Singulex: Next-Gen Immunodiagnostics

• Singulex says its next-generation immunodiagnostic assays are 100 times more sensitive than other current testing platforms, and they eliminate the problem of "not detectable" results.
• The company originally developed its Single Molecule Counting technology for research applications, but it is preparing to launch a product for clinical use.
• Singulex will pursue high-value applications for its Clarity IVD system, starting with an assay for acute myocardial infarction.
• A point-of-care system that provides results in 20 minutes could be commercially available in three to four years.

Singulex Inc., the privately held immunodiagnostics company based in Alameda, CA, is close to market launch of its Clarity in vitro diagnostic system. The fully automated platform is designed to bring the benefits of the company’s Single Molecule Counting (SMC) technology to hospital and reference laboratories.

Singulex bills its SMC technology as “the backbone of next generation immunodiagnostics, because it offers “unprecedented sensitivity and dynamic range in the precision measurement of biomarkers,” including proteins and nucleic acids. The company is already offering its SMC technology to clinicians through its state-of-the-art CLIA/CAP/NY-accredited clinical laboratory, which was set up at Singulex’s global headquarters in California in 2010. The lab provides physicians with data on cardiovascular and inflammatory biomarkers, allowing them to assess patients for risk of developing cardiovascular disease. As a result of this service, which generated $41 million in revenues in 2015, physicians are already appreciating the extra certainty that SMC testing can offer, says Singulex’s president and CEO, Guido Baechler. More than 3 million SMC tests have been processed since 2010.

The SMC technology allows biomarkers to be measured in blood samples at concentrations as low as 10 femtograms/ml, which is as much as 100 times more sensitive than other current testing platforms. This opens up a number of possibilities beyond the capabilities of conventional immunoassay-based tests: it has the potential, for example, to identify people who have a particular disease before the appearance of clinical symptoms, leading not only to earlier diagnosis and treatment, but also to the avoidance of unnecessary and expensive diagnostic procedures in those who have symptoms but who do not, in fact, have the disease. The technology can also be used to monitor the progression or recurrence of disease, or its response to treatment, again with greater sensitivity than with other methods. All these characteristics have the potential to lower health care costs as well as improve patient management.

Beginning With Research

The use of SMC as a clinical diagnostic tool evolved from Singulex’s development and commercialization of the technology as a life science research tool via the Erenna system, which the company introduced in 2008. The system includes fully integrated benchtop analytical instrumentation with dedicated software for automated data analysis. Singulex has developed research assays for a wide range of cardiovascular, infectious disease, oncology, autoimmune, neurology and nephrology markers. In addition, users may develop their own assays.

In July 2015 EMD Millipore, the life science business of Merck KGAA, headquartered in Darmstadt, Germany, acquired worldwide exclusive rights to Singulex’s SMC technology, including instruments, assays and services, for research applications. “This strategic agreement … puts our unique technology into the best possible hands to optimize its commercial scale-up and market potential,” Baechler said at the time.

Commenting on the deal to In Vivo, Baechler says the decision to out-license the research applications of the SMC technology was made for strategic reasons. “We realized that handling both the research and clinical applications of the technology was too much for one company,” he explains. The situation is similar to that at Roche in the early 1990s, he says, when the polymerase chain reaction (PCR) technology, originally developed by Cetus Corp., was commercialized by Roche for clinical diagnostic applications and by Perkin-Elmer Inc. for research applications. [See Deal]Baechler is in a position to know: between 1989 and 1994 and again between 1995 and 2008 he held executive and leadership positions at both Roche Diagnostics and Roche Molecular Diagnostics. He joined Singulex in February 2008 as vice president of diagnostics and then served as senior vice president of operations. He became president and CEO in 2013, replacing Philippe Goix, PhD, who had led the company since shortly after its founding in 2004.

Goix was at the helm in 2010 when three related whistleblower actions were brought against Singulex and Health Diagnostics Laboratory Inc. alleging that the companies had violated the False Claims Act by paying remuneration to physicians in return for patient referrals and billing federal health care programs for medically unnecessary testing. In April 2015, Singulex reached an agreement with the US Department of Justice to resolve the allegations, agreeing to pay $1.5 million.

No Non-Detectable Results

According to Singulex, the SMC technology uses standard immunoassay chemistry combined with a digital detection system. The capture step quantifies low-abundance biomarkers in biological fluids using paramagnetic microparticles coated with specific antibodies; the surface chemistry of the microparticles is designed to improve specific binding efficiency while minimizing non-specific binding. Following capture, the microparticles are magnetically separated and washed to remove unbound protein.

Each biomarker is translated into a signal by the addition of fluorescent dye-labeled detection antibodies, with each signal corresponding to a single biomarker molecule. The eluate is transferred to a reading plate where it is aspirated into the instrument’s capillary flow system. The interrogation space inside the capillary has a diameter of roughly 5 μm and is illuminated by a laser: single fluorescently labeled molecules generate intense flashes of light as they pass through the laser beam. These flashes are counted as digital events.

One of the benefits of being able to quantify biomarkers at such very low concentrations highlighted by Singulex is the ability to eliminate the problem of “not detectable” or “non-quantifiable” results, which can be difficult to interpret.

The instrument records the sum of all digital events counted; at high biomarker concentrations, when the digital counting mechanism becomes saturated, the sum of all photons recorded is computed via a proprietary algorithm. In this way a dynamic range of over four orders of magnitude is achieved.

