The field of cell and gene therapy is driving major advances in medicine, including marked successes in treating cancer, spinal muscular atrophy, vision loss, and sickle cell anemia. The development of biotherapeutics, however, carries specific contamination concerns. All biotherapeutics, whether originating from patient cells or developed in cell lines from bacteria, yeast, plants, insects, humans, or other animals, must be free of contaminants, such as mycoplasma, for safety reasons. Additionally, cell-line derived biotherapeutics, such as adeno-associated virus (AAV) therapeutics, must be free from host cell contamination.
Mycoplasma is a relatively common, insidious cell culture contaminant that is problematic for any research lab due to subtle impacts on cell function and responses. It is particularly problematic for cell-based vaccines, biologics, and gene therapies and potentially shuts down labs. In addition, several species are pathogenic—contaminated products can trigger infections in patients, and even non-pathogenic species may cause infections in immunocompromised individuals.
Residual host cell contamination forms a major concern for many biotherapeutics. DNA from host cells is systematically removed from products but complete DNA extraction can be difficult, with removal efficacy dependent on DNA concentrations and matrix conditions. As a result, trace amounts of host cell DNA typically remain. The presence of residual host cell DNA in products carries multiple potential health risks from infection to effects on genomic expression or oncogenic events in the case of immortalized cell lines.
In addition to the health risks posed by contaminants, releasing a contaminated product would have severe consequences for the responsible lab, likely resulting in shutdowns, major losses, and/or irreparable damage to the organization’s reputation.
Allowable limits for contaminants, including Mycoplasma and residual host cell DNA, are set by relevant regulatory agencies, like the World Health Organization, the US Food and Drug Administration, and the European Pharmacopeia. For therapeutics derived from human immortalized cell lines like HeLa and HEK293, the guidance calls for residual DNA fragment lengths of less than or equal to 200 bp—below functional gene length—and under 10 ng per dose. Challenges arise from attempting to measure these tiny fragments and trace concentrations with the accuracy and precision required of regulated labs.
The struggles with the status quo
The most commonly used solutions for determining the presence of or quantifying contaminants, namely culture tests, qPCR, and DNA quantification/qualification platforms like the BioAnalyzer, are limited in their speed, sensitivity, or accuracy at the scales required by current regulations.
Traditional culture-based tests are still considered by many to be the gold standard for Mycoplasma detection but take 28 days to obtain results. This is too long to wait for many applications in medicine, pharmaceuticals, and biotech, resulting in lengthy research delays, production delays, or late-stage mitigation strategies that culminate in considerable losses.
Both Mycoplasma and host cell contamination can be detected using qPCR, a technique that has grown in popularity over the last couple of decades. Results are obtained much faster than with cultures and the test is specific and moderately sensitive.
However, qPCR has a few limitations that affect its suitability for detecting trace contaminants. It has a high false-positive rate for Mycosplasma, which triggers unnecessary losses. Second, quantifying DNA at the levels expected in a finished product is challenging with poor resolution for low input DNA concentration (10 copies or less) and size (under 200 bp). Depending on the thresholds set for quantification cycles, trace levels of contaminating DNA may not trigger a positive hit. Accurate quantification is difficult to achieve, as the data is qualitative without a standard curve. Additionally, some of the more popular assays generate non-specific signals.
BioAnalyzers are also frequently used to look for host cell contamination as an inexpensive, rapid, and easy test. This analysis can’t differentiate between species present, though, instead quantifying total DNA in the sample. Accuracy is also low for small fragment sizes and low concentrations. Conversely, it won’t analyze high molecular weight DNA (greater than seven kb).
The need remains for accurate, reliable, and affordable detection and sizing of contaminating DNA that meets current regulatory standards.
Improving sensitivity and accuracy with ddPCR
Droplet Digital™ PCR (ddPCR) testing offers a few advantages over traditional formats, including increased accuracy, resolution, and specificity. With this technique, samples are partitioned into water-oil emulsion nanoliter droplets and the presence or absence of target DNA is recorded for each droplet. The starting concentration is then calculated using a Poisson distribution based on the proportion of negative droplets. This nanoscale presence/absence approach avoids issues with amplification bias or instrument variability that typically affect PCR.
Several additional benefits of ddPCR make it an ideal solution for mycoplasma and host cell DNA contamination testing. It offers higher sensitivity and specificity with a low false-positive rate, minimizing unnecessary losses and set-backs. Quantification of DNA, measured to femtograms and reported as the number of genome copies per reaction, is accurate and precise regardless of fragment size and without the need for standard curves. Workflows are also fast and simple with high throughput. Partitioning limits the effects of inhibitors within complex intermediates, so diluted samples can be input directly without DNA extraction steps, minimizing handling, additional steps, and variables and maximizing recovery. This also makes ddPCR technology an effective solution for all types of samples generated across development and manufacturing processes.
Existing ddPCR kits offered by Bio-Rad cover testing for key cell and gene therapy contaminants, including HEK293 residual DNA sizing and quantification, CHO and E. coli residual DNA quantification, and Mycoplasma detection. The Mycoplasma kit can detect 112 species with low cross-reactivity and makes direct calculation of GC: CFU (genome copies to colony forming units) easy with reported genomic copies per reaction. The HEK293 kits have high specificity (99.8 percent for quantification and 95 percent for sizing), and sizing provides the median fragment lengths and confidence intervals without reference curves. The reagents by Bio-Rad are also guaranteed free of DNA contaminants.
Support for regulatory adherence is critical in any methods used to determine or quantify contaminants in biotherapeutics. Bio-Rad’s solutions include software tools that assist with compliance with U.S. FDA 21 CFR Part 11 regulations, including the generation of audit trails with tracked protocol changes when using ddPCR instruments. In addition, auto-gating and auto-thresholding features in the software help minimize data handling and interpretation.
To learn more, visit www.bio-rad.com/cgtresources.
Limit of Detection (LOD), Limit of Quantification (LOQ), Limit of Blank (LOB), and detection ranges for HEK293 sizing and quantification, CHO and E. coli quantification, and Mycoplasma detection kits available from Bio-Rad.
|HEK293 Residual DNA Quantification Kit||HEK293 Residual DNA Sizing Kit||Mycoplasma DNA Detection Kit||E. coli Residual DNA Quantification Kit||CHO Residual DNA Quantification Kit|
|LOD||0.1 pg/µl||2 pg/well||<6 genomic copies/well; 1 CFU/ml||15 fg||1 fg|
|LOQ||1 pg/µl||N/A||N/A||≤30 fg/20 µl||≤15 fg/20 µl|
|Range||1 pg – 80 ng at R2?= 0.998||2 pg – 300 ng at R2?= 0.996/0.997||N/A||30 fg to 30 pg||3 fg to 3 pg|