A solution container having a coating and a method of making the coating

Abstract

The present application belongs to the technical field of solution container, disclose a kind of solution container with coating, comprising: container main body, for containing preparation;Hydrophobic coating, set in the inner wall of the container main body, for inhibiting protein aggregation, protein adsorbed in hydrophobic coating is difficult to desorb so that the insoluble microparticle in preparation is reduced.The present application utilizes the strong hydrophobic interaction between protein and hydrophobic surface, and protein adsorbed on the hydrophobic surface is difficult to desorb into solution, thereby inhibiting the aggregation of protein, and to a certain extent, reducing the generation of insoluble microparticle in preparation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of solution containers, and particularly relates to a solution container with a coating and a preparation method of the coating. BACKGROUND

[0002] Protein drugs can be divided into polypeptides and genetically engineered drugs, monoclonal antibodies and genetically engineered antibodies, and recombinant vaccines. Compared with previous small molecule drugs, protein drugs have the characteristics of high activity, strong specificity, low toxicity, clear biological function, and being conducive to clinical application. Due to low cost, high success rate, and safety and reliability, protein drugs have become an important part of pharmaceutical products.

[0003] However, proteins are biological macromolecules, which are extremely susceptible to external factors and can change the spatial conformation, resulting in protein aggregates and insoluble microparticles. Protein aggregation is usually achieved through a series of processes. First, the change in the internal structure of the protein leads to the formation of dimers or oligomers, and then the aggregates grow, and finally sub-visible or visible particles are formed. Their formation not only affects the efficacy of protein drugs, but also can cause serious adverse reactions. For example, in intravenous infusion, larger insoluble microparticles can cause blood vessel occlusion, granuloma, pulmonary hypertension, allergic-like and heat source-like reactions, and other serious adverse reactions. Marisa K. Joubert research shows that the aggregates of monoclonal antibodies prepared by stirring can induce immune responses by stimulating TLR-2 and TLR-4 receptors to induce the secretion of cytokines.

[0004] Proteins are biological molecules composed of amino acid subunits. Each amino acid has a side chain that increases or decreases charge depending on the pH of the surrounding environment and its own polarity / non-polarity properties. The charged regions can greatly facilitate the interaction of proteins with other molecules and surfaces, as well as their own tertiary structure (protein folding). Due to their hydrophilicity, charged amino acids tend to be located on the outside of the protein, where they can interact with surfaces. In terms of surface chemistry, protein adsorption is a key phenomenon that describes the aggregation of these molecules on the outside of materials. The ability of proteins to remain attached to the surface depends largely on material properties such as surface energy, texture, and relative charge distribution.

[0005] Medium borosilicate glass tubes for injection vials are relatively common antibody formulation storage containers, and protein adsorption on the surface of glass is a common phenomenon. When proteins come into contact with the surface of the inner packaging material, they are easily adsorbed, which can change the spatial conformation of the proteins and produce protein aggregates.

[0006] Therefore, effectively controlling and reducing the production of protein aggregates and insoluble microparticles is a very important link in the production and management of pharmaceuticals. Therefore, a new formulation container that can inhibit protein aggregation is needed. SUMMARY

[0007] The technical problem solved by the present application is that the currently used preparation storage container, when the protein contacts the surface of the inner packaging material, the protein is easily adsorbed to change the spatial conformation of the protein, resulting in protein aggregates, therefore, a new preparation container is needed, which has a coating inside to inhibit protein aggregation, which can effectively prevent protein aggregation and reduce the generation of insoluble particles.

[0008] The present application is a solution container with a coating, comprising:

[0009] A container body for containing the preparation;

[0010] A hydrophobic coating arranged on the inner wall of the container body for inhibiting protein aggregation, the protein adsorbed on the hydrophobic coating is difficult to desorb to reduce the insoluble particles in the preparation.

[0011] The present application utilizes the strong hydrophobic interaction between the protein and the hydrophobic surface, the protein adsorbed on the hydrophobic surface is difficult to desorb into the solution, thereby inhibiting the aggregation of the protein and reducing the generation of insoluble particles in the preparation to a certain extent.

[0012] In some embodiments of the present application, the hydrophobic coating is an octadecyltrichlorosilane coating (OTS), the protein adsorbed on the surface of the hydrophobic coating is difficult to desorb into the preparation solution, thereby reducing the insoluble particles in the preparation. During transportation, the denatured protein adsorbed on the hydrophobic surface is difficult to desorb, the hydrophobic surface can resist the generation of protein aggregates induced by fluid shear force to a certain extent, thereby greatly reducing the insoluble particles in the preparation.

[0013] In some embodiments of the present application, the container body is a bottle.

[0014] In some embodiments of the present application, the bottle includes a bottle cap, which is sealingly connected with the bottle and can be detached. During use, the bottle cap can be detached and installed to facilitate the loading or removal of the preparation solution into or from the bottle.

[0015] As a preferred, the bottle is a regular bottle structure. It can contain an accurate amount of preparation and avoid the residue of the preparation solution.

[0016] As a preferred, the bottle is a cylindrical bottle. Such a bottle has no edges and corners and is not easy to leave liquid medicine.

