OA21368A - Biochemical medical diagnostic kit. - Google Patents

Abstract

The invention relates to the deamidation rate measurement kit, which includes a stock solution of the compound Boc-Asn-Gly-OEt in deionized/distilled water, and a stock solution of the compound Boc-Asp-Gly-OEt in deionized/distilled water.

Description

DESCRIPTION

BIOCHEMICAL MEDICAL DIAGNOSTIC KIT

Technical Field

The invention relates to a kit suitable for biological samples in the field of in vitro diagnostics (biochemical medical diagnosis, etc.) or for use in other fields (pharmacy, chemistry, etc.).

In particular, the invention relates to a kit suitable for use to measure the effect of solutions on the deamidation rate of the proteins they contain.

State of the Art

Proteins are one of the basic structural and functional molécules that make up living organisms. Proteins are large organic compounds that are formed as a resuit of the binding of amino acids to each other in chains. Peptides are short polymers formed by linking aamino acids in a defined order. The bond between one amino acid residue and another is known as an amide bond or peptide bond. Proteins are polypeptide molécules (or contain multiple polypeptide subunits). The main différence is that peptides are short while polypeptides/proteins are long.

Asparagine (Asp, N) and glutamine (Gin, Q), two of the 20 different amino acids in the structure of proteins, hâve amide groups in their side chains. Removal of amide groups in proteins containing these amino acids is called protein deamidation. Protein deamidation can occur enzymatically or spontaneously. As a resuit of deamidation, changes occur in the structure and charge of proteins. As a resuit of deamidation, especially in long-lived proteins found in long-lived cells such as neurons, deamidated proteins may accumulate over time, causing structural and functional disorders in these proteins and therefore in the cells in which these proteins are présent. A great deal of scientific evidence supporting the rôle of protein deamidation in Alzheimer's, frontotemporal dementia and many other chronic diseases has been reported in the literature.

For this reason, it is necessary to détermine the factors or interventions that affect the deamidation rate, to develop strategies that can prevent/slow down deamidation, and to détermine the deamidation rates in individuals. In this way, it is expected that they can make very critical contributions to the prévention and postponmg of agmg and age-related diseases.

Deamidation is an important factor not only in aging, but also in the stability of proteincontaining pharmaceutical drugs (such as vaccines). It is necessary to measure the effect of the solutions in which these drugs are dissolved on the protein deamidation rate, and to make interventions to slow the deamidation rate when added to the solutions. In the state of the art, there are studies on this subject.

United States patent application number US5273886 The invention relates to methods and tools for the quantitative détermination of the isoaspartyl content of polypeptides by sélective méthylation of their fragments catalyzed by a protein L-isoaspartyl methyltransferase enzyme. Since the deamidation of asparagine side chains at certain protein sites and the resulting isoaspartate formation appear to be an important contributor to protein dégradation under mild conditions, the invention also relates to a method for quantifying protein dégradation associated with isoaspartate formation,

Although there are studies on this subject in the state of the art, they only allow to indirectly analyze the components found in the interior of pure proteins. Since it can only be used to measure the deamidation of purified proteins, it cannot be used for biological samples with more complex matrices such as blood plasma. There is still no test method that can be used to measure the effect of biological samples (sérum, plasma, saliva, cerebrospinal fluid, synovial fluid, urine, cell/tissue/organ extracts, etc.) of humans and other living things on the rate of protein deamidation. For this reason, there is a need to develop a test that has the potential to be used in medical biochemistry laboratories, to measure the effect of biological samples on the protein deamidation rate arid to detect interventions that affect the deamidation rate.

As a resuit, due to the negativities described above and the inadequacy of the existing solutions on the subject; It has been necessary to develop kits that measure the deamidation rate.

Aim of the Invention

The présent invention relates to a deamidation rate measurement kit that meets the abovementioned requirements, éliminâtes ail disadvantages and brings some additional advantages.

The primary aim of the invention is to develop a kit that measures the effect on the deamidation rate when added to solutions containing protein pharmaceuticals.

One aim of the invention is to develop a measurement kit that enables to détermine the factors affecting the deamidation rate, to examine the corrélation of individual différences in deamidation rate with aging and age-related diseases, and to detect diseases characterized by an increase in deamidation rate.

