Rapid Contamination Detection in Short Shelf-Life Biopharmaceuticals

At the Borderline of Sensitivity and Noise Detection of Nucleic Acid Traces – A PCR Kit Manufacturer Perspective Lisa Hollstein¹, Miriam Dormeyer², Robert Hertel*², Alexandra Müller Scholz*² ¹ Sartorius Stedim Biotech GmbH, August-Spindler-Straße 11, 37079 Göttingen, Germany ² Sartorius Lab Instruments GmbH & Co. KG, Otto-Brenner-Str. 20, 37079 Göttingen, Germany * Corresponding authors: Robert.Hertel@Sartorius.com and Alexandra.Mueller-Scholz@Sartorius.com Abstract The bio-pharmaceutical world has moved drastically towards short-lived personalized cell and gene therapy products in recent years. With the new quality control requirements, several new rapid microbial detection methods have been developed. USP <1071> states, “The ability to detect contamination, in real-time, prior to the administration of the shortlife product may be considered more important than detection of a single colony-forming unit (CFU) in the product.” However, the new methods strive to detect the holy grail of 1 CFU. Will this ever be possible, or is it even necessary? In this poster, we will walk you through state-of-the-art nucleic acid detection, elucidate the critical steps and highlight the many benefits of this approach. In doing so, we will address key requirements, such as the limit of detection and how to deal with “false” positives. As groundbreaking technology digital PCR (dPCR) allows a new level of precise quantification. We quantified the positive control (PC) of our Microsart® ATMP Sterile Release Kit that allows us now to shed light on the borderline between sensitivity and noise. We propose an open discussion on this advanced method and the importance of understanding standards in QC testing and release. 1. Advanced Therapy Medicinal Products (ATMPs) ATMPs¹ are a new class of complex medicinal products associated with viable cells and tissue. An example of a cell-based medicinal product is an “Ex Vivo Autologous Gene Therapy”, where cells of the patient are genetically modified ex vivo and administered back to the patient to fight the illness (Cell and Gene Therapy = CGT). Patient Native cells with defect Therapeutic Viral vector gene Recombinant viral vector Cells in culture Genetically modified viable cells Extraction Injection In vitro cell transfection In vitro cell culture 2. Sartorius’ Real-Time PCR-Based Sterile Testing Solutions Microsart® ATMP Sterile Release kit allows bacteria and fungal presence/absence tests within a matter of hours. The enclosed Microsart® ATMP Extraction kit enables the processing of two 1 mL samples and one negative extraction control (NEC). The extracted DNA can be further analyzed with the bundled real-time PCR kits, Microsart® ATMP Bacteria and Microsart® ATMP Fungi, enabling the fast and precise detection of bacterial and fungal contaminants by targeting ubiquities ribosomal genes. 3. Ribosomal Genes as Real-Time PCR Target Ribosomes translate genomic information into life-sustaining proteins. Thus, all living entities, including bacteria and fungi, have ribosomal genes, which can be targeted by real-time PCR. Biological contamination can be excluded by experimentally verifying the absence of the bacterial 16S and fungal 18S rRNA gene. However, the ubiquity of ribosomal genes is also a challenge. Omnipresent bacteria lead to a high basic load of bacterial DNA, which must be removed at great expense and, in some cases, can lead to false-positive results in PCR-based tests. 4. Challenges of a Real-Time PCR Test The Microsart ® ATMP Bacteria real-time PCR assay addresses the universal bacterial 16S rRNA gene and detects the presence of almost any bacterial DNA. Product sterility can no longer be guaranteed in the rare instance that the signal (Ct value) crosses a specified threshold. The question that arises is how to interpret the observation and what implications it holds for the Advanced Therapy Medicinal Product (ATMP), and ultimately, for the patient.2,3 ATMP non-released QC result pending Shelf life 48-72 h RFU Amplification Cycles 300 200 150 100 50 0 0 10 20 30 40 250 2x PC 2x NTC 2x NEC 2x Sample PC = Positive Control NTC = No Template Control NEC = Negative Extraction Control Threshold 5. A Quantified DNA Standard The dPCR quantified positive control (PC) can be used as a DNA standard to set a desired concentration to estimate the 16S rRNA gene copy number behind a Ct value (Cycle threshold value where the signal crosses the threshold) or can be used further as a positive control (PC). The Ct value as illustrated in Section 4 is about 38. With the help of the dPCR quantified PC, we now can estimate about 5-10 copies of 16S rRNA gene to be responsible for it. Assessing the potential contamination load becomes possible now. 6. 