[0017] Alternatively, the bottle is a rectangular bottle with smooth edges and corners. Such a bottle has a regular shape and smooth edges and corners and is easy to take.

[0018] As a preferred, the bottle is a glass bottle.

[0019] In some embodiments of the present application, the hydrophobic coating is evenly distributed on the bottom and side surfaces of the interior of the bottle, so that the inner surface of the bottle is entirely covered by the hydrophobic coating. This embodiment allows the hydrophobic coating to cover the inner surface of the bottle, so that the formulation solution in the bottle directly contacts the hydrophobic coating, and the protein adsorbed on the hydrophobic coating is not easily desorbed, and the protein aggregates will not fall into the formulation solution, thus avoiding the generation of insoluble particles.

[0020] In other embodiments of the present application, the hydrophobic coating is distributed on part of the inner surface of the interior of the bottle. In actual production, the hydrophobic coating can be arranged at a suitable position according to requirements.

[0021] Preferably, the hydrophobic coating is distributed on the side surface of the interior of the bottle at intervals to reduce costs.

[0022] Alternatively, the hydrophobic coating is evenly distributed on the bottom of the interior of the bottle and is distributed on the inner surface of the bottle at intervals.

[0023] Alternatively, the hydrophobic coating is evenly distributed on the bottom and side surface of the interior of the bottle at intervals.

[0024] The present application also discloses a method for arranging the hydrophobic coating on the inner surface of the bottle, comprising the following steps:

[0025] S1, configuring an oxidant solution, treating the interior of a glass bottle with the oxidant solution to obtain a glass bottle with a hydroxylated inner surface;

[0026] S2, preparing an octadecyltrichlorosilane solution, pouring the prepared solution into the glass bottle with the hydroxylated inner surface, and reacting at room temperature to obtain a glass bottle with an octadecyltrichlorosilane coating on the inner surface.

[0027] Preferably, the oxidant solution is a piranha solution.

[0028] Preferably, the piranha solution is prepared in a ratio of H2SO4:H2O2=3:1 (V / V).

[0029] Preferably, the glass bottle is filled with the piranha solution and placed in a 120°C oil bath for 3h; after the reaction is completed, the piranha solution in the glass bottle is poured out, the glass bottle is washed with a washing liquid, and is naturally dried.

[0030] Preferably, the washing liquid is deionized water, the glass bottle is washed with a large amount of deionized water, washed with anhydrous ethanol filtered by a 0.22μm polytetrafluoroethylene (PTFE) filter membrane, and naturally dried at 25°C to enrich the hydroxyl groups on the inner surface of the glass bottle.

[0031] As preferred, the octadecyltrichlorosilane solution of 0.6% (V / V) is prepared with toluene, the octadecyltrichlorosilane solution is reacted with the inner surface of the glass bottle in which hydroxylation occurs at room temperature for 12 hours, and then the glass bottle is cleaned with toluene, ethanol and deionized water respectively by ultrasonic cleaning for 30 minutes, and then the glass bottle is washed with filtered anhydrous ethanol, and then the glass bottle is naturally dried at 24°C to obtain the glass bottle with octadecyltrichlorosilane coating on the inner surface.

[0032] Advantages of the present application:

[0033] (1) By providing the octadecyltrichlorosilane coating on the inner surface of the glass bottle, the hydrophobicity of the inner surface of the glass bottle is enhanced, the proteins adsorbed on the coating are not easy to fall off, and the protein aggregates and insoluble particles in the preparation solution in the glass bottle are reduced, thereby reducing the risk of adverse reactions during drug use.

[0034] (2) The method for making octadecyltrichlorosilane coating in the glass bottle is simple, and pharmaceutical enterprises, laboratories and the like can easily make hydrophobic coating by using the method to solve the problem of protein aggregation in the production and management of drugs. BRIEF DESCRIPTION OF DRAWINGS

[0035] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings of the embodiments will be briefly introduced below. Obviously, the drawings described below only relate to some embodiments of the present application, but not limit the present application. Other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0036] Figure 1 The cross-sectional view of the present application shows the octadecyltrichlorosilane coating on the inner surface;

[0037] Figure 2 The schematic diagram of the overall structure of the bottle body of the present application;

[0038] Figure 3 The contact angle instrument detection results of the present application, (a) untreated glass bottle inner surface, (b) glass bottle inner surface treated with Otter solution, (c) OTS treated glass bottle inner surface;

[0039] Figure 4 The ATR-FTIR spectrum of the inner surface of the glass bottle of the present application, (a) untreated glass bottle inner surface; (b) OTS treated glass bottle inner surface;

[0040] Figure 5 The three-dimensional surface topography of the atomic force microscope of the present application, (a) untreated glass bottle inner surface; (b) OTS treated glass bottle inner surface;

[0041] Figure 6Langmuir adsorption model of IgG2 on the inner surface of glass bottle in the present application, (a) inner surface of untreated glass bottle; (b) inner surface of OTS treated glass bottle;

[0042] Figure 7 Protein aggregate produced by IgG2 subjected to mechanical stress treatment in untreated and OTS treated glass bottles in the present application, (a) sub-visible particle concentration; (b) average diameter of sub-visible particles; (c) Bis-ANS fluorescence increase;