To fulfill the above-described purposes, the invention is a deamidation rate measurement kit containing a stock solution in deionized/distilled water containing the compound Boc-AsnGly-OEt and a stock solution in deionized/distilled water containing the compound Boc-AspGly-OEt.

The structural and characteristic features of the invention and ali its advantages will be understood more clearly thanks to the detailed explanation written below, and therefore the évaluation should be made by taking this detailed explanation into considération.

Figures to Help Explain the Invention

Figure 1: Structure of the Boc-Asn-GIy-OEt compound

Explanation of References

Boc-

2 Asn

Gly

-Oet

Région being deamidated

Detailed Explanation of the Invention

In this detailed explanation, the deamidation rate measurement kit, which is the subject of the invention, is explained only for a better understanding of the subject and without causing any limiting effect.

The deamidation rate measurement kit, which is the subject of the invention, in its most basic form; contains stock solution in deionized/distilled water containing the compound Boc-Asn21368

Gly-OEt (preferably in the range of 0.000001 - 1000 mM, more preferably at the concentration of 0.25 mM) and stock solution in deionized/distilled water containing the compound Boc-Asp-Gly-OEt (preferably 0.000001 - 1000 mM) mM, more preferably at a concentration of 0.25 mM).

The Boc-Asn-Gly-OEt compound, a sample view of which is given in Figure 1, is used both as a calibrator and analytical standard in measurements, and as a deamidated element by being added to biological samples. Boc- (1) and -Oet (4) are for blocking Asparagine (Asn) (2) and glycine (Gly) (3) dipeptides respectively, as details shown in the figure. In the 10 deamidated région (5) shown in the figure, the “NH2” structure is removed by deamidation, while the OH” structure is attached instead. Thus, the Boc-Asn-Gly-OEt compound turns into the Boc-Asp-Gly-OEt compound and its molecular weight increases by about 1 (0.98) g/mol. Boc- (1) and -OEt (4) structures -in the structure of the Boc-Asn-Gly-OEt compound in solution- allow to block the binding of Asn (2) and Gly (3) dipeptides with unwanted Chemical 15 groups during the process. The Asn (2) and Gly (3) dipeptides are one of the structures with the fastest spontaneous deamidation known. For this reason, it is used in measurements as as the calibrator/analytical standard or the compound subject to deamidation after adding to biological samples. Since Boc-Asn-Gly-OEt is converted into Boc-Asp-Gly-OEt compound as a resuit of spontaneous deamidation, Boc-Asp-Gly-OEt compound is used as Calibrator and analytical standard in drawing the calibration curve. The Boc-Asp-Gly-OEt compound is not commercially available but has been specially synthesized. Deionized/distilled water allows to dissolve Boc-Asn-Gly-OEt and Boc-Asp-Gly-OEt compounds and is used as analytical blank.

In a sample application of the invention, the deamidation rate measurement kit developed includes the following steps of process: Préparation of stock solution and serial dilutions of Boc-Asn-Gly-OEt compound in deionized/distilled water; Préparation of stock solution and serial dilutions of Boc-Asp-Gly-OEt compound in deionized/distilled water; Création of the calibration curve on the instrument to be measured using serial dilutions of the Boc-Asn-Gly-

OEt stock solution; Création of the calibration curve on the instrument to be measured using serial dilutions of the Boc-Asp-Gly-OEt stock solution; aliquoting the biological sample whose effect on the deamidation rate will be tested in equal volumes in three different tubes; addition - preferably in the range of 1/1000 - 1/2 of the final reaction volume (more preferably in the range of 1/10) - of Boc-Asn-Gly-OEt and Boc-Asp-Gly-OEt (as an internai standard) 35 stock solutions to different tubes, and of deionized/distilled water to another tube as blank; at the beginning of the measurement, making measurements from ail three tubes, preferably by LC-MS method; after incubation making measurements of ail three tubes, preferably by LC-

MS method; subtraction of the measurement results m the tubes with added Boc-Asn-GlyOEt and Boc-Asp-Gly-OEt from the measurement in the tube with deionized/distilled water; détermination of deamidation rate by measuring Boc-Asp-Gly-OEt resulting from spontaneous deamidation in the tube with added Boc-Asn-Gly-OEt.

