ABSTRACT
OBJECTIVE: To develop and verify a magnetic beads-based extraction combined with quantitative PCR(q-PCR)method for determination of residual host cell DNA in recombinant human albumin products expressed in Pichia pastoris. METHODS: The residual Pichia pastoris host cell DNA in samples were extracted by magnetic beads-based extraction method and then determined by Taqman probe-based q-PCR. The residual DNA content was calculated according to the standard curve. The developed method was verified for accuracy and precision with different derivation albumin matrixes and concentrations, and the residual DNA of 3 batches of recombinant human albumin products expressed in Pichia pastoris were detected. RESULTS: The minimum detection limit of Pichia pastoris residual DNA by the developed method was 3 fgμL-1, the linear range was 3 fgμL-1-300 pgμL-1, and the correlation coefficient(r2) was 0.998 3. The recovery rates of spiked samples in rHA matrix were 93.58%(RSD 19.6%, n=4)at 100 fgμL-1 and 215.56%(RSD 42.9%, n=4) at 10 fgμL-1, respectively. The recovery rates of spiked samples in HSA matrix were 67.09%(RSD 6.9%,n=3)at 100 fgμL-1 and 113.40%(RSD 11.1%, n=3) at 10 fgμL-1, respectively. The residual Pichia pastoris DNA contents in 3 batches of recombinant human albumin products expressed in Pichia pastoris determined by the developed method were 5.98, 4.16, 4.49 fgμL-1(n=7) respectively and not more than 1 ng per 10 g protein. CONCLUSION: Magnetic beads extraction method combined with fluorescence quantitative PCR method solves the technical problem of quantitative determination of trace DNA in recombinant human albumin products with ultra-high concentration protein. The method is accurate and reproducible, and can be used for quantitative determination of DNA residue in recombinant human albumin expressed by Pichia pastoris.
ABSTRACT
Objective To study DNA quantification and STR typing of samples pre-treated with pyrami-don. Methods The blood samples of ten unrelated individuals were anticoagulated in EDTA. The blood stains were made on the filter paper. The experimental groups were divided into six groups in accor-dance with the storage time, 30 min, 1 h, 3 h, 6 h, 12 h and 24 h after pre-treated with pyramidon. DNA was extracted by three methods: magnetic bead-based extraction, QIAcube DNA purification method and Chelex-100 method. The quantification of DNA was made by fluorescent quantitative PCR. STR typing was detected by PCR-STR fluorescent technology. Results In the same DNA extraction method, the sample DNA decreased gradually with times after pre-treatment with pyramidon. In the same storage time, the DNA quantification in different extraction methods had significant differences. Sixteen loci DNA typing were detected in 90.56% of samples. Conclusion Pyramidon pre-treatment could cause DNA degradation, but effective STR typing can be achieved within 24 h. The magnetic bead-based extrac-tion is the best method for STR profiling and DNA extraction.