Albumin: The Natural Choice for Diagnostic Tests

Author: Helen Rawsthorne Ph.D., Senior Science Manager, Albumedix

The current global pandemic has shined a light on the importance of diagnostic testing, especially for COVID-19. As the coronavirus continues to sweep across the globe, it has never been more vital to focus efforts on identifying those infected with the disease to delay and halt the spread of infection. A coordinated effort is needed to accelerate research and, whilst vaccine roll-out is still only in the early stages, the world is more cognizant of how reliable diagnostic testing is a valuable tool to save lives.

With the number of new diagnostic tests being developed being at an all-time high and commercial successes making global headline news, the industry is seeing great progress. But this promising growth comes with underlying challenges. The most well documented of these is reducing costs while maximizing productivity, however one area that is becoming increasingly important, especially with regards to COVID-19 testing is reliability, faster testing and speed of results.

Many well-established diagnostic tests for COVID-19 are available1. These include, 1) reverse transcription polymerase chain reaction (RT-PCR) – the current standard test, 2) loop-mediated isothermal amplification (LAMP) – a simple, but less developed test, 3) lateral flow – hand-held single-use assays providing results in as short as 15 minutes, and 4) enzyme-linked immunosorbent assay (ELISA) – technically simple assays that are easily read and offer relatively high throughput. More tests gain emergency approval each day and new techniques continue to make their way through the development pipeline, fueled by huge leaps in medical understanding, increasing government expenditures, and the rising number of COVID-19 cases.

Many of these diagnostic tests are becoming smaller and more rapid, but can more be done to improve the accuracy, reliability and speed of the results?

Challenges in Diagnostic Tests

PCR, RT-PCR, LAMP and RT-LAMP are the most common ways of testing for pathogens.

Polymerase chain reaction (PCR) is used to "amplify" small segments of DNA. Once amplified, the DNA produced by PCR can be used in many different laboratory procedures, including the detection of viruses. Reverse transcription PCR (RT-PCR) is used when the starting material is RNA. In this method, RNA is first transcribed into complementary DNA (cDNA) by reverse transcriptase from total RNA or messenger RNA (mRNA). The cDNA is then used as the template for the PCR reaction. RT-PCR is used in a variety of applications including pathogen detection.

LAMP (Loop-mediated Isothermal Amplification) is a single tube technique for the amplification of DNA. It provides a low-cost alternative to polymerase chain reaction (PCR) technology to detect certain diseases. It involves the design of assay primers and use of a strand-displacing polymerase to allow rapid amplification at a constant temperature without the need for thermal cycling (required for PCR). A typical LAMP reaction mixture contains 6 primers which target 8 regions on the bacterial or viral genome. In RT-LAMP, RNA sequences are detected instead of DNA. Reverse transcriptase is added to the LAMP mixture, to convert the viral RNA into complementary cDNA that will be used for amplification. This procedure has provided great help in diagnosing a multitude of RNA viruses.

One crucial element of all these tests is the amplification efficiency. The full potential of diagnostic PCR is limited, in part, by the presence of inhibitors in complex biological samples that reduce the amplification efficiency.2 Inhibitors may also affect the sensitivity of the assay or even lead to false‐negative results.3

Different amplification facilitators, such as casein and formamide, can be used to enhance DNA amplification. Organic solvents, including DMSO and formamide, have often been employed as additives to increase the efficiency of amplification of high GC content (GC > 60%) DNA sequences. Bovine serum albumin (BSA) has been used as an additive in several applications, including restriction enzyme digestions as well as in PCR amplification of templates from environmental samples that contain potential inhibitors, such as phenolic compounds. It is thought that BSA binds to inhibitors thus reducing their detrimental effect on amplification.

In certain scenarios, chemical additives or co-solvents may be included in the buffer to improve amplification specificity by reducing mis-priming and to enhance amplification efficiency by removing secondary structures. PCR additives and enhancing agents have been used to increase the yield, specificity and consistency of PCR reactions. These additives may have beneficial effects on amplification, increasing DNA polymerase stability.