[0043] Figure 8 Particle size distribution of sub-visible particles produced by monoclonal antibody solution subjected to mechanical stress treatment in the present application, (a) untreated glass bottle; (b) OTS treated glass bottle;

[0044] Figure 9 Morphology of sub-visible particles produced by monoclonal antibody solution subjected to mechanical stress treatment in untreated and OTS treated glass bottles in the present application, (a) in untreated glass bottle, (b) in OTS treated glass bottle;

[0045] Figure 10 Contour plot between particle size and roundness of IgG2 sub-visible particles in the present application, (a) untreated glass bottle; (b) OTS treated glass bottle; Contour plot between particle size and intensity of IgG2 sub-visible particles in the present application, (c) untreated glass bottle; (d) OTS treated glass bottle;

[0046] Figure 11 SE-HPLC profile of soluble IgG2 in the present application, (a) unstressed IgG2, (b) stressed IgG2 in untreated glass bottle, (c) stressed IgG2 in OTS treated glass bottle;

[0047] In the figure, 1, bottle body; 2, bottle cap; 3, hydrophobic coating. DETAILED DESCRIPTION

[0048] The technical solutions in the embodiments of the present application will be described clearly and completely below through specific, specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Those skilled in the art can easily understand other advantages and effects of the present application from the content disclosed in the present application. The present application can also be implemented or applied through other different specific embodiments. In the case of no conflict, the following embodiments and features in the embodiments can be combined with each other. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0049] The embodiments and examples of the present invention will now be described in detail with reference to the accompanying drawings.

[0050] Example 1

[0051] like Figures 1-2 As shown in the illustration, this specific embodiment provides a coated solution container, comprising: a container body for containing a formulation; and a hydrophobic coating 3 disposed on the inner wall of the container body for inhibiting protein aggregation. Proteins adsorbed on the hydrophobic coating 3 are difficult to desorb, thus reducing the number of insoluble particles in the formulation. This invention utilizes the strong hydrophobic interaction between proteins and hydrophobic surfaces, making it difficult for proteins adsorbed on the hydrophobic surface to desorb into the solution, thereby inhibiting protein aggregation and reducing the generation of insoluble particles in the formulation to a certain extent.

[0052] In some embodiments of this application, the hydrophobic coating 3 is an octadecyltrichlorosilane coating (OTS). Proteins adsorbed on the surface of the hydrophobic coating 3 are difficult to desorb into the formulation solution, thus reducing the number of insoluble particles in the formulation. During formulation management and transportation, denatured proteins adsorbed on the hydrophobic surface are difficult to desorb. The hydrophobic surface can resist the formation of protein aggregates induced by fluid shear force to a certain extent, thereby significantly reducing the number of insoluble particles in the formulation.

[0053] In some embodiments of this application, the container body is a bottle 1, and the bottle 1 includes a bottle cap 2, which is sealed to the bottle 1 and is detachable. During use, the bottle cap 2 can be removed and installed to facilitate the filling or removal of the formulation solution into the bottle 1.

[0054] Bottle 1 is a regularly shaped bottle structure. It can hold accurately measured doses of formulation while avoiding residue of the formulation solution.

[0055] Preferably, the bottle body 1 is a cylindrical bottle. This type of bottle body 1 has no sharp edges and corners, making it less likely for medicine liquid to remain.

[0056] Alternatively, the bottle body 1 is a rectangular bottle with rounded corners. This type of bottle body 1 has a regular shape, rounded corners, and is easy to handle.

[0057] In this embodiment, bottle 1 is a glass bottle.

[0058] In some embodiments of this application, the hydrophobic coating 3 is uniformly distributed on the bottom and sides of the inside of the bottle 1, so that the entire inner surface of the bottle 1 is covered by the hydrophobic coating 3. In this embodiment, by covering the inner surface of the bottle 1 with the hydrophobic coating 3, the formulation solution inside the bottle 1 directly contacts the hydrophobic coating 3. After the protein is adsorbed on the hydrophobic coating 3, it is not easy to desorb, thereby reducing the generation of insoluble protein particles.

[0059] In other embodiments, the hydrophobic coating 3 is distributed on the inner surface of the bottle body 1. In actual production, the hydrophobic coating 3 is arranged at a suitable position according to requirements.

[0060] Alternatively, the hydrophobic coating 3 is distributed on the side surface of the bottle body 1 to reduce cost.

[0061] In another embodiment, the hydrophobic coating 3 is uniformly distributed on the bottom surface of the bottle body 1 and is distributed on the inner surface of the bottle body 1.

[0062] In some embodiments of the present application, the hydrophobic coating 3 is distributed on 30%-100% of the inner surface area of the bottle body 1.

[0063] In some embodiments of the present application, the coating thickness is 0.001-0.005 mm.

[0064] Example 2

[0065] The present application also discloses a method for arranging the hydrophobic coating 3 on the inner surface of the bottle body 1, which is as follows:

[0066] Experimental materials:

[0067] 7 mL borosilicate glass tube for injection, monoclonal antibody (IgG2, 75 mg / mL), sorbitol, sodium acetate, acetic acid, Tergazyme enzyme, 4,4'-diamino-1,1'-binaphthalene-5,5'-disulfonic acid dipotassium salt (Bis-ANS), octadecyltrichlorosilane (OTS, analytical pure), 2,2-biquinoline-4,4-dicarboxylic acid disodium (BCA), sodium tartrate, 98% H2SO 4、 H2O 2、 Toluene, ethanol (analytical pure).

