Buz's Thoughts for Introduction and Wrap-up
There are significant cost savings to be had by reducing cold-chain requirements in bio-medical research. The goal of this forum is to determine which vendors have what products and give them and the scientists who could use them a chance to communicate, to help shape future products and hasten the use of those products. My job as a liaison is to reduce interagency redundancy and leverage strengths in our research efforts, saving our agencies time and money, and reducing tensions in the process. My job is also to bridge interagency silos to reduce redundancy and enhance speed to deployment. To that end, CDC and USAMRIID are preparing a joint proposal to DITRA for funding for a 2-4 yr project (conducted under Booz Allen auspices) for evaluating COTS (commercial off the shelf) products stabilizing nucleic acids, proteins, blood markers and cytokines, live bacteria and viruses, and also for an array of assay bio-reagents like PCR master mixes. This mini-forum seeks to actively search for new ideas for using biostabilizers. The goal is to organize a group-think for uses of reagents that stabilize DNA, RNA, proteins, tissues, live bacteria and 'viable' viruses at room temperature. There is wide interest, both at Ft Detrick and bio-industry-wide here in the biotech corridor of Frederick and Montgomery Counties, than can tapped into for this purpose. Name your favorite biological sample (viruses, bacteria, other microbes, parasites, insects, clinical samples, tissue or organ, [and some day whole animals, holy sci-fi!]), or biological reagent) and there is going to be a series of biostabilizers coming down the pipeline to preserve it at room temperature during collection, shipping, storage and analysis. I think we all might want to get on the elevator as near the ground floor as possible. We have the same concerns as many of you do, that these revised storage conditions must not be detrimental to downstream applications of the sample or reagent. There will always be pros and cons to any new technology, as no change is perfect or without risk, but we all have to find ways to reduce cold-chain space requirements and monetary and energy costs.
Biomatrica Abstract - Rolf Mueller
As sometimes happens, Biomatrica began with a freezer breakdown. That suggested a need for better ways to preserve biological samples. Biomatrica began investigating the world of anhydrobiosis and thermal adaptation through the thermal stress response. Starting with the knowledge that vitrification was centered on the unusual glass transition temperature of trehalose sugar; they looked for additional chemicals to be better biostabilizers for different applications. Biomatrica now uses structural chemistry information and an added library screening approach on top of "biostability" recombinatorial chemistry. Combining screening and synthetic chemistry is a very powerful approach to finding the answer to a question. Biomatrica has accumulated the most extensive library of biostability compounds in the world. They have two synthetic chemists synthesizing and derivatizing biostability molecules to be used in targeted biostability screens. Biomatrica has used this approach for extremely rapid product development to find the best biostability solution for many different sample types. Their approach is knowledge-based with a factor of un-predictable random targeting. Their capacity allows them to provide products that will reduces cost, increases reliable test results through biostability of sample and reagent, which expands global technology reach and decreases the logistics burden. Biomatrica will lay out the general principles of dry-state ambient temperature biosample preservation, and then they will describe details of the specific approaches that Biomatrica has taken to develop their technology into products based on those principles. The bottom line will be how the Biomatrica technology can be additionally developed, going forward, to support integration into a broad range of diagnostic and screening device platforms.
