1.Preparation
2.Sample Pre-treatment
3.PCR Reaction Mix Preparation
4.Instrument Detection
5.Data Analysis
| Milestones | Specifications | Deliverables |
| DNA Extraction & Purification | • Efficient lysis of Pichia pastoris host cells | • Purified DNA ready for qPCR analysis |
| • High-recovery DNA extraction using silica-column or magnetic bead technology | • QC report (A260/A280 ratio, yield) | |
| • Removal of PCR inhibitors (e.g., proteins, polysaccharides) | ||
| qPCR Assay Setup | • Optimized primer/probe set targeting Pichia pastoris-specific genomic sequences (e.g., AOX1, DDI1) | • Pre-validated qPCR protocol |
| • Included standard curve (3–3×10<sup>5</sup> fg/μL) for absolute quantification | • Standard curve with R<sup>2</sup> ≥ 0.99, efficiency: 90–110% | |
| • Master mix with hot-start Taq polymerase and dNTPs | • Positive/negative controls included | |
| Sensitivity & Specificity | • LOD: 3 fg/μL (1–2 copies/μL) | • Validation report per ICH Q2(R1), EP 2.6.21, and Chinese Pharmacopoeia guidelines |
| • LOQ: 10 fg/μL | ||
| • No cross-reactivity with E. coli, CHO, human, or other common host DNA | ||
| Compliance & QC | • ISO 13485-certified production | • Certificate of Analysis (CoA) |
| • SDS-PAGE/WB for protein contamination check (optional) | • Regulatory-compliant documentation (FDA/EMA/NDA submissions) | |
| • Inter-/intra-assay CV ≤ 15% | ||
| Turnaround Time | • Routine testing: 3 hours (extraction + qPCR) | • Customized reports (per client requirements) |
| • Bulk orders: 1–2 weeks | • Technical support (experimental design, data analysis) | |
| • Custom validation: 4–6 weeks |
Background:
A biopharmaceutical company developing a Pichia pastoris-based HPV vaccine required stringent residual DNA quantification to meet FDA/EMA guidelines (≤10 ng/dose).
Key Challenge:
High polysaccharide content in Pichia lysates caused PCR inhibition, leading to false-negative results with standard kits.
Needed ≤3 fg/µL sensitivity to ensure safety for clinical batches.
Optimization:
Used silica-column DNA extraction (included in kit) to remove inhibitors.
Validated assay with AOX1-targeted probes (3–3×10<sup>5</sup> fg/µL range).
Result:
Consistent detection at 2 fg/µL (below LOQ).
GMP-compliant report accepted for Phase III trial submission.
Background:
An academic lab expressed TNF-α in Pichia but lacked tools to quantify residual DNA in purified samples.
Key Challenge:
Low-abundance DNA (<10 fg/µL) in high-purity protein (>95% by SDS-PAGE).
Required low-budget solution with publication-ready data.
Optimization:
Used kit’s dilution-adjusted linear range (extended to 3×10<sup>6</sup> fg/µL).
Cross-validated with spike-recovery experiments (92–105% recovery).
Result:
Confirmed DNA ≤50 fg/dose (meets ICH limits).
Data published in peer-reviewed journal (Biotech Reports).
Background:
A contract manufacturer producing recombinant insulin in Pichia needed routine residual DNA testing for 50+ batches/year.
Key Challenge:
Existing kits had high variability (CV >20%) across operators.
Turnaround time (>5 hours) delayed production timelines.
Optimization:
Implemented automated extraction (KingFisher) + pre-plated qPCR master mix.
Trained team on standardized protocol (3-hour workflow).
Result:
Reduced CV to ≤12% (inter-assay).
Saved 40% time/cost vs. competitor kits (Thermo Fisher Cat# 4464336).
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info@hillgene.com 
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