[0068] Experimental instruments:

[0069] FlowCAM 8000 micro-flow imaging instrument, multifunctional enzyme label instrument (Multiskan EX) F500), Atomic force microscope (AFM, Dimension ICON), Fourier transform infrared spectroscopy (FTIR, Nicolet iS 10) with an Attenuated Total Refraction (ATR) accessory, Thermo. Automatic contact angle measuring instrument (OCA20), Dataphysics, Rotating mixer, High performance liquid chromatograph (Primaide), Automatic specific surface area porosity analyzer (BET, ASAP2460), Oil bath pot (DF-101S), High speed centrifuge (H3-20K), Microplate incubator (ST60-4).

[0070] Experimental method:

[0071] (1) Surface modification and interface characterization method

[0072] S1, rinse the glass bottle with deionized water and anhydrous ethanol in sequence, and dry naturally at 25℃;

[0073] S2, prepare an oxidant solution, treat the inside of the glass bottle with the oxidant solution, and obtain a glass bottle with hydroxylated inner surface;

[0074] S3, pour out the oxidant solution in the glass bottle, rinse the glass bottle with a rinse solution, and dry;

[0075] S4, prepare an octadecyltrichlorosilane solution, pour the prepared solution into the glass bottle with hydroxylated inner surface, and react at room temperature to obtain a glass bottle with octadecyltrichlorosilane coating on the inner surface.

[0076] S5, ultrasonic cleaning with toluene, ethanol, deionized water for 30min, then rinse the glass bottle with filtered anhydrous ethanol, and dry naturally at 24℃.

[0077] The oxidant solution in this embodiment is piranha solution. The oxidant solution in this embodiment is piranha solution. More specifically, the ratio of the piranha solution is H2SO4:H2O2=9:1 (V / V)-1:1 (V / V); during use, the glass bottle is filled with the piranha solution and placed in a pre-set temperature oil bath pot for a predetermined time. Preferably, the preferred ratio of the piranha solution is H2SO4:H2O2=3:1 (V / V).

[0078] Wherein, the pre-set temperature is 100-150℃, preferably, the pre-set temperature is 120℃.

[0079] The predetermined time is 2-5h, preferably, the predetermined time is 3h.

[0080] The flushing liquid is deionized water, the glass bottle is flushed with a large amount of deionized water, and the glass bottle is flushed with anhydrous ethanol filtered by a 0.22μm polytetrafluoroethylene (PTFE) filter membrane, and then naturally dried at 25℃ to enrich the hydroxyl groups on the inner surface of the glass bottle, so as to realize the hydroxylation of the inner surface of the glass bottle.

[0081] In this embodiment, the octadecyltrichlorosilane solution with a predetermined concentration is prepared by using toluene, the prepared solution is filled into the hydroxylated glass bottle, and the reaction is carried out at room temperature for 12h.

[0082] The predetermined concentration is 0.1%-1%(V / V), preferably 0.6%.

[0083] Experimental test:

[0084] S1, prepare glass bottles of the experimental group and the control group with the same material;

[0085] S2, obtain the functional group information of the inner surface of the glass bottle;

[0086] S3, the inner surface of the glass bottle of the experimental group and the control group is tested for 3 times, and the average value is taken.

[0087] Specifically, the experimental group is the glass bottle with the hydrophobic coating 3, and the control group is the glass bottle without the hydrophobic coating 3. The two kinds of glass bottles are flushed with filtered anhydrous ethanol and deionized water; and then dried.

[0088] In this embodiment, the above two kinds of glass bottles are dried by nitrogen flow.

[0089] In this embodiment, the Fourier transform attenuated total reflection infrared spectrometer (ATR-FTIR) is used to characterize the functional group information of the octadecyltrichlorosilane (OTS) coating surface, and the 2000-4000cm -1 The spectrum is collected, and the atomic force microscope (AFM) is used to observe the surface morphology of the glass sheet.

[0090] (2) Adsorption of IgG2 on hydrophilic and hydrophobic surfaces

[0091] In order to compare the saturated adsorption amount of IgG2 on the hydrophilic and hydrophobic surfaces:

[0092] Firstly, the saturated adsorption amount of the unmodified glass surface is determined, the glass powder is prepared, the glass bottle is ground into powder, the powder is collected through a 75μm sieve, washed, dried and used, the specific surface area of the glass powder is measured by BET, and the specific surface area is 0.30m 2 / g. Then, the saturated adsorption amount of the OTS modified glass surface was measured, using glass microbeads with a diameter of 0.5 mm as the OTS modified material, and the modification method of the glass microbeads was referred to the surface modification and interface characterization method. The surface area of the glass microbeads was estimated to be 0.005 m 2 / g.