IXI Abstract -Mike Hogan
IXI is specifically focused on the use of solid matrices for ambient-temp biosample preservation, compared to Biomatrica, who is focused on hydrogel reagents. IXI is actively developing direct coupling of ambient-temp sample collection as the primary sample input into its own LoC devices and in parallel the development of ambient temp reagent stabilization internal to its LoC cartridges. Recent upward trends in energy costs have brought a resurgence of green laboratory technologies - the transportation of samples from remote areas, and the long term storage or bio-banking of biological samples for future analysis can require considerable energy. IntegenX offers a broad and complete array of technologies to allow room temperature transport and storage of biological samples. An overview of the various products: GenTegra DNA and RNA, GenPlate, The Personal Archive and more will be explained as well as their place in the workflow of sample transport and storage. Recent studies proving the efficacy of these products will also be presented, as proof that this room temperature storage solution truly works. IntegenX will describe how they have approached the same general challenges associated with dry-state, ambient temperature biosample preservation: mainly via the use of solid support materials science. We will then briefly describe products and technologies IXI has already developed based on such solid matrices, then move-on to a discussion of how IntegenX could develop its technology to support collection, transport and integration into a broad range of diagnostic and screening platforms. I believe that Biomatrica and IntegenX have each converged to different and potentially complementary solutions to the same generalized ambient-temperature preservation problem. It will be interesting and instructive for the assembled group to begin to think about how these two "semi-orthogonal" approaches can be developed, perhaps simultaneously, into a new set of standards for ambient temperature bio-sample collection, management and use: with a special eye to their direct coupling as an integrated component of screening and diagnostic testing devices. They will then lead a discussion on the following key elements: 1). the role that ambient temperature biopreservation can play in support of medical screening and diagnostics, rather than for biobanking: which has driven much of the field of ambient temperature biosample preservation to date. 2). there is an opportunity, right now, to define a new set of medical sample collection and preservation standards. The opportunity is much like the way that the vaccutainer technology became the standard for sample collection in medical diagnostics in the latter half of the 20th century. This work that can be done in the next 24-36 months, in collaboration with medical device developers, and could define a new set of sample preservation standards of that kind, based instead on ambient temperature biopreservation. 3). each of the two approaches (BM vs. IXI) may be optimal for individual sample types (blood, serum, saliva, aerosol) or optimal for a different analyte category (DNA, RNA, protein, enzymes, virus, bacteria). The key going forward will be to determine the right tool for the job. Recent upward trends in energy costs have brought a resurgence of green laboratory technologies - the transportation of samples from remote areas, and the long term storage or bio-banking of biological samples for future analysis can require considerable energy. IntegenX offers a broad and complete array of technologies to allow room temperature transport and storage of biological samples. An overview of the various products: GenTegra DNA and RNA, GenPlate, The Personal Archive and more will be explained as well as their place in the workflow of sample transport and storage. Recent studies proving the efficacy of these products will also be presented, as proof that this room temperature storage solution truly works.
DSD Abstract - Tim Minogue
Evaluation of Methods to Ensure Clinical Specimen Stabilization in Austere Environments
Tim Minogue, Diagnostics Systems Division, USAMRIID, Ft Detrick MD
The US military is increasingly mobilizing personnel to austere environments that preclude the ability to adequately support laboratory diagnosis of infectious disease in the field. This is in part due to the fact that microbial culture or extraction of highly purified nucleic acids is required for confirmative diagnosis of the majority of infectious diseases. The bulk of DoD's diagnostic platforms for the field environment are molecular based and require purified nucleic acids. Unfortunately, nucleic acid extraction using conventional methods is extremely difficult in these types of environments due to limitations in equipment availability, power supply, and proper cold-chain management. To mitigate these challenges, the Diagnostic Systems Division's current efforts are focused on evaluating technologies that will stabilize biomolecules contained within clinical specimens for adequate transport to OCONUS or CONUS reference laboratories. To that end, we have evaluated the temporal and thermostable properties of Biomatrica's CloneStable and RNAStable products in terms of their ability to prevent degradation of biomolecules required for downstream testing. In addition to these commercially available products, we have also evaluated custom stabilization formulas made in collaboration with Biomatrica for their ability to temporally and thermally stabilize biothreat agents in relevant clinical matrices. The impact on biomolecule stabilization using these commercially available and custom reagents will be discussed in detail. Lastly, we are also evaluating simplified sample processing technologies, such as FTA Elute Cards, that will support nucleic acid stabilization. FTA Elute Cards' simplicity is based on the chaotropic properties of proprietary reagents impregnated on the card. Using this technology, there is no need for extraction prior to application, the applied clinical sample can be shipped at room temperature, and follow-on extraction of nucleic acids is a one step process that does not require the use of a purification kit. Results from these evaluations will also be discussed.
ATCC/BEI Abstract - Kurt Langenbach
Assessing Novel Room Temperature Nucleic Acid and Protein Storage Medium
Kurt Langenbach, Maria Mayda, Robert Anderson and Tim Stedman, BEI Resources/ATCC, Manassas, VA
ATCC is a global biological resource center specializing in the management and distribution of a broad range of biological agents including their derivatives, aligned reagents and supporting products. Over the past 13 years ATCC has gained significant and measurable experience through the management of several government contracts, including NIAID-supported specific research agendas such as the Malaria Research and Reference Reagent Resource Center (MR4), the Biodefense and Emerging Infections Research Resource (BEI Resources), and the Microbiology and Infectious Diseases Biological Resource Repository (MID-BRR). This broad experience has shown us that biological materials, by their nature, are susceptible to degradation and deterioration during collection, storage and distribution. For this reason, improving the stability of these materials is crucial. Over the years we have successfully implemented traditional methods for long term storage of biological materials, including DNA, RNA, proteins, cells and tissues, through laboratory freezers at -20°C, -80°C, or in liquid nitrogen. However, we recognize that emerging novel technologies may offer several benefits, including: improving upon material shelf life, reducing shipping costs of biomaterials (by removing blue ice and dry-ice requirements), potential for providing material in a quantitative format or as a convenient single-use product. As part of a technology assessment program, we have begun exploring novel preservation techniques for use with nucleic acids and proteins. Our studies allow us to evaluate multiple aspects of the technology, including impacts on processes, costs and stability, including maintenance of appropriate functional characteristics. Insights gathered from this and other studies offer an opportunity for ATCC to step out from traditional biological sample storage concepts, presenting us with advantages such as a reduction in space, energy usage, long-term cost, as well as also greatly facilitating transfer and shipping of samples.
