Method for determining the amount of the damaged form of the protein

Abstract

The method for determining damaged forms of proteins based on the adsorption onto hydrophobic microparticles constitutes a methodology for the quantitative and qualitative determination of damaged therapeutic proteins with consequent implications for the development and validation of protein-based medications. The methodology facilitates the quantitative determination of an amount of a damaged / inactive form of proteins in the presence of active / functional forms. The determination of damaged protein forms requires an amount of a specimen in the order of magnitude 10-6 grams; this facilitates analysing various external conditions suitable for the storage of protein-based medications. The system comprises uncharged hydrophobic microparticles to which proteins bind through passive adsorption, depending on their conformation (active and damaged forms). The adsorption mechanism is primarily based on hydrophobic interactions between the hydrophobic fraction of the adsorbed protein and the hydrophobic surface of the microparticles. Damage to a native form of a protein causes the exposure of hydrophobic protein fractions on the surface. Compared to a native protein, such a damaged form binds to microparticles at an increased rate, and based on this difference it is possible to determine concentration of both these forms side by side.

Description

[0001] Method for determining the amount of the damaged form of the protein

[0002] Field of the invention

[0003] The invention relates to the qualitative and subsequently quantitative determination of the amount of damaged form of proteins in the presence of their active / functional forms, which may be applied in the development and validation of protein-based therapeutics.

[0004] Background of the invention

[0005] Current therapies are based on intensive use of monoclonal antibody-based (mAbs) therapeutic protein molecules. The complexity of the mAb structure leads to instability and subsequently protein degradation. Factors affecting protein instability have 2 different key mechanisms - chemical and physical. Unlike low molecular weight medications, proteins are extremely sensitive to the external environment. At relatively mild exposure to elevated temperature, loss of ordered structure occurs due to partial or complete unfolding of proteins. This leads to the formation of a damaged antibody form, which has a direct effect on the antibody's therapeutic function. Therefore, factors leading to protein damage should be eliminated as much as possible when storing drugs; however, prolonged storage or a required formulation of a medication (subcutaneous administration of antibodies requires solutions with a higher concentration) may lead to the increased formation of damaged forms of antibodies. The presence of damaged forms is intensively monitored since such forms may induce a protective - immune - response in a patient, leading to the elimination of not only the damaged but also the native forms of proteins. Such a response of an organism may subsequently cause disappearance of the medication therapeutic effect; in an extreme case, it may also lead to an exaggerated allergic reaction of the organism called anaphylaxis. The above clearly indicates that the determining the presence and amount of damaged forms of therapeutic proteins is vital for the safe application of medication. At present, pharmaceutical companies determine the presence of damaged forms of proteins using gel filtration and light scattering. The purpose of the invention is to disclose a method for determining the amount of the damaged form of a protein in a tested protein sample through passive adsorption onto the surface of hydrophobic uncharged particles.

[0006] Summary of the invention

[0007] The invention relates to a procedure for determining an amount of a damaged form of a protein in a sample. The method comprises passive adsorption of proteins onto the surface of hydrophobic uncharged microparticles which depends on a conformational state / form of a protein - the presence of a damaged or a native form.

[0008] The invention discloses a method for determining an amount of the damaged form of a therapeutic protein in a specimen; the method comprises the following steps:

[0009] - addition of hydrophobic microparticles at a concentration of 0.1 to 2% v / v to a test sample of a solution of at least one protein;

[0010] - incubation of the test protein together with hydrophobic microparticles at a temperature of 20 to 30°C, more preferably at a temperature of 20 to 28°C, and most preferably at a temperature of 25°C with continuous stirring for a minimum of 60 minutes in order to facilitate passive adsorption of proteins onto the surface of hydrophobic microparticles;

[0011] - separation of hydrophobic microparticles with the bound proteins having an altered form;

[0012] - determining an amount of the bound proteins.

[0013] The test sample is a sample of a therapeutic protein which may be an antibody or an enzyme and in which an amount of a damaged from of a protein is to be determined.

[0014] The invention also discloses a method for the removal of the damaged form of a therapeutic protein in a sample; the method comprises the following steps:

[0015] - addition of hydrophobic microparticles at a concentration of 0.1 to 2% v / v to a test sample of a solution of at least one protein; - incubation of the tested protein together with hydrophobic microparticles at a temperature of 20 to 30°C, more preferably at a temperature of 20 to 28°C, and most preferably at a temperature of 25°C with continuous stirring for a minimum of 60 minutes in order to facilitate passive adsorption of proteins having a damaged form onto the surface of hydrophobic microparticles;

[0016] - separation of hydrophobic microparticles with the bound proteins.