[0093] The content of the protein was measured by the method of BCA:

[0094] First, the BCA working reagent was configured, which was composed of two solutions with A:B = 50:1 (V / V). The A solution (PH = 11.25) was composed of 0.03 mol / L BCA, 0.19 mol / L Na2CO3, 8.3 μmol / L sodium tartrate, 0.1 mol / L NaOH, and 0.11 mol / L NaHCO3. The B solution was composed of 0.016 mol / L CuSO4·5H2O.

[0095] Then, protein solutions with different concentrations were configured. 75 mg / ml IgG2 stock solution was precisely diluted to 10 mg / mL. Then, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 1.2, 1.6, 2, and 3 mL of the 10 mg / ml stock solution were precisely taken into 10 mL volumetric flasks, which were then diluted to the calibration line with auxiliary materials filtered by 0.22 μm polyether sulfone PES membranes. The corresponding concentrations were 0.02, 0.05, 0.1, 0.2, 0.5, 1, 1.2, 1.6, 2, and 3 mg / mL, respectively.

[0096] The content of the IgG2 adsorbed on the glass surface was measured by the content loss method. 500 mg of unmodified glass powder was put into 1 ml of IgG2 solution with a concentration of 0.02-3 mg / mL. 1 g of OTS modified glass microbeads was added into the IgG2 solution with a concentration of 0.02-3 mg / mL. The mixture was mixed in a head-to-head manner on a rotary mixer at 10 rpm for 10 min at 24°C. The sample was centrifuged at 20000 g for 10 min to separate the glass powder from the solution.

[0097] 180 μL of BCA working reagent and 20 μL of supernatant of the sample were taken into a white 96-well plate, mixed uniformly, and incubated in a 37°C incubator for 30 min. Then, the sample was cooled to 24°C, and the absorbance value was read at 562 nm using an enzyme marker. The absorbance value of the IgG2 solution at the above concentrations was measured in the same way to determine the saturated adsorption amount of the IgG2, and the data was fitted to the Langmuir adsorption isotherm to estimate the saturated adsorption amount of the IgG2 on the glass surface.

[0098] (3) The IgG2 solution in the two glass bottles was given mechanical stress using a rotary mixer

[0099] To simulate the effect of fluid shear stress on the stability of the mAb formulation in the glass vials with hydrophilic and hydrophobic surfaces during transportation, the IgG2 solution in the two types of glass vials was subjected to mechanical stress using a rotating mixer.

[0100] First, a buffer solution without IgG2 was prepared, which consisted of 4.6% sorbitol and 17 mmol / L sodium acetate, and the pH was adjusted to 5.2 using 0.1 mol / L acetic acid.

[0101] Then, the IgG2 stock solution at 75 mg / mL was precisely diluted to 1 mg / mL using the buffer solution. 3 ml of the IgG2 solution at a concentration of 1 mg / mL was added to untreated glass vials and OTS glass vials, respectively, and the buffer solution without IgG2.

[0102] The glass vials were fixed on the rotating mixer at 4°C and rotated head-to-head at a speed of 10 rpm for 12 h, and a static group was set up, with three groups for each experiment.

[0103] (4) Microflow imaging method for analysis of sub-visible particles

[0104] The concentration and morphological parameters of particles with a diameter of ≥2 μm in the glass vials after mechanical stress treatment were measured using FlowCam. 300 μL of sample from the glass vials was injected into the flow cell at a flow rate of 0.2 mL / min, and the number of sub-visible particles with a diameter of ≥2 μm was continuously observed and counted using a high-speed camera. Between each sample test, the flow cell was cleaned using 0.22 μm polyether sulfone (PES) filter membrane filtered deionized water and Tergazyme enzyme, and the flow cell must be rinsed with deionized water before each sample test.

[0105] (5) Size exclusion-high performance liquid chromatography

[0106] SE-HPLC was used to determine the content of soluble IgG2 monomers and soluble aggregates in the glass vials before and after mechanical stress treatment. The IgG2-containing sample was centrifuged at 20,000 g for 10 min to remove insoluble aggregates, and the supernatant was analyzed. A guard column and a G3000SWxL column were used; the mobile phase was 0.1 mol / L sodium sulfate, 0.05 mol / L sodium dihydrogen phosphate, and 0.05 mol / L disodium hydrogen phosphate; the flow rate was 0.8 mL / min; the detection wavelength was 280 nm; and the collection time was 20 min. To quantify the level of soluble protein, the total area under the single peak was compared.

[0107] (6) Exogenous fluorescent dye method

[0108] Polar responsive exogenous fluorescent dye Bis-ANS can detect aggregation and structural changes of protein molecules very sensitively, and the fluorescence emission intensity of the dye increases when the dye binds to hydrophobic sites. Add 150 μL of the sample to be tested and 50 μL of Bis-ANS in a black 96-well plate, and the final concentration of Bis-ANS is 2 μmol / L. Perform the test at 24°C using an enzyme marker, with an excitation wavelength of 390 nm and an emission wavelength of 490 nm. Take the IgG2 at rest as a control, and calculate the fluorescence increase of the IgG2 sample after mechanical stress treatment.

