Single-use system (SUS)-based manufacturing of enzymes and reagents suitable for PCR

Thermo Fisher Scientific has developed a manufacturing process using Single Use System (SUS)-based technology with extensive quality control testing to drastically reduce the risk of DNA contaminates in the PCR reagents for commercial supply. The result is the same high level of performance and lot-to-lot consistency that is expected for our standard conventional PCR reagents. These reagents are the perfect choice for PCR assays where ambiguous or false-positive results are simply not acceptable.

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If you would like to discuss sample availability, please contact us at MDxenzymes@thermofisher.com

Benefits of DNA-Free PCR Reagents

  • Manufactured by SUS to be:
    • Free of bacterial DNA
    • Free of plasmid DNA
    • Free of human DNA
  • Same optimal performance as standard conventional enzymes
  • Manufactured in an ISO 13485 certified facility

Watch how SUS technology minimized the risks of DNA contamination in production of PCR reagents.

Conventional enzyme manufacturing–challenges

The manufacturing process for commercially available DNA polymerases typically consists of multiple steps handled by a scientist or operator in an open environment (Figure 1). In addition, one might find that equipment may be shared for the manufacture of other proteins and enzymes. As a result, the purity of a preparation must rely heavily on efficient decontamination steps between processes. Contaminating DNA commonly found in enzyme preparations can occur during one or all of these many steps throughout the manufacturing process. Not surprisingly, when tested, these DNA polymerases have been shown to contain detectable levels of DNA contaminants [2]. This poses an unacceptable risk for commercial-scale production of DNA polymerase that Thermo Fisher Scientific has addressed with SUS-based manufacturing.

Figure 1. A conventional manufacturing workflow for recombinant enzymes illustrates the risk of DNA contamination. The typical process for enzyme preparation includes repeated opportunities for potential DNA contamination from an open environment to handling by an operator between steps. There is also a potential risk for the carryover of DNA contaminants when utilizing equipment that is shared with other manufacturing processes for the preparation of other enzymes and proteins.
 

Closed SUS-based enzyme manufacturing–process solutions

At Thermo Fisher Scientific, we have uniquely adapted our SUS technology for the commercial-scale production of recombinant enzymes (Figure 2). All stages of SUS-based manufacturing are performed within a closed environment and rely on sterile single-use bags, tubing and connectors throughout production. All buffers and washing solutions are prepared in single-use bags and filtered for sterilization. With closed SUS-based manufacturing, the probability of DNA contamination has been reduced to a negligible risk level.

Highlights

  • Entirely closed system throughout the manufacturing process–no exposure to environment and human operators
  • Sterile single-use bags, tubing and connectors–no potential cross-contamination from common-use equipment
  • Manufacturing in facilities that meet Class D and Class C cleanroom standards–dedicated purpose-built space prevents exposure to DNA contaminants
  • Product filling and finishing is contained and meets Class B cleanroom standards–controlled and dedicated space helps to deliver a DNA-free product
  • Additional proprietary manufacturing process and purification steps for DNA decontamination–removes traces of exogenous DNA and DNA from host cells

Figure 2. Closed SUS-based process for the manufacture of recombinant enzymes. This process represents a completely closed system that relies on single-use bags, tubes and connectors, which help reduce the potential for DNA contamination to a negligible risk level whether from the environment or the operator. Moreover, because it is single-use and equipment or materials are not shared, it eliminates this as a source of cross-contamination.
 

DNA-Free enzymes–verified by quality control

PCR reagents manufactured utilizing our SUS-based technologies are subjected to rigorous quality control testing. This includes application-specific, functional testing and analytical testing for purity using highly sensitive qPCR assays.

Functional assay

A proprietary qPCR assay is utilized to evaluate enzyme activity and to validate that the DNA-Free enzyme retains the same functional characteristics as enzymes produced by conventional methods.

Purity test

DNA-Free enzymes are analyzed following the highest standards to ensure the absence of nucleases and DNA contamination per the specifications below (Table 1).

Table 1. Purity requirement for DNA-Free Taq DNA polymerase.

Purity test Requirement
Taq DNA polymerase purity Exonucleases and endonucleases: undetected
RNases: undetected
DNA contamination detection Bacterial gDNA (16S rRNA): ≤0.01 copy/enzyme unit
Human gDNA (Alu): ≤0.001 copy/enzyme unit
Plasmid DNA (ori1): ≤0.01 copy/enzyme unit

DNA-Free manufacturing services–now available

Thermo Fisher Scientific offers a wide range of DNA polymerases and reverse transcriptases with high quality and superior performance for your PCR- or qPCR-based assays. Now, we introduce Invitrogen Platinum Taq DNA polymerase, DNA-Free, manufactured utilizing a closed SUS-based technology. Platinum Taq DNA polymerase, DNA-Free provides the benefit of robust amplification with stringent hot-start technology, and is verified to purity levels that are orders of magnitude cleaner than other commercially available DNA polymerases. This will bring unparalleled confidence to your commercial scale, PCR-based assays. If you are interested in more information about Platinum Taq DNA polymerase, DNA-Free, or other DNA-Free enzymes, please contact us and we will work with you to meet your requirements.


References

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