16S rRNA Gene Copies ≠ Genome Copies ≠ CFU Bacterial genomes usually contain several 16S rRNA copies. A metabolically active bacterium usually holds several genome copies per cell, enabling fast cell division. Daughter cells often stick to each other and can form a conglomerate of several cells. Thus, already one colony forming unit (CFU) can contain a double/triple digit number of 16s rRNA gene copies6 . The following Table depicts the average number of 16S rRNA gene copies of QC-relevant bacterial strains (USP<71>) with the corresponding genome equivalent when 5, 10 or 50 16S rRNA gene copies are detected. Genome equivalents considering the detected 16S rRNA gene copies USP <71> Compendial bacteria species 16S cp/genome 5 cp 10 cp 50 cp Bacillus subtilis 10 0.5 1 5 Clostridium sporogenes 9 0.6 1.1 5.6 Pseudomonas aeruginosa 4 1.3 2.5 12.5 Staphylococcus aureus 7 0.7 1.4 7.1 7. Microbial Colony Forming Units – CFU The smallest unit leading to a bacterial colony is called the “colony forming unit or CFU”. In an ideal case, one bacterial cell is sufficient to form a CFU. However, in real life, bacterial cells often aggregate together in several units or even form a biofilm with a consortium of bacteria. Consequently, a CFU consists of several cells, with very species-specific numbers 4,5 . 8. At the Borderline of Sensitivity and Noise Microbial Colony-Forming Units (CFUs) display diverse characteristics influenced by growth conditions and division status. This inherent genomic diversity poses challenges for precise detection. Moreover, a single genome can have differing numbers of 16S rRNA gene copies, further complicating detection efforts. Regulatory guidelines, such as the United States Pharmacopeia (USP<1071>), suggest a detection limit of at least 100 CFUs for risk-based release assessments. But what does this mean for the experiment? Using our Microsart® Validation Standard 99 CFU P. aeruginosa to spike a sample, we detect it with a Ct value of 30. If P. aeruginosa was the only possible contaminant to expect, one might risk stating the observed contamination is uncritical (Sections 4 and 8). However, rarely is the contaminant known, and its exclusivity guaranteed. Thus, the question arises of how many 16S rRNA gene copies are the basis of an observed PCR signal and what is the worst-case scenario for a potential contaminant. In the most challenging scenario, an organism may possess one genome copy with only one 16S rRNA gene copy. Consequently, detecting 10 16S rRNA gene copies in a real-time PCR would correspond to 10 genome copies (GC) per PCR and 100 GC per 1 mL investigated sample when using our Microsart® ATMP Sterile Release kit. We addressed these questions using our new quantified PC to replicate the worst-case scenario. By knowing the 16S rRNA gene copies underlying specific Ct values (Section 5), we could estimate that our sample was contaminated with about 10 copies of a 16S rRNA gene (Sections 4 and 8). With such information, one can now assess the GC of relevant contaminants (Section 6) and potential CFUs (Section 7). In light of these findings, critical questions arise: Should we employ worst-case scenarios to define the boundary between sensitivity and noise in microbial detection? How relevant is such a cut-off in a clinical context? How would you address this borderline? Find further details of all tips, tricks, applications and products by contacting your local Sartorius representative. sartorius.com 4. Paeschke et. al., 2017 5. D’Apolito et. al., 2020, https://doi.org/10.1016/j.biologicals.2020.01.001 6. Větrovský and Baldrian 2013, https://doi.org/10.1371/journal.pone.0057923 References: 1. Goula et. al., 2020, https://doi.org/10.14740/jocmr3964 2. Cundell et. al., 2023, https://doi.org/ 10.1128/jcm.01654-22 3. Panch et. al.,2018, https://doi.org/10.1016/j.bbmt.2018.08.003 RF U Amplification Cycles 300 200 150 100 50 0 0 10 20 30 40 250 Signal Threshold 2x PC 2x NEC 2x NTC 2x Sample 2x PC 50cp 2x PC 25cp 2x PC 10cp 2x 99 CFU P. aeruginosa* * Microsart® Validation Standard 99 CFU P. aeruginosa Is the contamination in the investigated sample of relevance? 20 25 30 35 40 45 5 10 25 50 PC Ø 38.86 Ø 38.52 Ø 36.64 Ø 35.32 Ø 25.13 CT 16S rRNA gene cp/PCR Clostridium sporogenes Pseudomonas aeruginosa Bacterioides vulgatus Escherichia coli Pseudomonas protegens Serratia marcescens Propionibacterium acnes Bacterioides fragilis Enterobacter cloacae Klebsiella pneumoniae Clostridium perfringens Yersinia enterocolitica Staphylococcus aureus Staphylococcus epidermidis Streptococcus pyogenes Kocuria rhizophila Bacillus cereus Klebsiella pneumoniae Clostridium perfringens Enterococcus faecalis Kocuria rhizophila Kocuria rhizophila Kocuria rhizophila Klebsiella pneumoniae Clostridium perfringens