[0017] According to a preferred embodiment, hydrophobic microparticles are polystyrene microparticles, polypropylene microparticles, polyethylene microparticles, polycarbonate microparticles, fluoropolymer microparticles, silicon microparticles or carbon microparticles, most preferably polystyrene microparticles.

[0018] Diameter of hydrophobic particles ranges from 0.1 to 40 pm, preferably 1 to 10 pm.

[0019] Hydrophobic particles may have a round (spheroid) or oval shape.

[0020] Hydrophobic particles should be chemically inert; that is they should not chemically react with the tested therapeutic proteins.

[0021] Hydrophobic microparticles should be uncharged (without an electrostatic charge which would reduce the hydrophobic nature of the microparticles).

[0022] According to another preferred embodiment, a concentration of a tested therapeutic protein ranges from 0.05 to 1 mg / ml, depending on the protein type. The type of a therapeutic protein is determined by its molecular weight. A molecular weight of a protein used determines its concentration in a specimen. For lower molecular weight proteins, a lower protein concentration is applied; for proteins with a higher molecular weight, a higher protein concentration is applied.

[0023] The separation step may be carried out through centrifugation, chromatography, electrophoresis or sedimentation. The determination of an amount of the bound proteins may be carried out using spectrophotometric methods, chromatography or gravimetry; subsequently, the amount of bound proteins is determined as the difference between the baseline protein concentration in the sample and a protein concentration after the incubation with microparticles in the sample.

[0024] Native form therapeutic proteins bear on their surface predominantly hydrophilic amino acids. Denaturation of proteins causes damage to their native form - exposure of hydrophobic protein fractions to the surrounding environment; among other effects, the protein becomes inactive. The modification of the physical nature of the protein surface may be used to detect an amount of the damaged protein form in the sample. While native proteins tend to interact with hydrophilic surfaces, damaged protein molecules prefer interactions of the exposed hydrophobic fractions with hydrophobic surfaces. Following the incubation with hydrophobic polystyrene microparticles, the amount of unbound protein in the sample is determined by spectrophotometric methods.

[0025] The adsorption mechanism is primarily based on hydrophobic interactions between the hydrophobic fraction of the adsorbed protein and the hydrophobic surface of the microparticles. Damage to a native protein form causes the exposure of hydrophobic protein fractions on the surface. Compared to a native protein, such a damaged form binds to microparticles at an increased rate, and based on this difference it is possible to determine concentration of both these forms side by side.

[0026] A procedure for the determination of damaged forms of therapeutic proteins, such as antibodies or enzymes, by means of adsorption onto hydrophobic microparticles, constitutes a method for the quantitative and qualitative determination of damaged therapeutic proteins with consequent implications for the development and validation of protein-based medications.

[0027] The method may be applied to a wide range of proteins. It is particularly useful in the screening, development and validation of therapeutic antibodies where it is required to test a large number of factors and various mAb candidates. This method facilitates the identification of a candidate with increased adsorption onto a surface, allowing detection of damaged proteins. Due to a low consumption of the used material and sample, this procedure is economical, cost-efficient and technically easy to implement.

[0028] In a preferred embodiment, this method may also be used to remove the damaged (i.e. unsuitable) proteins from a tested specimen.

[0029] Compared to the currently used methods, the adsorption onto hydrophobic surfaces of microparticles facilitates the quantification of the amount of inactive forms of proteins in the presence of active forms using only a small amount of the sample (in the order of IO’5grams). The simplicity of this method facilitates the cheap and fast determination of damaged forms as early as in the first stage of the damaging process - protein unfolding. The currently applied methods (gel filtration and light scattering) can only monitor the subsequent stages in which unfolded proteins begin to accumulate and form dimers or even oligomers.

[0030] Brief description of the drawings

[0031] Fig. 1 contains graphs showing amounts of molecules of different proteins (A - HSA protein; B - Rituximab protein; C - Trastuzumab protein) bound to 1 microparticle in a native form (the dark-grey bar) and in a damaged form (the light-grey bar).

[0032] Fig. 2 shows a visual projection of microparticles after binding native forms and heatdamaged forms of a fluorescent-labelled HSA protein (A - native HSA form; B - heat- da maged HSA form).

[0033] Fig. 3 shows the reproducibility of the method - repeated measurements of the amount of HSA molecules in a native form (dark-grey bars) and a damaged form (light-grey bars) bound to 1 polystyrene microparticle.

