A breakthrough protein immuno-PCR quantitation platform designed for sensitive and robust protein quantitation with 4 orders of magnitude of dynamic range

The detection, characterization, and removal of process impurities are critical in biopharmaceutical production. Current methods to quantify proteins (e.g., ELISA) typically suffer from narrow dynamic range, low sensitivity, laborious workflows, lack of dilution linearity, and poor robustness when used with complex matrices. Numerous replicates are required to obtain valid results, while extensive dilutions to mitigate complex matrices further compromise sensitivity.

The ProteinSEQ™ Protein Quantitation System is an immuno–PCR-based method that addresses these challenges, providing greater confidence and quality of data throughout the quantitation process. Quantitative PCR (qPCR) delivers outstanding sensitivity and broad dynamic range. The ProteinSEQ Protein Quantitation System is a breakthrough immunoassay platform that leverages the analytical benefits of TaqMan™ qPCR readout and the selectivity of immunochemistry.

  • PCR sensitivity: quantitation to as low as 0.2 ng/mL generic host cell protein (HCP)
  • Wide dynamic range: 4 orders of magnitude
  • Robustness: Dilution linearity and efficiency in complex matrices
  • Semi-automation: 2-hour walk-away time

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The technology underlying ProteinSEQ Protein Quantitation Systems is based on the Applied Biosystems™ TaqMan™ Protein Expression Assay. This is a homogenous assay that utilizes two antibodies conjugated to an oligonucleotide via a straptavidin–biotin linkage. When the two antibodies bind to their protein target, their proximity permits ligation of their respective oligonucleotides, which can serve as templates for real-time PCR amplification and quantification (Figure 1).

Figure 1. TaqMan Protein Expression Assay Technology. The TaqMan Protein Expression Assay is a homogeneous assay based on two antibodies, each conjugated to an oligonucleotide through a streptavidin—biotin linkage. When the two conjugated antibodies bind to their target protein and are in proximity, the oligonucleotides can be ligated, serving as the template for real-time PCR amplification and quantification.

The software solution for mycoplasma detection, protein analyses and residual DNA

The Applied Biosystems™ AccuSEQ™ Real-Time PCR Detection Software supports the unique needs of analytical testing of contaminants and impurities during the biopharmaceutical manufacturing process, as well as routine qPCR assays.

AccuSEQ software is part of the integrated workflow for Mycoplasma detection, residual Protein A quantitation, and residual DNA quantitation. Automated analysis tools enable one-click processing of complex Ct, amplitude (DV), and melting temperature for the MycoSEQ™ Mycoplasma detection assay according to user specifications. Nonlinear curve-fitting enables rapid analysis of ProteinSEQ Protein Quantitation System data. Next-generation algorithms deliver accurate quantitation data for the resDNASEQ™ Residual Host Cell DNA Quantitation.

Key features

  • Interfaces with the Applied Biosystems™ 7500 Fast Real-Time PCR instrument
  • Nonlinear curve-fitting of ProteinSEQ™ assay data
  • Single software platform for multiple SEQ real-time PCR assays
  • Supported and fully tested for Windows™ 7 operating system
  • Helps enable 21 CFR Part 11 compliance
ProteinSEQ Protein Quantitation System
Applied Biosystems™ SEQ Analytical SOlutions for Pharmaceutical Manufacturing
Product and Process Impurities Analysis