October 15, 2014 (Vol. 34, No. 18)

Vasco Liberal, Ph.D. associate director Biomatrica
Louis Chen, Ph.D. technical support scientist Biomatrica
Michael Vengrow marketing specialist Biomatrica

Chemical Stabilization Provides Significant Benefits over Existing Technologies

Diagnostics is moving increasingly toward decentralized, point-of-care, reduced complexity, and rapid sample-to-answer formats. This trend is driven by improving standards of care and reducing time to results while maintaining assay accuracy. Simultaneously, companion diagnostics are increasing the number of assays available to meet the needs of patient subpopulations characteristic of personalized medicine.

To succeed in the increasingly diverse diagnostics market, manufacturers require new technologies for stabilizing assay samples and reagents to ensure the accuracy of diagnostic tests outside of traditional clinical settings.

Cold storage is the most widely established approach to stabilization of biological samples and assay reagents, and is the gold standard benchmark for other stabilization technologies. However, low temperatures do not preserve all reagents and samples equally. This is especially important in molecular diagnostics, where specific storage conditions are needed for optimal preservation of diverse reagents and analytes. Moreover, cold chain preservation requires significant energy consumption and transportation costs, and is susceptible to failure, making it less suitable for the demands of globalization, decentralization, and personalized medicine.

Lyophilization has been adopted by some manufacturers to eliminate the need for cold chain logistics. This has allowed manufacturers and laboratories to move toward serving decentralized markets, leading to wider availability of diagnostic testing. However, adoption has been limited by challenges such as relatively high cost, inconvenient scalability, and limitations in assay devices that can be lyophilized. Additional limitations include long processing times and susceptibility of lyophilized reagents to degradation at standard humidity conditions. These drawbacks have led scientists to seek alternative technologies for assay biomaterial stabilization. 


Chemical Preservation

To meet the stabilization needs of the next-generation of diagnostics, Biomatrica has developed a technology platform for chemical preservation of bioassay materials, including proteins and nucleic acids. In contrast to cold chain preservation and lyophilization, this technology does not require low temperatures, stringent humidity conditions, or lengthy optimization processes (Figure 1).

Biomatrica stabilizers tolerate a wide range of ambient temperatures, up to 40% relative humidity, and are easily scaled from small development batches to large production lots. Additionally, they can be used with assay reagents in nearly any format, from standard plates and vials to specialized cartridges and cassettes characteristic of next-generation diagnostics.


Figure 1. Comparison of relative costs and risk of failure using cold chain, lyophilization, and chemical stabilization of diagnostic assay reagents.

Case Studies

As case studies of Biomatrica’s chemical stabilization technology, we show the stabilization of several Focus Diagnostics’ Simplexa® Direct kits. The Simplexa Direct system enables qPCR amplification, detection, and differentiation of RNA or DNA directly from patient samples, without nucleic acid extraction. Simplexa Direct chemistry was previously formatted as −20°C frozen reagent aliquots thawed prior to use. The objective of this study was to develop assay reagents stable at 2°C to 30°C. This would facilitate more economical and readily available reagent storage and distribution for pandemic preparedness and stockpiling in resource-limited settings such as developing countries.

Here, we show optimization of two Simplexa assays utilizing Biomatrica technology: Simplexa® Anthrax Direct and Simplexa®  Ebola/Marburg Direct. Simplexa Anthrax Direct detects the Bacillus anthracis chromosome (Target 1) and its plasmids pXO1 (Target 2) and pXO2 (Target 3) from environmental samples or whole blood.


All sensitive assay reagents (Taq DNA Polymerase, Scorpion probes, primers, and dNTPs) were formulated in a single mix and screened using Biomatrica’s stabilizer library. The optimal stabilizer formulation was added to the assay reagents, and the mix was dried down into single-use reaction tubes through simple centrifugal evaporation. Positive control samples (plasmid DNA stabilized with Biomatrica’s DNAstable® Plus) were used to test the performance of the stabilized reagents over time at different storage temperatures (Figure 2).

