Our promise to you:
Guaranteed product quality, expert customer support.
24x7 CUSTOMER SERVICE
CONTACT US TO ORDER
Genetic stability testing confirms that an engineered strain's genetic construct remains intact and functional from MCB through WCB to EOPC. This service offers three complementary approaches: plasmid retention rate by plate counting, gene copy number by qPCR, and structural verification by sequencing and restriction mapping. It meets ICH Q5B requirements for two-time-point comparison and supports IND/BLA submissions.
Plasmid retention rate is the most direct indicator of genetic stability in engineered strains. The strain is serially passaged under non-selective pressure, meaning without antibiotics or nutrient supplements that maintain the plasmid. At regular intervals, samples are plated on both selective and non-selective agar plates. The proportion of colony-forming units on selective versus non-selective plates is calculated to estimate the frequency of plasmid loss.
Plasmid copy number, or PCN, is the average number of plasmid DNA molecules per bacterial cell. PCN directly affects recombinant protein expression levels. Changes in PCN represent another important manifestation of genetic instability. Even if the plasmid is not completely lost, a significant drop in copy number can reduce yields and cause batch-to-batch inconsistency.
We determine PCN using SYBR Green or TaqMan probe qPCR with absolute quantification based on a standard curve. Relative quantification using the ΔΔCt method with an MCB sample as calibrator is also available.
Plasmid retention rate confirms plasmid presence or absence but cannot detect structural variations such as deletions, rearrangements, or point mutations that cause gene function loss without plasmid loss.
For structural verification, we extract plasmid DNA and sequence key regions (gene of interest, promoter, marker, origin) by Sanger or NGS, comparing to the original map. Restriction enzyme mapping compares fragment patterns from MCB and EOPC/WCB by gel electrophoresis to detect large insertions, deletions, or rearrangements.
For integrated expression systems (e.g., P. pastoris AOX1), we use Southern blot or long-range PCR to verify integration site integrity and copy number stability.
ICH Q5B Section 4 requires genetic stability assessment at MCB recovery and at the limit of in vitro cell age (EOPC), including restriction mapping, coding region sequencing, and copy number determination. ICH Q5D Sections 4.1 and 4.2 add requirements for expression construct characterization and extrachromosomal element integrity.
| Test Item | Method Type | Regulatory Basis |
| Plasmid retention rate | Industry standard | ICH Q5B / Q5D |
| qPCR copy number | Industry standard | Škulj et al. 2008 |
| Restriction mapping | Industry standard | ICH Q5B |
| Plasmid sequencing | Industry standard | ICH Q5B / Q5D |
For detailed technical protocols, passage scheme design specific to your strain, or customized service pricing, please contact our technical team. Free technical consultation and pre-evaluation of testing strategies are available.
Q1: Is genetic stability testing mandatory for cell bank release?
Yes. ICH Q5B requires genetic stability assessment for production cell substrates. For microbial systems, this has become standard for IND submissions. Start baseline testing after MCB establishment and complete EOPC testing before IND.
Q2: How many passages do I need to test?
Cover the maximum in vitro cell age. ICH Q5B requires two time points: MCB recovery and EOPC. We recommend validating 1.2–1.5 times your production passage number. For early projects, 50 passages is a conservative benchmark.
Q3: My strain uses an integrated expression system with no plasmid. How is genetic stability tested?
Use Southern blot, long-range PCR, or qPCR for integration sites. ICH Q5B requires verification of integration site integrity and copy number. We recommend starting with qPCR for rapid screening, then confirming abnormalities with Southern blot.
Q4: If my strain shows 100% plasmid retention after 50 passages under non-selective pressure, does that mean it is absolutely stable?
Not necessarily. 100% retention does not rule out a drop in copy number (e.g., 50 to 2 copies per cell) or structural variations like promoter deletions that preserve plasmid maintenance but impair function. Combine qPCR for copy number and sequencing of key regions for full confirmation.
Copyright © 2026 Creative Biogene. All rights reserved.