Technology & Processes

Technology & Processes

Lyophilisation

Lyophilisation (ly·oph·i·li·sa·tion) n. The creation of a stable preparation of a substance by rapid freezing and dehydration of the frozen product under high vacuum.

Under controlled temperature and pressure, water sublimes moving from the solid into the vapour state. In the presence of protectants, many biological materials may be frozen and using a carefully controlled change in temperature and pressure, >95% of the water may be removed. This leaves the biological material both intact and active. This process is termed lyophilisation and the specialised devices used to perform this process are termed freeze dryers.

Freeze-drying protectants include sugars that are naturally found in extremophiles, organisms that can withstand environmentally harsh conditions such as low temperature (cryobiosis) and drought (anhydrobiosis). Along with a combination of anti-oxidising agents and bulking materials they form the “excipient” or “carrier” material for the dried biologic. They are usually the largest material by mass in the lyophilised product and form a “cake”, retaining the dried biologic ingredient in a stabilized state.

Fluorogenics has developed proprietary excipient formulations and lyophilisation processes to deliver its Cirrus™ and Cyxi™ amplification product ranges. The same technology may also be applied to other molecular biology reagents. Fluorogenics’ approach to lyophilisation has unique features including:

  • Applied to a wide variety of molecular biological reagent types, including complex mixtures
  • Rapid dissolution and ideally suited to laboratory process automation
  • Long shelf-life at ambient temperature which is desirable for warehousing, transport and end-user storage of the product

Polymerase Chain Reaction

Polymerase Chain Reaction n. an in vitro technique for rapidly synthesizing large quantities of a given DNA segment – abbreviation PCR.

The Polymerase Chain Reaction (PCR) is a Nobel Prize winning technology. It underpins a variety of scientific processes including the decoding of genomes and human identification through DNA fingerprinting. It is a core method for both life science research and the detection of both hereditary and infectious disease.

A purified enzyme from a thermostable bacterium (Thermus aquaticus) isolated from hot springs is used in the laboratory to copy specific segments of target DNA. The reaction specificity is controlled by oligonucleotide primers, short pieces of synthetic DNA specific for the target, combined with careful changes in temperature. The process is cyclic, the copied DNA forming the substrate for further replication (amplification).

PCR thermal cyclers are automated instruments that heat and cool the reaction test tube facilitate the process. Single molecules may be amplified in 30-40 cycles of the reaction to greater than 1×1012 copies, by inference of the amplified material, the target DNA can then be identified. Real-time thermal cyclers incorporate a fluorimeter and use additional fluorescent oligonucleotides/ fluorescent DNA-binding dyes in the PCR reaction to monitor the amplification as it occurs. This greatly simplifies the process within modern laboratories.

Anything that contains target DNA may be identified providing its sequence is known and oligonucleotide primers/probes can be designed against unique regions for its differentiation from other targets. The PCR process has fuelled a growing industry, particularly in the supply of novel reagents and instrumentation for its rapidly expanding number of research, diagnostic and commercial applications.

The synergy of lyophilisation and PCR.

By harnessing our expertise in biological reagent stabilization with our knowledge of PCR assay design and optimisation, Fluorogenics is playing its part to broaden the global application of PCR by freeing the reagents and process from its traditional cold supply chain requirements. CirrusTM lyophilised PCR products offer a number of benefits to both the reagent supply industry and the end-user:

Ambient temperature stability. Unlike conventional PCR reagents that require both cold transport/ cold storage and typically expire within six months, lyophilized reagents can be stored at ambient temperature for up to two years minimizing cost, packaging and environmental impact.

Convenience. Freeze-thawing and assembly of multiple PCR reagents creates the potential for pipetting errors, batch-to-batch inconsistency and poor result reproducibility by virtue of their labour-intensive nature. Using pre-formulated lyophilised reagents that contain all the components needed for PCR offers simplicity and minimizes hands-on time; essential for improved laboratory throughput and time-sensitive results.

Contamination prevention. By minimizing the number of reagent handling steps needed in reaction set-up, sample handling and pipetting steps are reduced, lowering the risk of sample degradation, cross-contamination and critically, human error.

Robustness. By using single-source, single-batch core PCR reagents pre-formulated for immediate use, batch-to-batch variation between individual reagents is reduced, providing consistency and reproducibility for quantitative applications.