells may reflect a similar amount of light as a larger single cell, making the objects indistinguishable.
In other words, two cells can be misidentified as a single cell if assumptions are not properly managed. This not only reduces the probability of clonality under these specific circumstances, it also reduces the confidence of the method in general, which has significant consequences for regulatory requirements of the FDA.
The Role of Imaging in Monoclonality Assurance
So what does the FDA require in terms of monoclonality? As it turns out, it’s pretty loosely defined. One guideline provided by ICH Q5D states, “For recombinant products, the cell substrate is the transfected cell containing the desired sequences, which has been cloned from a single cell progenitor.”
There are other guidelines, but overall, the FDA leaves this topic broadly defined in order to accommodate a variety of processes. The prevailing view within the industry though is that indirect methods such as limiting dilution and flow cytometry require direct visual measurements (i.e. imaging at the microplate level) to provide assurance of monoclonality. In the absence of visual confirmation, the industry expects that a second round of single cell isolation will likely be required.
As a reference point, a typical flow cytometry experiment can achieve clonal probabilities of ~98-99%, which is usually not sufficient evidence for the FDA because these probabilities are based on indirect measurements.
Therefore, the presence of single cell-containing wells on a microplate are typically validated by directly measuring them visually using an automated, label-free imager (such as the CloneSelect Imager). The CloneSelect Imager (CSI) is specifically designed to quickly capture images of single cells in a microplate (<90 sec for an entire 96-well plate) using label-free technology.
This is not to say that cellular imaging of single cells at the microplate level is a fool-proof method for assurance of monoclonality. As with any technology, there are limitations that must be taken into consideration.
Label-free identification of single cells in a well is a manual and labor-intensive process that often leads to errors. Cellular debris can easily be misconstrued for cells by the untrained eye. The vast microplate surface also introduces its own set of complexities.
Imagine being tasked with finding a golf ball inside an Olympic-sized swimming pool (equivalent to finding a single cell in the well of a 96-well microplate) — there is a decent chance you won’t find it. Now multiply that by 96. And that’s just one microplate.
Despite these limitations, it is well-established that providing direct images at the microplate level as evidence of clonality eliminates the need for a second round of cloning, thereby improving productivity 2-fold and reducing timelines by several weeks.
Single-cell Printing: An Improved Technology for Single Cell Isolation and Imaging
The benefits of improved clonal outgrowth rates and high single-cell deposition efficiencies combined with visual assurance of monoclonality are fairly obvious, but developing the instrumentation to support these features had been challenging until recently. The CloneSelect™ Single-Cell Printer™ (SCP) by Cytena and Molecular Devices accomplishes all of these feats.
The SCP uses a microfluidics-based system to image and isolate a single cell immediately prior to gently depositing it into an individual well of a microplate. In doing so, the system achieves a typical clonal outgrowth efficiency of > 70%, approximately three times more efficient than traditional methods. Images captured on the SCP are only 200 microns in size as well, simplifying the process of finding and identifying single cells while also minimizing the need to scan an entire well for the absence of a second cell. Revisiting our analogy, finding a single cell with the SCP is like finding a golf ball in a bath tub.
The additional benefit of capturing a sequence of single-cell images on the SCP prior to deposition is that it provides direct evidence that a given cell line is monoclonal (i.e. SCP images provide high assurance of clonality).
As discussed previously, providing assurance of clonality through direct evidence is critical for regulatory agencies because it minimizes the risk inherent to indirect methods. However, although images from the SCP provide confidence that a given cell line is monoclonal, it is unknown whether they will provide sufficient evidence to the FDA for assurance of monoclonality. Not only because FDA guidelines are loosely defined, but also because it’s a relatively new technology with limited data.
We have observed a probability of monoclonality >98% (data unpublished) based on correlating images captured with the SCP to images captured with the CSI. Considering that this probability is similar to the probability observed with flow cytometry and that flow typically requires imaging to assure clonality, the CSI will likely serve a similar role in the SCP workflow.
However, the CSI adds substantially more value to the SCP workflow when compared to flow cytometry because it provides a second, independent and direct measurement of clonality, thereby increasing both probability and assurance of monoclonality. In addition, the CSI can also characterize the growth pattern and morphology of candidate clones, further complementing the cell line development workflow downstream of single-cell printing.
Ultimately, the combination of the CloneSelect Single-Cell Printer and CloneSelect Imager not only saves time and money by improving the efficiency of single cell depositing and viability post-deposit, but also reduces timelines by providing the image evidence needed to support a single round of cloning. They say that a picture is worth a thousand words, but in this case, it’s probably worth thousands of dollars.
Steve Wiltgen is the Product Manager, Bioproduction Development at Molecular Devices, LLC, where he is responsible for the marketing strategy for the Bioproduction suite of products including the ClonePix™ and QPix™ systems. He obtained his Bachelor’s of Science degree in Mathematics and Biological Sciences from the University of Nebraska-Lincoln and his Ph.D. in Neurobiology and Behavior from the University of California, Irvine where his research focus was developing a novel super-resolution imaging technique to look at ion channel function. The single-cell printing technology is developed by Cytena GmbH. To learn more about the technology, please visit moleculardevices.com/CloneSelectSCP.