One of the benefits of being able to quantify biomarkers at such very low concentrations highlighted by Singulex is the ability to eliminate the problem of “not detectable” or “non-quantifiable” results, which can be difficult to interpret. As well as facilitating comparisons between endogenous and elevated analyte levels, this capability facilitates a number of different types of investigation, leading to:

• more accurate stratification of populations;
• pharmacokinetic studies extended over longer periods;
• more accurate microdosing studies;
• study of disease progression even when biomarker levels are low; and
• discovery of new uses for existing biomarkers.

The Clarity System

Singulex says that the Clarity system will be the most sensitive IVD immunoassay system for routine diagnostics. Singulex began development in 2014, in collaboration with the Swiss laboratory automation specialist Tecan Group Ltd. The concept combines Singulex’s SMC technology and customized consumables with Tecan’s Freedom EVO liquid handling platform to create a fully automated benchtop analyzer targeted at clinical diagnostic laboratories. It also incorporates a customized one-piece detection vessel designed and manufactured by Sony DADC BioSciences GMBH (currently being acquired by STRATEC Biomedical AG). The aim of partnering with Tecan was to ensure the development of an instrument of the highest quality while at the same time reduce the time to market, Baechler explains.

The Clarity system will feature ready-to-use assay reagents that do not require special handling prior to use. Other benefits will include an advanced workflow with built-in touch screen with a simple graphical user interface, on-board capacity for 12 assays, throughput of more than 400 tests/shift, capacity to hold 48 primary sample tubes and a continuous inventory of on-board resources.

The first placements of the instrument for premarket evaluation are currently underway in Spain and the UK, with the aim of generating sufficient data to support European approval by the beginning of 2017. Three evaluation sites in the US are also being rolled out, again with the intention of generating data for an FDA approval submission in early 2017. The company will be pursuing the 510(k) route for the Clarity system. Baechler reports Singulex has already had a number of positive conversations with the FDA to help guide this process.

High-Value Diagnostics Applications

Part of Singulex’s strategy for the launch of the Clarity system will be to focus on high-value applications. The first of these will be an assay for cardiac troponin I. Low concentrations of cardiac troponin I measured on presentation to the emergency room have been shown to have a high negative predictive value for the identification of acute myocardial infarction (AMI). Last year, the European Society of Cardiology established a new guideline to rule out AMI in patients presenting with chest pains (2015 ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation, European Heart Journal 2016;37:267–315): patients with cardiac troponin I levels of less than 5 pg/ml can be safely ruled out as not having AMI. Unlike other platforms, the Clarity system is capable of measuring down to this level of sensitivity.

The average saving achieved by incorporating cardiac troponin I testing into the diagnostic workup of suspected cardiac patients (and thereby eliminating unnecessary scintigraphy studies) is about $364 per patient. Applying this model to the whole of the US would save around $2.5 billion per year, Singulex says.

Baechler says that applying the Singulex cardiac troponin I assay also helps avoid the unnecessary use of nuclear stress testing in cardiac patients, which uses a relatively high dose of radiation and is expensive. Over 90% of nuclear stress tests currently carried out are negative, which adds significant unnecessary cost to the health care system. Studies carried out at the University Hospital of Basel, Switzerland, have shown that the average saving achieved by incorporating cardiac troponin I testing into the diagnostic workup of suspected cardiac patients (and thereby eliminating unnecessary scintigraphy studies) is about $364 per patient. Applying this model to the whole of the US would save around $2.5 billion per year, Singulex says.

Looking further ahead, Baechler says Singulex is developing a point-of-care system that will permit SMC testing to be carried out at the patient’s bedside and provide results within 20 minutes: commercial introduction in Europe is provisionally planned for the end of 2019, subject to the necessary approvals, and for 2020 for the US. The instrument will be based on a dry blotting system, and basic feasibility has already been established using cardiac troponin I as the biomarker. The company is now concentrating on miniaturizing the optics involved with the aim of developing a microfluidic cartridge “about the size of a coffee cup.” Its strategy is to create the market for SMC testing with the Clarity system, and then to further exploit the market with the point-of-care system.

In support of these developments, Singulex owns a broad IP portfolio. The company holds six issued and four filed patents relating to the Erenna system (in the US and in the rest of the world), and the Clarity system is supported by 11 issued and nine pending patents. In addition, the firm has a portfolio of 10 issued patents and 26 filed and/or published patents relating to individual biomarker applications: the focus of this portfolio is primarily cardiovascular, but it also includes rheumatoid arthritis and kidney disease markers.

New Money, New Market

Privately held since its founding in 2004, Singulex attempted to go public in 2012, but withdrew the IPO three months after filing, citing poor market conditions. [See Deal]It has received multiple rounds of financing over the years from backers including OrbiMed Advisors, Prolog Ventures, Fisk Ventures and JAFCO.

A new investor signed on in May 2016, when Grifols SA, a leading producer of therapeutic plasma products with a presence in more than 100 countries, announced a $50 million equity investment in Singulex, thereby increasing its stake in the US company to 20%.[See Deal] The deal gives Grifols, based in Barcelona, Spain, an exclusive worldwide license for the use and sales of Singulex’s SMC technology for the screening of blood and plasma, and represents a major advancement in further ensuring the safety of blood and plasma products, the two firms said in an announcement.

Along with revenues from its lab business and future Clarity system sales (subject to approval of the device), Baechler anticipates that partnerships, like the one with Grifols, will play a major role in financing the company’s ambitious plans for the future, such as the point-of-care system. “We continually explore additional external funding opportunities, although we don’t have specific plans for additional rounds of funding at this time," he adds.