[0034] Fig. 4 shows the absorbance at 630 nm for the specimens of HSA solutions with various ratios of native vs. damaged protein forms before the incubation with microparticles (dots) and after the incubation with microparticles (squares). Fig. 5 shows a difference in absorbance before and after binding of HSA molecules to microparticles for sample solutions with various ratios of a native vs. damaged protein forms.

[0035] Fig. 6 shows the calibration line for the determining the percentage of a damaged form of HSA protein relative to an amount of HSA bound to microparticles.

[0036] Examples of embodiment

[0037] Example 1

[0038] Passive adsorption of a native protein onto a hydrophobic surface of polystyrene microparticles with a diameter of 1 pm is shown on the HSA (human serum albumin) protein and therapeutic antibodies - Rituximab and Trastuzumab. Proteins were prepared in phosphate-buffered saline (PBS) with a pH value adjusted to 7.4. The concentration of the prepared proteins was 0.25 mg / ml. Polystyrene microparticles were added to the protein solutions in such an amount that the resulting solution contained 1% of microparticles. Before addition, the microparticles were vortexed for 10 minutes. Protein solutions were incubated with microparticles for 60 minutes at a temperature of 25°C with continuous stirring. After the incubation, the solutions were centrifuged at 20,000 x g and 25°C for 20 minutes.

[0039] The concentrations before and after binding of HSA and antibodies to microparticles were analysed by applying the Bradford assay. The amount of the bound native protein was calculated as a difference between the baseline concentration and the protein concentration in solutions after the incubation with microparticles. After a conversion to 1 microparticle, 2,056,700 molecules of native HSA, 670,203 molecules of native Rituximab and 917,534 molecules of native Trastuzumab were bound, as shown in Fig. 1A-1C.

[0040] Example 2

[0041] Solutions of native HSA proteins and therapeutic antibodies - Rituximab and Trastuzumab - at a concentration of 0.25 mg / ml (PBS; pH 7.4) were heated in a thermoblock for 5 minutes at a temperature of 95°C to prepare solutions of proteins with a damaged form. Afterwards they were placed on ice. Polystyrene microparticles were added to the solutions in such quantity that the resulting solution contained 1% of microparticles. Before addition, the microparticles were vortexed for 10 minutes. Protein solutions were incubated with microparticles for 60 minutes at a temperature of 25°C with continuous stirring. After the incubation, the solutions were centrifuged at 20,000 x g and 25°C for 20 minutes. The concentrations before and after binding of HSA and antibodies to microparticles were analysed by applying the Bradford assay. The amount of the bound damaged protein was calculated as a difference between the baseline protein concentration and the concentration after the incubation with microparticles. After conversion to 1 microparticle, 3,713,140 molecules of damaged HSA, 1,946,455 molecules of Rituximab and 2,177,136 molecules of Trastuzumab were bound, as shown in Fig. 1.

[0042] In laboratory assays, this method showed higher adsorption of damaged proteins onto hydrophobic surfaces of hydrophobic microparticles when compared to that of a native protein, as shown in Fig. 1.

[0043] Example 3

[0044] A visual representation of a difference in the adsorption of native and damaged forms is shown in Fig. 2. The HSA protein solution (PBS; pH 7.4) was incubated with a 15- fold excess concentration of chromophore - Abberior STAR ORANGE (NHS ester) for 2 hours, and the staining reaction was stopped by adding hydroxylamine. Unbound chromophore was removed from the solution using a PD10 column (Sephadex G-25), and a concentration of the protein with bound chromophore was determined using the Bradford reagent. A HSA protein sample with a concentration of 0.15 mg / ml was heated in the thermoblock for 5 minutes at a temperature of 95°C to produce protein in the damaged form. Polystyrene microparticles were added to the native protein solution and the damaged protein solution in such quantity that the resulting solution contained 1% of microparticles. Before addition, the microparticles were vortexed for 10 minutes. Protein solutions were incubated with microparticles for 60 minutes at a temperature of 25°C with continuous stirring. Microparticles with bound native / heat- damaged protein were visualised using a STED microscope (STEDYCON, Abberior Instruments) - an excitation laser 561 nm, a STED laser 775 nm and a detector 616 nm. In the presence of heat-damaged forms of a fluorescently labelled HSA protein, it was possible to observe microparticles with higher signal intensity and brighter contours compared to those of microparticles incubated with native HSA; this was caused by a larger amount of protein with a damaged form having bound to the hydrophobic surface of microparticles.