NIH NCI-Frederick Repository Abstract - Mark Cosentino
Need Title of Talk
I have spent the past 2 years working full time with OBBR on their projects. The BRN Symposium is focused primarily on having the projects that the BRN funds provide the audience with a yearly update. Some of these projects focus on fixatives and stabilizers, but not from a commercial product development perspective, or from a room temperature storage perspective. This Forum is designed to generate discussion in a more targeted fashion. The BRN Symposium tends to have limited discussion, and because the topics are very diverse, so is the discussion. This Forum will be different from the BRN Symposium and something that is really needed. Hopefully, having a product development slant will identify possibilities for new collaborations. The following concepts will be addressed: 1.Everybody wants to redirect money, energy and space usage away from cold-chain maintenance for sample preservation. 2. However, nobody in their right minds wants to go back and add a stabilizing reagent tube-by-tube to their existing inventories of samples and organisms, because: 1. It has an immediate short-term cost, often unbudgeted, and is a nuisance and morale killer (Question - do we need a rentable robotic system?) 2. There is an unknown amount of risk of detrimental effects to the samples for unforeseen downstream applications 3. There is a risk of contaminating and cross-contaminating samples 4. There is a risk of misplacing samples 3. The benefits are: 1.There can be significant complex long-term cost and space savings 2. There can be actual improved stability over cold-chain for some markers 3. There can be improved access to samples and ease of paring down collections 4. There is a chance to simultaneously add new RFID, bar-code or similar inventory tracking technology to older sample collections 5. You no longer need to be concerned with freeze/thaw degradation of samples, which in some cases might reduce the need for aliquots 4. What does the repository community want to see in this new biostabilizer technology? What are their short term priorities and long term goals? 5. Ideas you have for such an end-users' presentation?
NCI Genetics Branch Abstract - Daniel Edelman
Long-Term Stability of Total RNA in RNAStableŽ as Evaluated by Expression Microarray
Daniel C. Edelman, Holly Stevenson, Yonghong Wang, Sharon Oghi, Rolf Muller, Paul S. Meltzer, CCR
Storage of normally labile RNA in laboratories is accomplished through ultra-low freezing of the nucleic acids. This however requires expensive freezers, a lack of convenient storage, and increased shipping costs. Biomatrica (San Diego, CA) has created RNAstableŽ, a stabilization reagent that is used to store RNA in a dehydrated state at room temperature (RT) which protects the RNA from degradation. Previous studies have demonstrated that the integrity of RNAstable-protected RNA is maintained for at least four weeks and is suitable for quantitative PCR and expression microarray analysis. Our objective was to investigate the functionality of RNA stored in RNAstableŽ at extended time periods through use of Illumina and Agilent RNA expression microarrays. We observed in Bioanalyzer electropherograms that RNA extracted from 293 cells stored at RT in RNAstableŽ for 4.5 mo and 11.5 mo is similar in quality to RNA stored at -80°C. Illumina expression array QC metrics and gene expression patterns from RNAstableŽ-protected RNA correlated well with those of freezer controls. In contrast, when RNA was stored at RT but without RNAstableŽ, there were increased levels of degradation and a decrease in relatedness to both -80°C control and RNAstableŽ-protected RNA. Significantly, when RNA was stored in RNAstableŽ at 45°C for 4.5 months, equivalent to 22 months RT storage, RNA quality and expression microarray performance was similar to RNAstableŽ-protected RNA at RT and -80°C controls. In addition to evaluating mRNA, a comparison of the storage conditions at 10.5 months of the miRNA expression was performed on Agilent Human miRNA v3 arrays. Here too we only found strong concordance when miRNA was stored in RNAstableŽ or in the -80°C controls. We conclude that use of RNAstableŽ holds promise as an effective stabilization reagent for total RNA and might be useful in situations where shipping and storage options are resource limited.
