• For highest specificity, cost efficient and fast analysis, we opted to remove cellular noise comprising red blood cells, platelets and white blood cells as much as possible thereby introducing pre-enrichment of nucleated cells and enrichment of rare cells (4log).
• In terms of sensitivity, we chose and advanced enrichment methodology under the strict obligation to prevent losses of desired cells greater 10% throughout the assay.
• In terms of transferability, precision and reproducibility, we act according to the paradigm of hands-off as much as possible and are on process of automating the complete diagnostic assay.
• In terms of costs, we are developing our automation in a highly professional yet low cost environment, have developed simplest possible assay modalities, minimized company dependencies in particular with respect to expensive consumables and orient our R&D optimization towards cost efficiency, then being able to offer highly competitive prices that would redefine the sense of scalability and accessibility of invitro-diagnostic tests.
SanoLibio is proud to add itself to the few that are taking the challenge of advancing the CD45 negative selection assay. We have invented a new and patented approach in magnetic particle cell separation technology that we call dynamic magnetic labelling (DML) and have presented the first and manual Version in 2018 in form of a scientific publication (Schreier et al. 2017, reference article: https://translational-medicine.biomedcentral.com/articles/10.1186/s12967-016-1108-1). Meanwhile, our efforts have resulted in the automated version being our Walderbach Platform that allows remedy of before mentioned drawbacks of current CD45 negative selection assays. Our Walderbach Platform can be considered the fastest, cheapest and highest enrichment-performing assay worldwide. In short, we have achieved 4log enrichment with 90% Recovery of desired cells in less than 3h for a fraction of the current sample prices. Unique is also the ability to control the enrichment with respect to white blood cell noise with the intention to save costs.
• It may have been already understood that the paradigm of our technology is to minimize loss of desired cells that carry vital information. Additionally, the assay part of enrichment is non-biased towards any cell abnormality (normality is considered to be a CD45 receptor expression cell =leukocyte). Consequently, we may have achieved the highest possible sensitivity at the individual level and will be able to facilitate meaningful clinical research in the field of early stage cancer diagnostics, screening as well as individualized cancer therapy. We understand that SanoLibio alone will hardly impact liquid biopsy advancement and therefore, intends to distribute its potential technology worldwide by sales and beneficial biomarker and invitro-diagnostic partnerships. Therefore, advancement is achieved in community.
• Besides the issue of sensitivity, our enrichment technology has gained and will gain more interest due to the high reduction of cellular noise comprising red and white blood cells. This level of noise reduction in the CD45 negative selection assay allows significant faster analysis therefore reduces costs and time and consequently increases sample throughput.
• Moreover, our own research and work with our technology is dedicated to a more wholly biomarker analysis that can be understood as the analysis of the rare cell spectrum. In other words, we are currently discovering the relationship between rare cell profiles and certain health conditions with each individual sample tested. Using our enabling CD45 negative selection assay, we are one the few that are able to discover and characterize the so far little investigated rare cell spectrum. Though disputed, it is our hypothesis and in fact, observation that smallest tissue irregularities (incl.-circumvented hyperplasia) are represented as a measurable altered profile within the rare cell spectrum. Consequently, this may facilitate research on pre-cancerous lesions and thereby enters the field of screening by liquid biopsy.
SanoLibio is engaging in cancer liquid biopsy. Liquid biopsy has proven potential to improve and evolve cancer diagnostics to the point of replacing exploratory tissue biopsy. We envision to develop our own cancer in-vitro diagnostic solutions within two phases of development. The first phase is dedicated to technology and methodology. The second phase is focused on the clinical translation of our liquid biopsy assay in any supportive market.
When taken the term in its original meaning, it is likewise an examination of tissue removed from a living body to discover the presence, cause, or extent of a disease in this case cancer. The tissue in case of liquid biopsy is blood. Tissue biopsy investigates cells and its behavior and so is the case in liquid biopsy. Besides the ease of access, the advantages of liquid biopsy over tissue biopsy are obvious as the degree of diagnostic information elicited by liquid biopsy may equate, complement or even exceed that of tissue biopsy. However, in contrast to tissue biopsy, the proportions of normal to abnormal cells in liquid biopsy are vastly imbalanced for the obvious reason of sampling a streaming bodily fluid instead of selected tissue regions that contain the abnormality at great abundance. Therefore, the abnormal cells in the blood are called circulating rare cells and comprise a spectrum of different cell types at concentrations often lower than 1 cell per 1 mL with many of them still awaiting discovery. The most commonly targeted rare cell in liquid biopsy is the circulating epithelial cell that is presumed to be a circulating tumor cell (CTC) in the setting of a cancer patient. As a side note, we have already contributed to this so-called rare cell mining having found immature bone marrow cells of the erythroid cell line circulating the blood stream in healthy donors (Schreier et al. 2019, reference article: http://atm.amegroups.com/article/view/21928/21464). Moreover, the term liquid biopsy has been extended to investigations of free circulating DNA and RNA as well as exosomes and may exclude serum protein analysis. SanoLibio directs its research and development towards whole cell analysis, as only cells bare the complete set of diagnostic information as well as suffice diagnostic confirmation. The liquid biopsy of blood cells addresses and opens new questions in cancer research and concerns many applications of which each is equally important to develop, such as cell line development, pharmacodynamics investigations, individualized therapy, prognosis and diagnosis from pre to post treatment scenarios.