[0045] Example 4

[0046] The reproducibility of this method was analysed by 20 independent measurements of passive adsorption of a native and a heat-damaged HSA protein onto the hydrophobic surface of polystyrene microparticles. After achieving the maximum saturation of the surface of microparticles with protein (incubation for 60 minutes at a temperature of 25°C), 1,747,579 ± 299,268 molecules of native HSA (standard deviation of a data set: o = 357,429; standard error of the mean: 79,923.5) and approximately a 5-times larger amount of molecules (9,410,327 ± 451,858) of heat-damaged HSA (standard deviation of a data set: o = 539,965, standard error of the mean (SEM): 120,740) were bound to 1 microparticle, as shown in Fig. 3.

[0047] Example 5

[0048] For the purpose of the quantitative determination of an amount of a damaged protein in a sample, the prepared samples of native and damaged HSA were mixed at various ratios (native:damaged 10:0; 9:1; 1:1; 1:9; 0:10). Polystyrene microparticles were added to the protein solutions in such quantity that the resulting solution contained 1% of PS microparticles. Before addition, the microparticles were vortexed for 10 minutes. The protein solution was incubated with microparticles for 60 minutes at a temperature of 25°C under continuous stirring. After the incubation, the solution was centrifuged at 20,000 x g and 25°C for 20 minutes. The concentrations before and after HSA bound to microparticles, as shown in Fig. 4, were analysed by applying the Bradford assay. The amounts of the bound protein for the individual solutions with various ratios of the amounts of HSA in native / damaged forms was calculated as a difference between the baseline protein concentration and the HSA concentration in the solution after the incubation with microparticles, as shown in Fig. 5. The measured data for the amount of HSA bound to microparticles for the individual ratios were used to construct a calibration line representing the amounts of the bound HSA for the individual ratios of native to damaged forms of protein and a percentage of a damaged form of HSA, Fig. 6. The correlation coefficient of the linear calibration curve was 0.999.

[0049] Industrial applicability

[0050] The present invention may be applied in the pharmaceutical industry, with implications for the development and validation of protein-based therapeutics.

Claims

Claims1. A method for determining an amount of a damaged therapeutic protein form in a sample, characterized in that the method comprises the following steps:- addition of hydrophobic microparticles at a concentration of 0.1 to 2% v / v to the test sample of a solution of at least one protein;- incubation of the test protein together with the hydrophobic microparticles at a temperature of 20 to 30°C, more preferably at a temperature of 20 to 28°C, and most preferably at a temperature of 25°C with continuous stirring for a minimum of 60 minutes, in order to facilitate passive adsorption of proteins having a damaged form onto the surface of the hydrophobic microparticles;- separation of the hydrophobic microparticles bearing the bound proteins;- determining an amount of the bound proteins.

2. A method for removing a damaged form of a therapeutic protein in a sample, characterized in that the method comprises the following steps:- addition of hydrophobic microparticles at a concentration of 0.1 to 2% v / v to the test sample of a solution of at least one protein;- incubation of the test protein together with the hydrophobic microparticles at a temperature of 20 to 30°C, more preferably at a temperature of 20 to 28°C, and most preferably at a temperature of 25°C with continuous stirring for a minimum of 60 minutes, in order to facilitate passive adsorption of proteins having a damaged form onto the surface of the hydrophobic microparticles;- separation of the hydrophobic microparticles with the bound proteins.

3. The method according to claim 1 or 2, characterized in that the hydrophobic microparticles are polystyrene microparticles, polypropylene particles, polyethylene particles, polycarbonate microparticles, fluoropolymer microparticles, silicon microparticles or carbon microparticles, preferably polystyrene microparticles.

4. The method according to claim 1, 2 or 3, characterized in that a diameter of the hydrophobic particles ranges from 0.1 to 40 pm, preferably from 1 to 10 pm.

5. The method according to any of the preceding claims, characterized in that a concentration of the test protein ranges from 0.05 to 1 mg / ml, depending on the protein type.

6. The method of claim 1 or claim 2, characterized in that the separation step is carried out through centrifugation, chromatography, electrophoresis or sedimentation.

7. The method according to claim 1, characterized in that the step of determination of an amount of the bound proteins is carried out using spectrophotometric methods, chromatography or gravimetry, and subsequently the amount of the bound proteins is determined as a difference between the baseline protein concentration in the sample and the protein concentration in the sample after the incubation with microparticles.

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