CytoInspect™ DNA Biochip for Mycoplasma Detection by Greiner Bio-One GmbH
Manufacturer Greiner Bio-One GmbH | Model: Mycoplasma, Mycoplas | Available Worldwide
(1.) The Problem: Mycoplasma contamination of cell cultures represents one of the major problems encountered in facilities that are involved in the development and manufacture of biopharmaceutical products. Undetected contamination endangers the quality and safety of drugs being produced in cells. (2.) The Solution: Greiner Bio-One GmbH provides a fast and sensitive microarray based system for the detection of mycoplasma contaminations to improve efficiency, quality and safety in the manufacturi... Read more
CytoInspect™
(1.) The Problem:
Mycoplasma contamination of cell cultures represents one of the major problems encountered in facilities that are involved in the development and manufacture of biopharmaceutical products. Undetected contamination endangers the quality and safety of drugs being produced in cells.
(2.) The Solution:
Greiner Bio-One GmbH provides a fast and sensitive microarray based system for the detection of mycoplasma contaminations to improve efficiency, quality and safety in the manufacturing of biopharmaceutical products. The CytoInspect™ enables the detection and identification of 41 mycoplasma species representing over 99 % of all reported contaminations, hence enabling a solid root cause analysis. In addition, a universal probe tests for the presence of any mycoplasma species (including Acholeplasma sp., Spiroplasma sp. and Ureaplasma sp.).
(3.) The Technology:
The technology of the CytoInspect™ is based on the well-established Greiner Bio-One HTA (High Throughput microArray) slide that allows the analysis of 12 samples by 12 microarrays.
One microarray has a total of 225 individual DNA measuring points distributed over an area of about 16 mm2. All requested controls, as described in the European Pharmacopoeia (EP) monograph 2.6.21, have been implemented into the system in a “one tube only” reaction. These controls enable the monitoring of the presence and homogeneity of each individual DNA measuring point, the hybridisation, the DNA extraction and the PCR reaction.
Due to the use of fluorescently labelled PCR primers and on-chip controls, the analysis is performed using laser wavelengths of 532 nm and 635 nm.
(4.) The Workflow:
The workflow of the CytoInspect™ starts with the extraction of DNA from cell culture supernatant or other sources. The purified DNA has to be as pure as possible and free from any PCR inhibitors. For this purpose, a CytoInspect™ DNA Extraction kit was developed. The kit has been validated for the extraction of mycoplasma DNA from different matrices (cell cultures of up to 108 cells/mL, cell supernatants, and allantoic fluids). It can process volumes from 500 µL to 50 mL.
For the detection, fluorescently labelled PCR primers amplify the 16S-23S rRNA intergenic transcribed spacer (ITS) region of mycoplasma DNA. A touchdown PCR protocol is used to maximise the sensitivity and specificity of primer annealing and amplification.
The PCR Master Mix contains dUTP which is integrated into the final PCR product. Treatment with uracil-N-glycosylase (UNG) eliminates carry-over contaminations from previous PCRs.
The amplified and labelled PCR products are hybridised to the CytoInspect™ DNA microarray comprising of probes that allow the identification of the different mycoplasma species. Upon hybridisation and washing, the analysis is performed using the CheckScanner™ and the CheckReport™Software.
(5.) Regulatory Guidelines:
The CytoInspect™ has been externally validated in accordance with the Good Manufacturing Practice (GMP) and regulatory guidelines for Nucleic Acid Amplification Tests (NAT) such as ICHQ2(R1). The experiments and parameters assessed were based on the guidelines found in the EP monographs 2.6.7 and 2.6.21. The most relevant testings included the following parameters:
• Limit of detection (LoD), ≤ 10 CFU/10 mL
• Specificity
• Robustness.
Mycoplasma contamination of cell cultures represents one of the major problems encountered in facilities that are involved in the development and manufacture of biopharmaceutical products. Undetected contamination endangers the quality and safety of drugs being produced in cells.
(2.) The Solution:
Greiner Bio-One GmbH provides a fast and sensitive microarray based system for the detection of mycoplasma contaminations to improve efficiency, quality and safety in the manufacturing of biopharmaceutical products. The CytoInspect™ enables the detection and identification of 41 mycoplasma species representing over 99 % of all reported contaminations, hence enabling a solid root cause analysis. In addition, a universal probe tests for the presence of any mycoplasma species (including Acholeplasma sp., Spiroplasma sp. and Ureaplasma sp.).
(3.) The Technology:
The technology of the CytoInspect™ is based on the well-established Greiner Bio-One HTA (High Throughput microArray) slide that allows the analysis of 12 samples by 12 microarrays.
One microarray has a total of 225 individual DNA measuring points distributed over an area of about 16 mm2. All requested controls, as described in the European Pharmacopoeia (EP) monograph 2.6.21, have been implemented into the system in a “one tube only” reaction. These controls enable the monitoring of the presence and homogeneity of each individual DNA measuring point, the hybridisation, the DNA extraction and the PCR reaction.
Due to the use of fluorescently labelled PCR primers and on-chip controls, the analysis is performed using laser wavelengths of 532 nm and 635 nm.
(4.) The Workflow:
The workflow of the CytoInspect™ starts with the extraction of DNA from cell culture supernatant or other sources. The purified DNA has to be as pure as possible and free from any PCR inhibitors. For this purpose, a CytoInspect™ DNA Extraction kit was developed. The kit has been validated for the extraction of mycoplasma DNA from different matrices (cell cultures of up to 108 cells/mL, cell supernatants, and allantoic fluids). It can process volumes from 500 µL to 50 mL.
For the detection, fluorescently labelled PCR primers amplify the 16S-23S rRNA intergenic transcribed spacer (ITS) region of mycoplasma DNA. A touchdown PCR protocol is used to maximise the sensitivity and specificity of primer annealing and amplification.
The PCR Master Mix contains dUTP which is integrated into the final PCR product. Treatment with uracil-N-glycosylase (UNG) eliminates carry-over contaminations from previous PCRs.
The amplified and labelled PCR products are hybridised to the CytoInspect™ DNA microarray comprising of probes that allow the identification of the different mycoplasma species. Upon hybridisation and washing, the analysis is performed using the CheckScanner™ and the CheckReport™Software.
(5.) Regulatory Guidelines:
The CytoInspect™ has been externally validated in accordance with the Good Manufacturing Practice (GMP) and regulatory guidelines for Nucleic Acid Amplification Tests (NAT) such as ICHQ2(R1). The experiments and parameters assessed were based on the guidelines found in the EP monographs 2.6.7 and 2.6.21. The most relevant testings included the following parameters:
• Limit of detection (LoD), ≤ 10 CFU/10 mL
• Specificity
• Robustness.
Product Overview
CytoInspect™ DNA Biochip for Mycoplasma Detection
Manufacturer Greiner Bio-One GmbH | Available Worldwide
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