Biomarkers

Atheris offers its biomarker discovery, validation and quantification expertise to pharmaceutical and biopharmaceutical companies for prognosis, diagnosis and for the follow-up of a medical treatment (profiling).

In this regard, the identification of specific molecules which are selective, specific and characteristic of a disease state, is crucial to facilitate the diagnosis, the prognosis of the disease’s evolution and the monitoring of the efficiency of therapeutic approaches. Moreover, it is expected that the identification of biomarkers will provide a better comprehension of the disease mechanisms at the molecular level, which may assist in the design of novel therapeutic approaches. The methodology we developed and established at Atheris to discover, validate and follow-up selective and reliable biomarkers is based on an original proprietary label-free peptidomics strategy coupled to our specifically developed statistical tools. It allows fast and efficient identification and follow-up of biomarkers in complex biological mixtures.

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Peptidomics - PPMF

Wind Of Change

Mass spectrometric technologies aiming at the discovery of protein biomarkers have been emerging at a rapid rate over the last two decades. Only recently the focus of proteomic approaches was laid on the low molecular fraction of plasma and new so-called peptidomic techniques were developed for this pool of potential biomarkers.

Our label-free differential approach, which we refer to as PPMF (peptide and protein mass fingerprints) has been developped in order to offer a robust and validated method for biomarker discovery and profiling starting with a small number of samples. Again, our strategy is transversal and complementary to conventional approaches.

While classical proteomics involves reduction-alkylation-proteolysis coupled to 2D-SDS-PAGE, off-gel or 2D-LC separation techniques with MS and MS/MS analysis and automated database matching for protein identification, PPMF involves the following features:

• Due to the rarity of available samples in many cases and to the complexity of the human plasma peptidome, the generation of high quality data sets is one of our primary objectives.
  
  
• We work on intact human or animal matrices (plasma, serum, cerebrospinal fluid, urines, solid tissues, tumors, etc.) with no chemical or enzymatic degradation. This preserves metabolites intact and the sample complexity is kept as low as possible.
  
  
• Our approach is based on a simple pre-analytical preparation procedure prior to fractionation and two-dimensional mass spectrometric (MS) analyses. On-line LC-ESI-MS and off-line MALDI-TOF-MS were found to provide complementary data.
  
  
• Intact PPMF profiles are generated that allow to compare healthy versus control sets of samples, or series of samples from the same patient collected over time to follow-up the PPMF profile.
  
  
• The PPMF technology offers unambiguous detection of small peptides, secondary metabolites and degradation fragments, which is otherwise impossible to achieve.
  
  
• Potent biostatistical tools are used to analyse the thousands of molecular masses detected in each sample, and those that reveal consequent folds of differential expression with relevant p-values are selected as biomarker candidates.
  
  
• The candidate are then isolated and their structure is elucidated using classical biochemical techniques with database matching, which can be coupled to Edman or de novo MS/MS amino acid sequencing in case of subsisting doubts.
  
  

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PPMF Profiling

Furthermore, our approach offers new analytical opportunitites in the frame of a medical treatment or during drug development. Profiling of the intact peptide and protein fingerprints over time for one patient can be achieved. Even if no specific biomarker is followed, this allows to highlight those signals that evolve over time.

This does not only allow to identifiy novel biomarkers, representative of a reaction to a condition. It is also of particular interest to follow-up a medical state, especially with the emerging personalized medicines. We believe that our technology will have major impact to assess the putative sensitivity of individuals to different options for medical treatments.

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For each identified biomarker that is found relevant, specific bioassays are developed, optimized and validated for a reliable, cost- and time-effective follow-up. Since mass spectrometry was used to identify the biomarker, LC-MS and LC-MS/MS methods are usually well suited, and we use our expertise in the field to this end, typically based on FDA standards.

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Validation

The relevance of identified biomarker candidates is first evaluated in silico, to evaluate if the corresponding molecule has a known link to the disease or not yet. Bioassays are then developped and validated (see above). The last step, which often is the most challenging, consists in the validation of the biomarker on large sets of samples, in different biological matrices. The biomarker has to be specific to the disease when compared to healthy samples, but its specificity versus other pathologies (which can sometimes be closely related) and versus different types of medical treatments (a biomarker can be induced by another drug used to treat other problems) remains a major challenge.

Newly discovered biomarkers are then used to develop and validate clinical assays for prognosis, for diagnosis and for the follow-up of medical treatments.

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