Quanta Biosciences Abstract - Dave Schuster
Published work from others will be blended with performance data on our Quanta SuperMixes (related to their ability to overcome inhibitors, etc.) to develop the story of how performance optimization often has a strong component of stability optimization. To a certain extent, amplification reagents with at least limited room temperature stability are simply a serendipitous outcome of engineering better master mixes. Combined approaches, such as the Solis Biodyne peptide tag on the enzymes, along with the innovations we've built into our master mixes, bolstered with Biomatrica's additives, might produce an ideal solution to the problem. Unfortunately, collaborative research activities between independent for-profit companies, such as those needed to develop the next generation of PCR master mixes, are often as difficult to work out as the science itself.
Solis BioDyne Abstract - Olev Kahre
A Novel Approach for Increasing Stability of Polypeptides at Ambient Temperatures
Solis BioDyne was created in 1995, selling various PCR and qPCR reagents manufactured in our production site in Estonia. We have entered a very competitive market with 2 key sales arguments that have allowed us to grow and improve every year: Competitive pricing for our products, and the development of a unique technology for increasing the stability of Solis BioDyne´s polymerases at ambient temperature, as well as at -20C [patent pending: "Compositions for Increasing Polypeptide Stability and Activity, and Related Methods.": US2011/ 0142792 A1]. This novel technology allows enzymes to handle ambient and elevated temperatures (so far tested at up to +35°C) for at least 1 month, without any loss of activity. This stability also extends to Solis BioDyne´s real time master mixes. The technology consists of a genetic recombinant modification of the enzyme's cloned gene which genetically fuses it with an amino acid (AA) tag coding sequence. This amino acid tag is what allows the enzyme to remain stable at ambient temperatures for prolonged times. We will present our experimental results for different polymerases and real-time PCR master mixes, and we will show examples for both end-point (intercalating-dye-based) PCR and probe-based qPCR. We will lead a discussion of our experiences, where we will comment on the advantages and disadvantages of this technology for PCR polymerases and master mixes. We will also provide a short overview and the results of our approach for applications of this technology for non-polymerase polypeptides, such as dCTP deaminase, hLIF and other proteins of interest. We are looking to move forward in this technology field by seeking collaboration partners who seek to gain from our experiences, who can benefit from our PCR-related products, and those who could adapt them for use in the diagnostic and therapeutic markets. We are also looking for companies and organizations who would be interested in incorporating our novel stabilizing technology into other types of polypeptides.
Concrete benefits of this peptide-stabilizing technology:
- Low logistical costs, as peptide reagents with this modification can be shipped worldwide via regular courier at ambient temperatures (no dry-ice needed)
- Reduced concern about the cold-chain, as a limited exposure to ambient temperature will not adversely affect the activity of the products
- PCR-based diagnostic kits manufacturers and researchers using Solis BioDyne´s enzymes also benefit from this increased stability, which is especially valuable in tropical and poorly developed areas, where the cold chain is extremely problematic (due to complicated logistics and/or custom transit times, or equipment malfunction).
- Although the technology is currently commercialized only in the form of modified Taq DNA polymerases, it is in no way limited to them. Successful experiments have already shown that the technology is widely applicable to increase the stability of other peptides.
Bioneer Abstract - Vince Prezioso and Dr. Park, Bioneer CEO, inventor of AccuPower Technology
Three approaches to Stabilizing qPCR Detection Kits: AccuPower, PyroHotStart and RocketScript
Biostabilization technologies have been sought in order to improve shelf-life of enzymatic reagents in molecular biology applications. We will present three approaches that Bioneer have taken in this area. The first approach, called AccuPower Technology, uses a novel stabilizer which allows the lyophilization of any PCR reaction master mix. AccuPower is currently used in Standard, HotStart and High Fidelity PCR reactions, as well as reverse transcription and DNA ligation reactions. Our AccuPower Technology is amenable to virtually any enzymatic reaction mix. Our second approach, called PyroHotStart, involves the use of thermostable enzymes to improve and stabilize HotStart PCR technologies. Finally, we have employed protein recombinant engineering to increase the stability of peptides; this is best exemplified by the MMLV reverse transcriptase used in our RocketScript Reverse Transcriptase Mix. Each of these separate technologies will be described in depth, and we will describe how we have combined them in various products to produce long-lasting room temperature-stable enzyme mixtures.