As to improve, one needs to understand the problem. Despite the well-accepted potential, liquid biopsy clinical research and translation is now ongoing since 2 decades without impacting clinical routine as well as public health as far as the western world is concerned. When looked in detail, there is a certain volatility of liquid biopsy technology as can be expected in times of market introduction and diagnostic novelty. Furthermore, the vast amount of meaningful clinical research on liquid biopsy has been performed using only a few different technologies that would support transferability. The most common tool and declared gold standard is the CellSearch system that however, yields low sensitivity in particular at early stage carcinomas and is comparatively costly. It is well to say that sensitivity and costs are the two most distinguished problems in liquid biopsy. It has occurred to us and presumably to others in the field that the so-called oncological translation of liquid biopsy biomarkers is in parts hampered by insufficient technology. We have come to understand that the liquid biopsy biomarker is associated with the technology as a function of selection to the point that the biomarker becomes inseparable to the technology in use. In case of CellSearch, we may well talk about the CellSearch circulating tumor cell (Cellsearch CTC) thereby, denoting a subset of circulating epithelial cells. Consequently, any means of selection as well as flaws in technology would affect the biomarker usefulness and hinder liquid biopsy advancement. Reasonably, one would seek to minimize selection and flaws throughout the diagnostic assay. Bearing in mind the technology problem, SanoLibio is currently laying great emphasis on the development of its own diagnostic technology and methodology that would fit the purpose of advancing liquid biopsy technology with respect to accuracy, transferability, reproducibility and costs.
In our current and first phase, we are developing an automated fluid handling platform going by the name “Walderbach” and is in its basics a platform for automated ultra-high enrichment of circulating rare cells based on magnetic cell separation technology to serve the purpose of liquid biopsy. Although, our Walderbach may well be trained to carry out all kinds of different cell separation assays (e.g. positive selection of hematopoietic stem cells) in the future, it has been specialized to perform the so-called CD45 negative selection assay. There is substantial interest in the CD45 negative selection assay in particular in the field of liquid biopsy with respect to cancer pharmacodynamic as well as clinical and biological research. Therefore, our Walderbach will be initially marketed as a liquid biopsy rare cell enrichment platform for the research sectors in academia, pharma and diagnostics. The technological finalization and then commercialization of our Walderbach Platform will mark the end our first project phase and the begin of our activity in the invitro-diagnostic test development. Moreover, we have already begun to develop our own diagnostic platform going by the name RarmaxTM that incorporates the application of our Walderbach and is essentially an immunofluorescence assay that reveals the entirety of the rare cell spectrum.
First of all, CD45 represents a marker found on the surface of every white blood cell only, hence the name common leukocyte antigen. In this assay, magnetic particles react with white blood cells specifically due to ligand binding to CD45 and thereby selecting and likewise magnetizing all the white blood cells in the sample cell suspension. Consequently, at least 99.98% of the cells in the suspension are in theory magnetically separable and a miniscule fraction of nucleated cell events being part of the rare cell spectrum would remain in solution after magnetic separation.The current technological approach in liquid biopsy mostly follows the established paradigm of positive identification therefore, beginning and ending with positive selection of a specific biomarker. The most prominent liquid biopsy marker is the circulating epithelial cell that was found to be specifically associated with carcinoma patients, already excluding any other type of malignancy. Later on, its specificity and more importantly its sensitivity in particular at early stage was softened and questioned being forced to identify and translate new cellular biomarkers in liquid biopsy. Naturally, marker multiplexing increases sensitivity and meanwhile, an abundance of cellular tumor biomarkers is known to research creating the problem of how to enrich multiple markers by positive selection by equally efficient means. One solution is simply not to enrich, such as done by Epic Sciences, or the Kuhn Lab at Scripps, yet having to deal with extreme large amounts of noise that requires elevated levels of technological sophistication and prevents adaption to existing analytical procedure. A second solution assumes marked differences between cell parameters of circulating tumor cells and normal white blood cells, such as size, membrane charge, cell density, flexibility and some more. The assumptions have their justification due to positive clinical research outcome yet do not constitute a complete solution to the problem for there is often considerable parameter overlap between circulating tumor cells and white blood cells, thus loss of information. The third solution is the CD45 negative selection assay, a completely unbiased or non-selective method towards the selection of any kind of non-hematopoietic and CD45 negative hematopoietic cells therefore, yielding so called untouched and viable cells of the rare cell spectrum. In conclusion, the CD45 negative selection assay is in fact the best solution to gain access to the rare cell spectrum and to the problem of multiple marker enrichment and reveals the abnormality without limitation caused by underlying or overt diseases in each and every individual and moreover, provides a complete insight into the relationship between cancer and its tumor cell shedding behavior on the individual level.
