Transgene copy number comparison in recombinant mammalian cell lines: critical reflection of quantitative real-time PCR evaluation

Abstract

Nucleic acid quantification is a relevant issue for the characterization of mammalian recombinant cell lines and also for the registration of producer clones. Quantitative real-time PCR is a powerful tool to investigate nucleic acid levels but numerous different quantification strategies exist, which sometimes lead to misinterpretation of obtained qPCR data. In contrast to absolute quantification using ampliconor plasmid standard curves, relative quantification strategies relate the gene of interest to an endogenous reference gene. The relative quantification methods also consider the amplification efficiency for the calculation of the gene copy number and thus more accurate results compared to absolute quantification methods are generated. In this study two recombinant Chinese hamster ovary cell lines were analysed for their transgene copy number using different relative quantification strategies. The individual calculation methods resulted in differences of relative gene copy numbers because efficiency calculations have strong impact on gene copy numbers. However, in context of comparing transgene copy numbers of two individual clones the influence of the calculation method is marginal. Therefore especially for the comparison of two cell lines with the identical transgene any of the relative qPCR methods was proven as powerful tool.

Bioprocessing of Recombinant CHO-K1, CHO-DG44, and CHO-S: CHO Expression Hosts Favor Either mAb Production or Biomass Synthesis

“…qPCR was performed in 2‐step mode and all samples were measured in triplicates of three biological samples in two technical runs. Data evaluation and calculations based on the 2−ΔΔCq method were previously described . Data normalization was performed using both reference genes.…”

Evaluating the bottlenecks of recombinant IgM production in mammalian cells

“…Primers and probes targeting protein disulfide isomerase (PDI: GenBank AF364317.1), xbox binding protein (XBP1: NCBI refseq: NM001244047.1; this primer probe set was designed to detect the spliced variant only) and 78 kDa glucoseregulated protein (GRP78: GenBank M171169.1; also known as binding immunoglobulin protein, BiP; summarized in Table 1) were used in qPCR analysis on a MiniOpticon TM System (BioRad, Hercules, CA, USA), using TaqMan probe principle. Calculation of gene copy numbers and transgene transcript levels was done relative to beta-actin housekeeping gene (Pfaffl 2001;Sommeregger et al 2013). …”

Prospect and Competence of Quantitative Methods via Real-time PCR in a Comparative Manner: An Experimental Review of Current Methods

“…Nucleic acid quantification is an applicable issue for the description of mammalian recombinant cell lines and also for the recording of producer clones [25]. Quantitative real-time RT-PCR is broadly and progressively used in different kinds of gene quantification strategies, due to its wide range of dynamic quantification, satisfactory reproducibility and great thoroughness.…”

“…Pfaffl's method and its other derived formula with efficiency correction are more preferable substitutions for quantification relative to a reference gene. The efficiencies are calculated via different methods like 1:10 dilution series [25] or mathematical models based on single sample fluorescent curve [20]. Despite similar values obtained from one sample efficiency corrected models and the 2 -ΔΔCt method, due to efficiency differences, the formula in the former method is suggested to be employed.…”