Naval Research Labs Abstract - Scott Walper and Ellen Goldman
Ellen Goldman and Scott Walper will present a brief introduction to the intrinsically stabile single-domain antibodies (sdAb) from camelids and sharks. They will offer a review of the work that they have done selecting and characterizing ricin and Bacillus anthraces binding sdAbs, and conclude with their preliminary work done using them in conjunction with the immune-PCR proximity ligase assay (PLA) in terms of potential future directions for uses of thermal stabile antibodies.
Xoma Abstract - Milan Tomic
Although Xoma does not work on biostabilizers per say, they do develop therapeutic antibody formulations that could benefit from their application. The stability of our lyophilized multi-mAb products (three antibody cocktail) against BoNT is >3 mo at 50°C. We have results that show our antibody constant region backbone infers stability to multiple mAbs in a standard formulation, and this characteristic speeds development time and cuts costs (only the variable regions are changed, maintaining target specificity and affinity). Current standards for stability of antibody formulations are - 30°C for >3 to 5 years; however, our latest information is that we should target for ~7 years of stability to met our client's demands. There is a need for standardized markers indicating the molecular integrity of products such as ours. We have begun to explore using marker/indicator peptides (from antibody tryptic digests) which are unique and can be used for ID as well as quantification in complex mixtures. To address the future challenges and directions of our field, we ask, "How can our needs drive the direction of biostabilization research?" There are some interesting companies that work with spray-drying therapeutic antibodies and other proteins; the interesting thing with this approach is that these lyophilized proteins do not behave like the typical lyophilized powders, but can be combined and compounded, much like tablets and pills. The other area of interest and enablement here at Xoma is the analytical technology required for characterization of multi-antibody mixture(s); antibody-specific probes and complex analytical techniques that can result in well-characterized antibody mixtures for therapeutics or diagnostics.
National Cancer Institute MicroBiome Working Group Abstract - Roberto Flores
Effect of Delayed Freezing on Microbial Composition in Human Feces: Lessons for Epidemiological Studies. Roberto Flores1,2, Jianxin Shi3, James J. Goedert1, Rashmi Sinha4 [1Infections and Immunoepidemiology Branch; 2Cancer Prevention Fellowship Program; 3Biostatistics Branch; 4Nutritional Epidemiology Branch. National Cancer Institute, Rockville, MD] Evaluating how differences in the microbiome affect disease risk in population studies is needed. However, collecting samples in field studies is challenging. Of paramount importance is to optimize collection and handling so that specimens yield highly stable and reproducible data. Our aim was to assess the effect of delayed freezing on two parameters: the reproducibility of 16S rRNA pyrosequencing and taxonomic analysis of DNA extracted from human fecal specimens, and the stability of fecal bacterial enzymatic bioactivity involved in deconjugation of carbohydrate moieties (β-glucuronidase and β-glucosidase). Eight volunteers were recruited, each of whom self-collected eight aliquots of feces that were immediately mixed with RNAlater. Four aliquots were immediately chilled on frozen gel packs and then frozen in liquid nitrogen within 3 hours of collection. To simulate field settings, the other four aliquots were left at room temperature for 3 days prior to freezing. DNA extraction and 16S rRNA gene PCR and pyrosequencing were performed. Taxonomic assignment using RDP classifier and indices of alpha and beta diversity were compared for rapid vs. delayed freezing. Specimens collected in PBS were used for protein extraction and activities of β-glucuronidase and β-glucosidase were measured by real-time kinetics. Parameters of enzymatic stability and decay were tested by incubating the samples at room temperature (RT) at different intervals before freezing up to 48 hr. No differences for indices of alpha diversity (rarefaction curves) were observed by time to freezing. However, by taxonomic analysis, we found that the mean number of OTU for the phylum Firmicutes was increased 47% in the specimens left at RT (p=0.0003). Six genera within the phylum Firmicutes contributed to the difference. In relation to enzymatic activity, substantial (30-40%) decay was observed within 4 hours at room temperature for both enzymes tested. Irrespective of time delay, bacterial enzymatic activity was most reliably measured in fecal specimens collected in PBS at neutral pH without protease inhibitors. Both acidic pH and protease inhibitors reduced the enzymatic activity by 50% compared to PBS alone.
Conclusion: Delayed freezing of fecal specimens may introduce large artifacts even for DNA from specimens collected in RNAlater. To minimize enzymatic decay, fecal samples should be collected in PBS alone, at neutral pH, and frozen within 2 hours post collection. Development and evaluation of methods to fully inhibit bacterial growth and stabilize biomolecules are needed to advance population studies of the microbiome.