Traditionally, the CD45negative selection assay was marketed already since 2 decades however, was not intended to perform rare cell enrichment as required for liquid biopsy. As a consequence, commercial solutions suffer from extremely high costs due to the enormous amount of target cells being white blood cells that require unforeseen expenditure of costly magnetic particles and respective complementary reagents. Moreover, the achieved enrichment factors may not exceed 2log for reasons of high white blood cell carry over as well as considerable loss of desired rare cells. In the know of these technological flaws and great potential of this assay, a few groups specialized in the development of respective assays that would increase the enrichment factor of the CD45negative selection assay in range of 3 to 4log. One group is the Zborowski Group, Cleveland Clinic that continues to report 3log enrichment with a quadrupole magnetic flow sorter device. Another group is the famed Toner group of Massachusetts General Hospital that has been working on liquid biopsy chip technology ever since its re-introduction in the 1990s and presented a last version being the CTC-iCHIP in 2017 and performing a complete enrichment at 4log within a couple of hours. However, it has been reported that the chip also includes the size exclusion principle in parallel with CD45negative selection technology and it has been admitted that a 30% loss of desired cells may occur in case of small cells. Similar to commercial suppliers, the costs per sample may not be competitive.
DML is SanoLibio’s core technological innovation that allows major improvements with respect to assay time, cost and quality in magnetic cell separation technology. We introduced this term as to describe our invention and represents an active form of incubating magnetic particles with biological material. It should be noted that past innovation in magnetic cell separation technology was largely focused on the magnetic separation unit or apparatus as to increase magnetic strength and thus increase separation efficiency. In other words, in depth investigations on the reaction of cells with magnetic particles during incubation has been neglected. Therefore, conventional methodology sees to incubate biological material with magnetic particles by mixture and resting or occasional agitation. In contrast, DML means to incubate cells and magnetic particles in the presence of changing magnetic fields as to guide particle cell contact, hence the name dynamic magnetic labelling. It has been observed that reaction kinetics are significantly enhanced and consequently reducing incubation times to 2 or 3 minutes (conventional methods require 5min to 2h). Furthermore, it has been observed that non-specific binding is largely reduced so that recovery of desired cells is independent of magnetic particle amount, which then allows highest enrichment. We have in parts realized the potential of this technology in form of our Walderbach Platform and will continue to develop the CD45 negative selection assay as well as other assays based on our platform.
We have proven to ourselves that the assay and the Walderbach works following scientific method and have good reason to believe that any repeat of the experiment will achieve the expected outcomes and keep on validating our system. Admittedly, for the obvious reason of bias, this prove stands little chance of conviction. We are currently in the process of proving feasibility to others in the field firstly having introduced the existence and potential of our technology in 2018 as well as reporting already new findings of rare cells upon application of our technology ((Schreier et al. 2019, reference article: http://atm.amegroups.com/article/view/21928/21464). Admittedly, peer reviewed publications even at higher impact may still cast doubts on technology, if not carried out and validated by independent third parties. Therefore, it is our intend to seek third party validation in 2019 and have initiated respective cooperation with Fraunhofer Institute. Furthermore, we seek complete transparency on this matter and published the SOP of the key enrichment experiment, denoted as MCF7-50 (reference will be uploaded soon). Moreover, we have invited an independent reviewer on site of our Partner Company in Thailand to review the conduct of this experiment, which shall be carried out in beginning May 2019. Also, we invite everybody to our Partner Laboratory in Bangkok to either attend and review or even perform own experimentation with our platform.
SanoLibio has been working in close partnership with Premise Biosystems, Ltd. located in Bangkok to continue the development of our first prototype of the Walderbach Enrichment platform. The technology as well as the method is now under patent application on EU level. Currently, the Walderbach is under scrutiny to validate the CD45 negative selection assay with standardized experiment setup (technical note will be uploaded soon) that will produce data to back up our enrichment goals. The in-house testing has produced several items of improvement for the next generation of the Walderbach Platform (Walderbach II) that is intended to be validated independently by Fraunhofer Institute. For this vital issue as well as the continuation of our project, we urgently seek seed funding. Meanwhile, the R&D in clinical research and development of our diagnostic platform RarmaxTM is continued in Thailand in cooperation with our Partner Premise Biosystems. In January 2019, we have initiated a pilot study to validate diagnostic feasibility of the RarmaxTM assay in cooperation with a local hospital in Bangkok.