[0109] Experimental results:

[0110] Interface characterization:

[0111] The piranha solution enriches the OH groups on the glass surface, and the OH exposed by the hydrolysis of the tail of OTS is dehydrated to Si-O bond on the glass surface, generating a monolayer with 18 carbon atoms at one end on the glass surface, increasing the hydrophobicity of the glass surface. As shown in Figure 3 , the contact angle of the untreated glass surface is 50°±1°, and the water contact angle of the surface treated by the piranha solution is 18°±1°. OH is a hydrophilic group, so the water contact angle is very small. The contact angle of the OTS treated surface increases to 90°±2°, indicating that the hydrophobicity of the OTS treated glass surface is significantly increased. Through ATR-FTIR, it can be seen that the OTS treated glass has obvious characteristic absorption peaks of methyl (-CH3) and methylene (-CH2-) at 2800 cm -1 , 2900 cm -1 (see Figure 4 ). Through AFM, it can be seen that the OTS treated glass surface is rougher (see Figure 5 ), and the roughness of the untreated glass surface is 0.448±0.086, and the roughness of the OTS treated surface is 1.282±0.117. Therefore, a hydrophobic glass surface is successfully prepared.

[0112] Adsorption of proteins on hydrophilic and hydrophobic surfaces:

[0113] The adsorption of IgG2 on hydrophilic and hydrophobic glass surfaces conforms to the Langmuir adsorption model ( Figure 6 ), and the saturated adsorption amount on the hydrophilic glass surface is about 1.3 mg / m2, and the saturated adsorption amount on the OTS glass surface is 9.5 mg / m2. Therefore, compared with the hydrophilic surface, the protein is more easily adsorbed on the hydrophobic surface.

[0114] Sub-visible particles:

[0115] IgG2 produced a large amount of sub-visible particles after mechanical stress stimulation in both untreated glass vials and OTS treated glass vials, with the particle concentration of 759598 ± 70092 particles / mL and 448470 ± 66821 particles / mL, respectively. Figure 7 a) The particle concentration produced in OTS glass vials was significantly lower than that in untreated glass vials.

[0116] The particle size of the particles produced in untreated and OTS treated glass vials was not significantly different, with the particle size mainly distributed in 1-5 μm. Figure 7 b) and the particle size distribution Figure 8 ) were not significantly different. However, from the perspective of particle morphology, the particles produced in untreated and OTS treated glass vials were different Figure 9 ) in morphology.

[0117] To characterize the particles produced in the two vials, we analyzed the morphological parameters of the particles given by FlowCam. The circularity is a shape parameter calculated from the perimeter and the area filled, with the value of 1.0 for a circle. The intensity refers to the average gray value of the pixels that make up the particle, Figure 10 The contour plots of diameter and circularity and the contour plots of diameter and intensity can effectively show that the morphological parameters of the particles produced in untreated and OTS treated glass vials are different. The adsorption of proteins on hydrophilic surfaces is generally mediated by electrostatic interactions, and when the protein and the surface have different charges, the adsorption increases, and when the charges have the same sign, the adsorption decreases.

[0118] Soluble protein monomers and oligomers:

[0119] The SE-HPLC results show that the peak time of IgG2 monomers is about 10.6 min, and the peak time of the proteins after mechanical stress treatment in the two glass vials is consistent with that of IgG2 monomers, and there is no peak of soluble oligomers, and the monomer content is not significantly lost, and the remaining monomer content is close to 100% Figure 11 ).

[0120] Exogenous fluorescence analysis of protein aggregates:

[0121] From the Bis-ANS fluorescence data Figure 7 c), the fluorescence increase of the aggregates produced in OTS vials is greater than that in untreated vials. This indicates that the structure of the aggregates produced in the two vials is different, and the hydrophobic sites of the aggregates produced in OTS vials are more exposed.

[0122] Experimental conclusions:

[0123] (1) The hydrophobicity of the OTS treated glass surface is obviously increased, the AFM result shows that the surface roughness is increased, and the infrared spectrum shows the characteristic peaks of -CH2- and -CH3-, which indicates that the OTS modification is successful.

[0124] (2) The monoclonal antibody in the OTS modified glass bottle is subjected to mechanical stress, simulating the influence of fluid shear force on the stability of the monoclonal antibody preparation during transportation. Compared with the untreated hydrophilic glass bottle, the content of insoluble particles of the monoclonal antibody in the OTS glass bottle is lower under stress induction, and the aggregates produced in the two bottles are obviously different.

[0125] (3) Due to the strong hydrophobic interaction between IgG2 and the hydrophobic surface, it is found that the hydrophobic surface can resist the production of IgG2 aggregates induced by fluid shear force within a certain stress range. This indicates that the hydrophobic surface also has a coating that can be used as an inner packaging material to protect proteins from the production of protein aggregates induced by mechanical stress.

[0126] Example 3

[0127] The difference between this embodiment and Example 2 is the surface modification and interface characterization method:

[0128] S1, configure an oxidizing agent solution, treat the inside of the glass bottle with the oxidizing agent solution to obtain a glass bottle with the inside surface subjected to hydroxylation;

[0129] S2, prepare an octadecyltrichlorosilane solution, pour the prepared solution into the glass bottle with the inside surface subjected to hydroxylation, and react at room temperature to obtain a glass bottle with an octadecyltrichlorosilane coating on the inside surface.