Several studies have shown the benefits of BSA to significantly enhance PCR amplification yield when used in combination with organic solvents, DMSO or formamide. BSA enhancing effects were obtained in several PCR applications, with DNA templates of high GC content and spanning a broad size range. When added to the reaction buffer, promoting effects of BSA were seen in the first cycles of the PCR, regardless of the size of the DNA to be amplified. Such results demonstrate a cost-effective alternative for increasing the efficiency of PCR amplification of GC-rich DNA targets over a broad size range.4

BSA has also been reported to reduce the adhesion of the DNA polymerase to glass capillaries used in certain real-time PCR instruments.5 This is important for reducing the loss of reagents through adsorption to tube walls.

Scientists at New England Biolabs demonstrated the ability of a colorimetric LAMP assay to detect RNA from the SARS-CoV-2 virus that causes COVID-19.6 With regards to LAMP techniques, challenges can arise when the starting sample is “dirty”. This often means that additional steps and processes are needed to prepare the sample, which can be time consuming. BSA can help to provide a faster signal as amplification is quicker. In addition, the testing material does not have to be as “pure”, as albumin binds the impurities in the reaction mixture, removing the need for additional purification steps in the process.

However, there are notable challenges with BSA, and indeed some human serum albumins (HSA). It is important to appreciate that not all albumins are created equally, and the differing product characteristics can impact overall performance, efficacy and safety.

BSA and HSA Challenges

Finding the right additive, which enhances the performance of the diagnostic test, is an integral part of the development and regulatory pathway. The ideal product should be pure and virus-free. Stability and batch-to-batch consistency are also paramount considerations, owing to their impact on performance, efficacy, and safety.

BSA and other recombinant albumins can suffer from poor batch-to-batch consistency. Also, BSA is derived from animals which can pose potential virus contamination risks. Albumin quality is essential to ensure batch-to-batch consistency, but also, albumin can be altered by the presence of impurities and/or aggregates. The purity of materials is crucial for developers building the next generation of advanced diagnostic tests while avoiding the risk of false results.

The natural properties of recombinant human albumins (rAlb) make them the natural choice for diagnostic test additives. As a naturally occurring protein, albumin is widely recognized as safe. In addition, human- and animal-free recombinant versions reduce the risk of unexpected pathogens to zero.

rAlb, a multi-functional excipient with validated stabilization properties, can play a key role in ensuring consistency and maintaining the stability and efficacy of a diagnostic test so that patients receive optimal results. Unlike plasma derived human serum albumin (HSA) and other recombinant albumins, Recombumin® offers a reliable albumin source with excellent batch-to-batch consistency and security of supply. Our products have extremely low levels of post translational modifications (PTMs), unsurpassed cGMP quality meeting USP-NF standards and have been commercially validated in marketed and late-stage clinical drug and vaccine candidates.

Therefore, rAlb is a natural choice when safety, efficacy and reliability are important.

Nature’s Own Coating

Only a highly pure albumin, like Recombumin®, facilitates the full exploitation of albumin’s potential while reducing variability and risk. The animal- and human-free product offers excellent batch-to-batch consistency and security of supply, providing safety, efficacy, reliability, and regulatory peace of mind.

Recombumin® offers a safer alternative to BSA and HSA. Driven by the desire for high quality, human- and animal-component free albumin products, structurally and functionally equivalent recombinant versions to HSA have been developed over the past decades. HSA prepared from plasma presents different genetic variants and exhibits varying degrees of N-terminal truncation, glycation and oxidation due to the extracted albumins’ extended residence time in the body. Unlike HSA, Albumedix rAlb products have a remarkably high and consistent quality profile.

There is often a need to avoid BSA or albumin from an animal source. Like BSA, recombinant human albumin has been shown to prevent adhesion of enzymes to reaction tubes and pipette surfaces. It also stabilizes some proteins during incubation. Recombumin® human albumin may also be used to deactivate PCR inhibitors.