Stability of positive control plasmid and assay reagents for Simplexa Anthrax Environmental and SimplexaAnthrax IVD assays was achieved. When stressed at different temperatures over time, the assays with stabilized reagents performed similar to Day 0 controls, both in terms of amplification cycle threshold (C(t)) (Figure 2A) and fluorescence intensity (Figure 2B) for all three targets.


Figure 2. Long-term stabilization of Simplexa Anthrax Environmental and IVD assay reagents at ambient and elevated temperatures. Assay reagents were stabilized with Biomatrica chemical stabilizers. Positive control plasmid stabilized with Biomatrica’s DNAstable Plus was used to validate the assays. (A) Real-time PCR amplification cycles for three different targets at different storage conditions, and comparison to Day 0. A negative change in C(t) values indicates an improvement from Day 0. (B) Examples of amplification curves for three different targets with the indicated reagent storage conditions tested with the Anthrax IVD assay; similar results were obtained for environmental assays. [Data for Figure 2 was kindly provided by Michelle Tabb and Regina Martin at Focus Diagnostics, Inc., a wholly owned subsidiary of Quest Diagnostics, Inc.]

The stabilized reagents were also tested using samples of extracted RA3 spores in soil (Anthrax Environmental) and B. anthracis Ames vegetative strain in whole blood (Anthrax IVD) (Table 1). Excellent performance of the assays was demonstrated for all targets. The assays with stabilized reagents were able to correctly call all tested samples using multiple targets (data not shown).

A similar approach was used for reagent stabilization of the Simplexa Ebola/Marburg Direct assay that detects and differentiates Ebola and Marburg viruses using reverse transcription-qPCR (RT-qPCR) (Table 1). A blend of low positive inactivated Ebola and Marburg strains was tested in whole blood using the stabilized reagent mix after storage at various temperatures. The stabilized assay demonstrated correct detection of all tested RNA targets at 14 days (Table 1) and 21 days (Data not shown).

The above case studies show that Biomatrica’s chemical stabilization of Anthrax and Ebola/Marburg assay reagents is effective at maintaining long-term ambient assay functionality, therefore providing a valuable alternative to cold storage for PCR/RT-PCR based assay reagents.

Beyond effectively preserving reagents under a wide range of conditions, Biomatrica has also developed stabilizers for protecting target analytes in patient samples, such as blood and saliva during sample collection, shipping, and storage. For example, RNAgard® Blood Tubes maintain RNA expression levels in blood samples essentially identical to those at sample collection time, even after 14 days of ambient storage (data not shown).


Table 1. Performance of Biomatrica-stabilized Simplexa Anthrax and Simplexa Ebola/Marburg assay reagents using blood or soil samples. Assay reagents were chemically stabilized (except the reagents stored at -20°C). Average C(t) values of three different targets for B. anthracis RA3, B. anthracis Ames targets, and Ebola/Marburg blend targets are indicated.[Data for Table 1 was kindly provided by Michelle Tabb and Regina Martin at Focus Diagnostics, Inc., a wholly owned subsidiary of Quest Diagnostics, Inc.]

Conclusion

Our results indicate the effectiveness of chemical stabilization as an alternative to cold chain and lyophilization for preserving diagnostic assay reagents and biological samples. Biomatrica’s chemical stabilization technology provides a reduced risk and cost solution for next-generation diagnostics, from immediate sample protection at the point of collection to stabilization of reagents in molecular assays. Chemical stabilization simplifies the manufacturing process relative to lyophilization. Moreover, it allows for distribution of assays to broader markets, facilitating more economical and readily available diagnostic supplies for point-of-care diagnostics in resource-limited settings.

Taken together, the manufacturing convenience, reduced cost, and ease of use of chemical stabilization technology will provide significant benefits for the next generation of point-of-care and companion diagnostics. 


























Vasco Liberal, Ph.D. (vliberal@biomatrica.com), is associate director of custom services, Louis Chen, Ph.D. (lchen@biomatrica.com), is technical support scientist, and Michael Vengrow (mvengrow@biomatrica.com) is marketing specialist at Biomatrica.

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