[0130] In this embodiment, the oxidizing agent solution is used to treat the inside surface of the glass bottle, so that the inside surface of the glass bottle has hydroxyl groups; the octadecyltrichlorosilane solution reacts with the inside surface of the glass bottle with hydroxyl groups, and the inside surface of the glass bottle forms an octadecyltrichlorosilane (OTS) coating.

[0131] Example 4

[0132] This embodiment adds a step based on Example 3:

[0133] S1, configure an oxidizing agent solution, treat the inside of the glass bottle with the oxidizing agent solution to obtain a glass bottle with the inside surface subjected to hydroxylation;

[0134] S2, pour out the oxidizing agent solution in the glass bottle, rinse the glass bottle with a rinsing solution, and dry;

[0135] S3, prepare an octadecyltrichlorosilane solution, pour the prepared solution into the glass bottle with the inside surface subjected to hydroxylation, and react at room temperature to obtain a glass bottle with an octadecyltrichlorosilane coating on the inside surface.

[0136] Specifically, the oxidizing agent solution in the embodiment is piranha solution. More specifically, the piranha solution is prepared in a ratio of H2SO4:H2O2=3:1 (V / V); during use, the glass bottle is filled with the piranha solution and placed in a 120°C oil bath for 3h.

[0137] wherein the rinsing liquid is deionized water, the glass bottle is rinsed with a large amount of deionized water, and then rinsed with anhydrous ethanol filtered through a 0.22μm polytetrafluoroethylene (PTFE) filter membrane, and naturally dried at 25°C to enrich the inner surface of the glass bottle with hydroxyl groups, thereby realizing the hydroxylation of the inner surface of the glass bottle.

[0138] Embodiment 5

[0139] The embodiment adds the following steps to Embodiment 3:

[0140] S1, an oxidizing agent solution is prepared, and the interior of the glass bottle is treated with the oxidizing agent solution to obtain a glass bottle with a hydroxylated inner surface;

[0141] S2, an octadecyltrichlorosilane solution is prepared, and the prepared solution is filled into the glass bottle with the hydroxylated inner surface, and reacted at room temperature to obtain a glass bottle with an octadecyltrichlorosilane coating on the inner surface.

[0142] S3, the glass bottle is ultrasonically cleaned with toluene, ethanol and deionized water for 30min, and then rinsed with filtered anhydrous ethanol and naturally dried at 24°C.

[0143] In the embodiment, a 0.6% (V / V) octadecyltrichlorosilane solution is prepared with toluene, and the prepared solution is filled into the hydroxylated glass bottle, and reacted at room temperature for 12h.

[0144] Embodiment 6

[0145] The embodiment adds the following steps to Embodiment 5:

[0146] S1, an oxidizing agent solution is prepared, and the interior of the glass bottle is treated with the oxidizing agent solution to obtain a glass bottle with a hydroxylated inner surface;

[0147] S2, the oxidizing agent solution in the glass bottle is poured out, the glass bottle is rinsed with a rinsing liquid and dried;

[0148] S3, an octadecyltrichlorosilane solution is prepared, and the prepared solution is filled into the glass bottle with the hydroxylated inner surface, and reacted at room temperature to obtain a glass bottle with an octadecyltrichlorosilane coating on the inner surface.

[0149] S4, the glass bottle is ultrasonically cleaned with toluene, ethanol and deionized water for 30min, and then rinsed with filtered anhydrous ethanol and naturally dried at 24°C.

[0150] The oxidant solution in this embodiment is piranha solution. The oxidant solution in this embodiment is piranha solution. More specifically, the piranha solution is prepared with the ratio of H2SO4:H2O2=3:1 (V / V); during use, the glass bottle is filled with the piranha solution and placed in a 120℃ oil bath for 3h.

[0151] The rinsing liquid is deionized water, the glass bottle is rinsed with a large amount of deionized water, and then rinsed with anhydrous ethanol filtered with a 0.22μm PTFE filter membrane, and naturally dried at 25℃ to enrich the hydroxyl groups on the inner surface of the glass bottle, thereby realizing the hydroxylation of the inner surface of the glass bottle.

[0152] A 0.6% (V / V) octadecyltrichlorosilane solution is prepared with toluene, and the prepared solution is filled into the hydroxylated glass bottle for reaction at room temperature for 12h.

[0153] Example 7

[0154] This embodiment adds a step before the step of Example 3:

[0155] S1, the glass bottle is sequentially rinsed with deionized water and anhydrous ethanol, and naturally dried at 25℃;

[0156] S2, an oxidant solution is prepared, and the interior of the glass bottle is treated with the oxidant solution to obtain a glass bottle with a hydroxylated inner surface;

[0157] S3, an octadecyltrichlorosilane solution is prepared, and the prepared solution is filled into the glass bottle with a hydroxylated inner surface for reaction at room temperature to obtain a glass bottle with an octadecyltrichlorosilane coating on the inner surface.

[0158] In this embodiment, the glass bottle is rinsed before modification of the inner surface of the glass bottle, and then dried, so that the impurities in the glass bottle are removed without affecting the subsequent reaction.