As a diagnostic test additive, Recombumin® is:

• Biologically stable
• Chemically inert
• Non-toxic
• Solvent free
• Comprehensive regulatory documentation
• USP-NF compliant
• GMP-grade manufacturing
• Supplied ready to use

Key Benefits of Recombinant Human Albumin for COVID-19 Tests

Recombumin® may enable more effective COVID-19 tests, including PCR, RT-PCR, LAMP and RT-LAMP diagnostic test kits via:

• Saving valuable time preparing samples; the purity of Recombumin® may allow amplification to occur with lower levels of template DNA, or from samples that are “dirtier”
• Preventing non-specific adsorption of the reagents to the PCR tube
• May act as an enhancer as it binds to PCR inhibitors
• May enabling the signal to be achieved quicker (LAMP, RT-LAMP)

Due to the unsurpassed purity of Recombumin® the binding sites for PCR inhibitors will be free (unlike BSA or plasma derived HSA), consequently making the test more effective. Recombumin® is confirmed to be virus free, eliminating the risk of false positives that might otherwise arise from BSA or HSA.

Regulatory Support

Albumedix has applied its scientific experience and technology to support a range of diagnostic testing applications.

Albumedix can support the registration of your diagnostic test by giving you access to our highly experienced regulatory support team. Our regulatory documentation and data can help alleviate the regulatory burden and satisfy requirements for regulators.

Working in a dedicated facility, we have GMP certification from the MHRA and our facility has been inspected by FDA and Health Canada in support of customer applications.  We have DMFs in many regions including the US and can provide support in answering regulators’ questions in relation to the use of albumin.  By providing these key resources and capabilities, Albumedix has played a vital role in helping diagnostic test manufacturers throughout the world develop their advanced technologies.

Towards the Future of Diagnostic Tests

The success of PCR and other diagnostic kits depends on several factors, with its reaction components playing critical roles in amplification. Recombumin® offers several advantages: binding to reaction inhibitors, eliminating additional sample cleaning steps as it combines impurities and coating the surface of tubes so that precious sample is not wasted. As new diagnostic tests continue to enter the market, our expertise and evidence will be instrumental in supporting the development of new kits. Our many years of experience in recombinant human albumin can be applied to such products and Albumedix is primed to support customers in developing COVID-19 testing kits.

This year, the biggest impact on our industry has obviously been the COVID-19 pandemic. As we are supplying customers developing diagnostics, medical devices, and vaccines in the fight against the coronavirus, we are vigilantly watching these developments. Solving challenges in cold-chain storage and transportation are central issues, important in both ensuring COVID-19 vaccines reach patients worldwide, but also an issue advanced therapy developers are addressing to make therapies scalable. An example is the increasing trend toward ‘thaw and inject’ of cellular therapies.

The availability of established diagnostic kits has enabled researchers to plug-and-play in the design of COVID-19 diagnostics. Such technologies took decades to optimize, but they are now playing an important role in identifying and managing the spread of COVID-19. Recombumin® provides an enhanced solution as part of this toolbox for dealing with this disease.

To learn more about recombinant human albumin for diagnostic tests, contact us today.

References

1. What tests could potentially be used for the screening, diagnosis and monitoring of COVID-19 and what are their advantages and disadvantages? https://www.cebm.net/covid-19/what-tests-could-potentially-be-used-for-the-screening-diagnosis-and-monitoring-of-covid-19-and-what-are-their-advantages-and-disadvantages/
2. Abu Al-Soud W, Rådström P. Effects of amplification facilitators on diagnostic PCR in the presence of blood, feces, and meat. J Clin Microbiol. 2000 Dec;38(12):4463-70. doi: 10.1128/JCM.38.12.4463-4470.2000. PMID: 11101581; PMCID: PMC87622.
3. PCR inhibitors – occurrence, properties and removal https://t.co/gEQViMkNQ8
4. Farell, E.M., Alexandre, G. Bovine serum albumin further enhances the effects of organic solvents on increased yield of polymerase chain reaction of GC-rich templates. BMC Res Notes 5, 257 (2012). https://doi.org/10.1186/1756-0500-5-257
5. Plante D, Bélanger G, Leblanc D, Ward P, Houde A, Trottier YL. The use of bovine serum albumin to improve the RT-qPCR detection of foodborne viruses rinsed from vegetable surfaces. Lett Appl Microbiol. 2011 Mar;52(3):239-44. doi: 10.1111/j.1472-765X.2010.02989.x. Epub 2011 Jan 11. PMID: 21219370.
6. https://international.neb.com/nebinspired-blog/loop-mediated-isothermal-amplification-allows-testing-to-be-performed-anywhere