Prolias Technology

Major unmet needs in biotechnology include the discovery of new therapeutic proteins and biomarkers of disease, identification of ligands for protein purification, and preparation of complex biological samples for proteomic and discovery analyses. Prolias technology enables all of these processes through use of proprietary small molecule libraries of ligands that allows the simultaneous separation, fractionation, and analysis of essentially all of the components in complex materials, including whole blood.

A major limitation in the study of biological samples is that they usually have a large range in protein concentrations and current techniques are unable to detect the desired trace proteins in the presence of abundant proteins. Recent attempts to overcome the "dynamic concentration range" limitation by immunodepletion of the abundant proteins have proved unsuccessful overall and add dramatically to the already existing high cost of sample preparation. Moreover, trace proteins bound to the abundant proteins may be lost during processing and those remaining are further diluted with buffer, necessitating the introduction of additional concentration steps. Furthermore, the protein and species specificity of the antibodies limits their broad application. Since millions of samples are processed annually for identification of a wide range of different analytes (chemicals, proteins, viruses, bacteria, etc) using diverse analytical formats (mass spectrometry, ELISA, activity, etc.), the solution to this universal problem must be rapid, cost-effective, versatile, robust, and reproducible.

Prolias' ProSpectrum Libraries™

The Prolias family of ProSpectrum Libraries consist of millions of peptide ligands synthesized on microscopic chromatography resin beads. The ligands are synthesized by solid-phase peptide synthesis, which results in millions of beads, each bearing millions of copies of a single ligand, and each bead theoretically different from every other bead. These beads, each of which has a protein binding capacity in the nanomolar range, are incubated with the starting material. The proteins and other components of the starting material, including viruses, bacteria and chemicals bind to individual beads according to the standard laws of affinity interactions.

Since the bound proteins are evaluated instead of the non-bound fractions there is no limit to the sample size that can be processed. Therefore increasing amounts of sample can be added to the library

Prolias uses these libraries and different starting materials in a broad range of applications, including sample preparation, ligand identification (Bead blot), and new therapeutic discovery (FIoNA).

Sample Preparation Using ProSpectrum Libraries™ (PSL-2)

Improving detection of trace proteins

One of the major problems in the discovery of new biomarkers and therapeutic proteins is the difficulty in detecting trace proteins in critical samples such as whole blood due to interferences from highly abundant proteins. Proteins are present in plasma at concentrations that vary from 40 mg/ml (albumin) to less than one pg/ml (cytokines and infectious prions) a difference in concentration of 10 orders of magnitude. Current analytical instruments and methods are unable to penetrate the signal from abundant proteins to detect trace proteins; therefore, samples must be processed prior to analysis. Prolias has developed methods to overcome this concentration range issue, without specifically depleting any proteins.

Prolias libraries can be used for proteomic sample preparation with a number of complex biological materials, including whole blood. The vast number of ligands contained in the libraries allows all of the components of the starting material to bind. The procedure is straightforward: the samples are incubated with the library and bound proteins eluted under a variety of conditions. This method has several advantages:

• Trace proteins are enriched and abundant proteins are depleted, resulting in a collection of proteins that contains most of the proteins in the original sample, but at a concentration range that can be analyzed by standard methods.
• Because the bound proteins are evaluated instead of the non-bound fractions, there is little limit to the sample size that can be applied to the library. This means that increasing amounts of sample can be added to the library until the signal from trace proteins rises above the limits of detection.
• Immobilization of the proteins on the library improves their stability and hence their detection
• Complex mixtures such as whole blood can be incubated with the library immediately upon collection, decreasing artifacts associated with plasma and serum preparation

1. Whole blood before contact showing abundant proteins
2. Unbound whole blood proteins after treatment with ProSpectrum Libraries™
3. Whole blood proteins bound to ProSpectrum Libraries™
• Compare proteins detectable in lanes 1 and 3
4. Molecular weight markers.
5. Plasma before contact showing abundant proteins
6. Unbound plasma proteins after contact with ProSpectrum Libraries™
7. Plasma proteins bound to ProSpectrum Libraries™
• Compare proteins detectable to lane 5 and 7

Improving the sensitivity of diagnostics

Another benefit to processing samples with ProSpectrum Libraries™ is an increase in the sensitivity of diagnostic assays while maintaining a dose-dependant response. This is achieved through both the enrichment of the trace analytes, which increases their signal, and the dilution of abundant proteins, which decreases the signal-to-noise ratio. Together these can improve the sensitivity of commercial assays, as well as allow whole blood to be used as a starting material for these assays. This can be especially useful in providing earlier detection of rising levels of troponin, for example to earlier diagnose myocardial infarction.

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Ligand Identification (Bead blot)

Prolias uses libraries of ligands to identify specific ligands that can be used to purify proteins or as targets in a patented technology called the Bead blot. In this method, ligands are identified to proteins in their natural state, without purification or derivization (diagram of the bead blot). This enables ligands to be identified under conditions in which the ligand will be used in practice, and decreases false positive results. The starting material is incubated with library (A) and the beads are immobilized in a matrix (B). Bound proteins are eluted from the beads by capillary action of a transfer buffer (C2) and captured on a protein-binding membrane (C1). All of the proteins that elute under the particular transfer conditions are captured on the membrane. The location of the protein of interest is detected on the membrane (D); the membrane and gel are aligned (E) and the bead that bound the protein is identified. The bead can be collected, the ligand sequenced and scaled up, and the protein purified on the same chromatography resin backbone as it was originally identified.

Ligands have been identified to a number of targets, including:

• Fibrinogen
• CRP in blood and plasma
• IL-2
• Prion protein - the bead blot was used to identify ligands that remove infectivity from blood. One of these has been developed into a CE-marked class IIB device.
• and more

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Discovery: Functional Identification of Novel Activities (FIoNA)

Prolias uses libraries of peptide ligands for discovery of new, active entities through a technology called Functional Identification of Novel Activities, or FIoNA. In FIoNA, the library is incubated with a starting material, unbound proteins are removed by washing, and the beads with bound proteins are analyzed in disease-relevant biological or biochemical assays, including assays for cell proliferation, cell death, and enzyme activity. All of the proteins are present in the assay, and all are simultaneously analyzed, but only those associated with a desired activity are selected (Figure from business plan of cell-based and enzymatic assay).

Beads associated with a desired activity are collected, the ligand sequenced by MS or Edman degradation, and a specific resin synthesized to purify the active protein. Discovery with FIoNA is unbiased, in that all proteins, regardless of sequence or functional class are present in the assay and therefore eligible for selection. The identity of neither the active protein nor of the ligand to which it binds are known at the outset.

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