[0159] Example 8

[0160] This embodiment adds a step based on Example 7:

[0161] S1, the glass bottle is sequentially rinsed with deionized water and anhydrous ethanol, and naturally dried at 25℃;

[0162] S2, an oxidant solution is prepared, and the interior of the glass bottle is treated with the oxidant solution to obtain a glass bottle with a hydroxylated inner surface;

[0163] S3, the oxidant solution in the glass bottle is poured out, the glass bottle is rinsed with a rinsing liquid, and dried;

[0164] S4, prepare octadecyltrichlorosilane solution, fill the prepared solution into the glass bottle with the inner surface of which is hydroxylated, and react at room temperature to obtain a glass bottle with an octadecyltrichlorosilane coating on the inner surface.

[0165] The oxidant solution in this embodiment is piranha solution. More specifically, the piranha solution is prepared in a ratio of H2SO4:H2O2=3:1 (V / V); during use, the glass bottle is filled with the piranha solution and placed in a 120°C oil bath for 3h.

[0166] The rinsing liquid is deionized water, and the glass bottle is rinsed with a large amount of deionized water, rinsed with anhydrous ethanol filtered through a 0.22μm polytetrafluoroethylene (PTFE) filter membrane, and naturally air-dried at 25°C to enrich the inner surface of the glass bottle with hydroxyl groups and achieve hydroxylation of the inner surface of the glass bottle.

[0167] In this embodiment, 0.6% (V / V) octadecyltrichlorosilane solution is prepared in toluene, the prepared solution is filled into the hydroxylated glass bottle, and the reaction is carried out at room temperature for 12h.

[0168] Example 9

[0169] This embodiment adds the following steps to Example 7:

[0170] S1, rinse the glass bottle with deionized water and anhydrous ethanol in sequence, and naturally air-dry at 25°C;

[0171] S2, prepare an oxidant solution, and treat the interior of the glass bottle with the oxidant solution to obtain a glass bottle with the inner surface of which is hydroxylated;

[0172] S3, prepare octadecyltrichlorosilane solution, fill the prepared solution into the glass bottle with the inner surface of which is hydroxylated, and react at room temperature to obtain a glass bottle with an octadecyltrichlorosilane coating on the inner surface.

[0173] S4, ultrasonically clean the glass bottle with toluene, ethanol, and deionized water for 30min, then rinse the glass bottle with filtered anhydrous ethanol, and naturally air-dry at 24°C.

[0174] The oxidant solution in this embodiment is piranha solution. More specifically, the piranha solution is prepared in a ratio of H2SO4:H2O2=3:1 (V / V); during use, the glass bottle is filled with the piranha solution and placed in a 120°C oil bath for 3h.

[0175] The flushing liquid is deionized water, the glass bottle is washed with a large amount of deionized water, washed with anhydrous ethanol filtered by a 0.22 μm polytetrafluoroethylene (PTFE) filter membrane, and naturally dried at 25°C to enrich the hydroxyl group on the inner surface of the glass bottle, thereby realizing the hydroxylation of the inner surface of the glass bottle.

[0176] In the example, a 0.6% (V / V) octadecyltrichlorosilane solution is prepared with toluene, the prepared solution is filled into the hydroxylated glass bottle, and the reaction is carried out at room temperature for 12 h.

[0177] The above examples are only used for understanding the present application. It should be noted that those skilled in the art can make several improvements to the present application without departing from the principles of the present application, and these improvements will also fall within the protection scope of the claims of the present application.

Claims

1. A solution container with a coating, characterized in that, include: The main body of the container, used to hold the formulation; A hydrophobic coating is applied to the inner wall of the container body to inhibit protein aggregation. Proteins adsorbed on the hydrophobic coating are difficult to desorb, thus reducing the number of insoluble particles in the formulation. The hydrophobic coating is an octadecyltrichlorosilane coating; the coating thickness is 0.001-0.005 mm.

2. The coated solution container according to claim 1, characterized in that, The main body of the container is a bottle, which includes a bottle cap. The bottle cap is sealed to the bottle and is detachable.

3. The coated solution container according to claim 2, characterized in that, The bottle body is a regularly shaped bottle-like structure.

4. The coated solution container according to claim 2, characterized in that, The hydrophobic coating is evenly distributed on the bottom and sides of the bottle body, so that the entire inner surface of the bottle body is covered by the hydrophobic coating.

5. The coated solution container according to claim 2, characterized in that, The hydrophobic coating is distributed on a portion of the inner surface of the bottle.

6. The coated solution container according to claim 2, characterized in that, The hydrophobic coating is evenly distributed at intervals on the bottom and sides of the bottle body.

7. A method for preparing a coated solution container, used to prepare a coated solution container as described in any one of claims 1-6, characterized in that, Includes the following steps: S1. Prepare an oxidizing agent solution and treat the inside of the glass bottle with the oxidizing agent solution to obtain a glass bottle with hydroxylated inner surface; S2. Prepare an octadecyltrichlorosilane solution, pour the prepared solution into the glass bottle whose inner surface has been hydroxylated, and react at room temperature to obtain a glass bottle with an octadecyltrichlorosilane coating on the inner surface.

8. The method according to claim 7, characterized in that, The oxidizing agent solution is a piranha etching solution.

9. The method according to claim 7, characterized in that, Prepare a 0.6% (v / v) solution of the octadecyltrichlorosilane using toluene.

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