The présent invention is based on the principle of spontaneous deamidation of Boc-Asn-GlyOEt compound in solution into Boc-Asp-Gly-OEt compound and measuring the resulting BocAsp-Gly-OEt compound by an appropriate analytical method. Boc-Asn-Gly-OEt solution is added to any sample whose effect on the spontaneous deamidation rate is desired to be measured. Spontaneous deamidation of Boc-Asn-Gly-OEt in this mixture, which is kept at a température between 1-100 °C for 1 minute - 1 month, is ensured. During the incubation period, as a resuit of the deamidation of the Boc-Asn-Gly-OEt dipeptide in the mixture, the — NH2 group [Figure 1:(5)] will spontaneously be released from the molécule, while the asparagine in the compound will be converted to isoaspartate or aspartate with the addition of the -OH group, and a molecular weight increase of approximately 1 (0.98) g/mol mass will occur. This change in mass, that is, the resulting Boc-Asp-Gly-OEt and Boc-lsoasp-Gly-OEt can be measured by mass spectrometry, as well as with ail kinds of optimized methods based on the different Chemical properties of the -NH2 group in the asparagine of the compound and the -OH group of the newly formed aspartate in the compound.

By measuring the effect of the biological samples from individuals/patients on the deamidation rate by using the deamidation rate measurement test, which is the subject of the présent invention, and by examining the corrélation of individual différences -to be detected and that affect the deamidation rate- with aging and age-related diseases, and if diseases characterized by an increase in deamidation rate are detected, the test is provided to be used as a marker to détermine the susceptibility to these diseases and for their early diagnosis.

Claims (8)

1. It is a deamidation rate measurement kit, and its feature is; it contains a stock solution in deionized/distilled water containing Boc-Asn-Gly-OEt compound and a stock solution

5 in deionized/distilled water containing Boc-Asp-Gly-OEt compound.

2. It is a kit according to claim 1 and its feature is; the stock solution in deionized/distilled water containing the Boc-Asn-Gly-OEt compound in the concentration range of 0.000001 -1000 mM.

3. It is a kit in accordance with claim 2 and its feature is; its concentration is 0.25 mM.

10

4. It is a kit according to claim 1 and its feature is; it is a stock solution in deionized/distilled . water containing Boc-Asp-Gly-OEt compound in the concentration range of 0.000001 1000 mM.

5. It is a kit in accordance with claim 4 and its feature is; its concentration is 0.25 mM.

6. It is a rate measurement method made with a deamidation rate measurement kit in 15 accordance with any of the daims 1 to 5, and its feature is;

- Préparation of stock solution and serial dilutions of Boc-Asn-Gly-OEt compound in deionized/distilled water;

- Préparation of stock solution and serial dilutions of Boc-Asp-Gly-OEt compound in 20 deionized/distilled water;

- Création of the calibration curve on the instrument to be measured using serial dilutions of the Boc-Asn-Gly-OEt stock solution;

- Création of the calibration curve on the instrument to be measured using serial dilutions of the Boc-Asp-Gly-OEt stock solution;

25 - Aliquoting the biological sample whose effect on the deamidation rate will be tested in equal volumes in three different tubes;

- Adding Boc-Asn-Gly-OEt stock solution to one of the sample tubes, Boc-Asp-Gly-OEt (as an internai standard) stock solution to the other, and deionized/distilled water to the last one as the blank, in the range of 1/1000 - 1/2 of the final reaction volume;

30 - Making measurements from ail three tubes at the beginning of the measurement;

- Measurement of ail three tubes after incubation;;

- Subtracting the measurement results in the tubes with added Boc-Asn-Gly-OEt and Boc-Asp-Gly-OEt from the measurement in the tube with added deionized/distilled water;

- Détermination of the deamidation rate by measuring Boc-Asp-Gly-OEt, which is 5 formed as a resuit of spontaneous deamidation in the tube with added Boc-Asn-Gly-

OEt,

7. It is a method according to claim 6 and its feature is; the measurements are made by LC-MS method.

8. It is a method according to claim 6 and its feature is; The reaction volume is 1/10.