Client Challenge
A biotechnology company developing a CRISPR-based genetic medicine needed a potency assay to confirm that its guide RNA drug substance retained its expected gene-editing activity relative to a reference standard. The assay needed to measure relative potency accurately and precisely across a defined activity range.
Avance Biosciences’ Solution
Avance Biosciences qualified a droplet digital PCR (ddPCR)-based cleavage assay that measures the relative potency of the sgRNA drug substance. The method:
- Forms a ribonucleoprotein (RNP) complex by combining the sgRNA with a recombinant Cas9 nuclease at a fixed molar ratio
- Incubates the RNP with a DNA template containing the target sequence to allow site-specific cleavage
- Serially dilutes the RNP to generate a dose-response curve for cleavage at each dilution step
- Detects cleaved versus uncleaved DNA copies by ddPCR, using a sequence-specific fluorescent probe
- Fits a four-parameter curve to the dose-response data and calculates relative potency against the reference standard
Qualification Protocol
The study evaluated specificity, accuracy, precision, linearity, and range. Quality control samples were prepared at multiple potency levels spanning a defined range relative to the reference standard, and tested independently by two operators.
Assay Qualification
Each parameter addressed a question a quality unit will ask of a potency method:
- Specificity: Does the assay detect only the intended target? Background and cross-reactivity signal in no-template and non-targeting control wells stayed within the predefined acceptance range, confirming the assay detects only its intended target.
- Accuracy: Does the assay return the expected relative potency? Measured potency for the quality control samples, across the qualified range, stayed within the predefined accuracy acceptance criterion.
- Precision: Does the result hold up across replicates and operators? Both intra-assay and inter-operator precision met their predefined acceptance criteria, demonstrating consistent results regardless of who ran the assay.
- Linearity and Range: Does potency scale predictably with concentration? The assay demonstrated strong linearity across its qualified potency range, consistent with predefined acceptance criteria.
Customer Outcome
- A qualified ddPCR-based potency assay measuring relative gene-editing activity for its guide RNA drug substance
- Documented specificity, accuracy, precision, linearity, and range, all meeting predefined acceptance criteria across the qualified range
- A designated assay control sample, with a system suitability range established for future routine testing
- A signed final qualification report
Client Impact
The client now has a qualified ddPCR-based potency method ready to support lot release and stability testing of its guide RNA drug substance.
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