Method for predicting stability of protein co-prescription preparation

Abstract

The invention discloses a method for predicting the stability of a protein co-prescription preparation, which is characterized in that when the second virie coefficients of the proteins of all active pharmaceutical ingredients in the co-prescription preparation are positive values and the cross virie coefficients of the proteins of different active pharmaceutical ingredients are also positive values, the co-prescription preparation has good long-term stability; when the second virie coefficient of the protein of at least one medicine active ingredient is a negative value and at least one cross virie coefficient of the protein of each heterogeneous medicine active ingredient is a negative value, the long-term stability of the co-prescription preparation is poor; according to the method, the stability of the co-prescription preparation is predicted through the second Wirie coefficient and the cross Wirie coefficient of the active pharmaceutical ingredient protein of the co-prescription preparation, and the prediction result is accurate; the second Virie coefficient and the cross Virie coefficient are measured by using the component gradient multi-angle light scatterometer, the sample does not need to be marked or subjected to other treatment, sample preparation and instrument operation are simple, and the detection speed is high.

Description

Technical Field

[0001] The present invention relates to the technical field of biomacromolecule preparations, and in particular to a method for predicting the stability of a protein co-prescription preparation. Background Art

[0002] In the past few decades, therapeutic antibodies have become one of the most important treatments for a variety of serious diseases, especially for inflammatory diseases and solid tumors. Due to their high specificity, long half-life and good storage stability, antibodies have become best-selling drugs in the biologics pharmaceutical market. Although therapeutic antibodies have achieved great success, one of their disadvantages is that a single antibody can only target one site of cancer resistance or viral mutation. In order to overcome this limitation, a combination formulation (Co-formulation), also known as a co-prescription formulation, was created. It usually contains two or three antibodies that can block primary and emerging secondary targets. Compared with single antibody formulations, co-prescription formulations are considered to have obvious advantages, such as better therapeutic effects and higher efficiency, thereby better meeting the needs of patients and pharmaceutical companies.

[0003] Each antibody in a co-prescription formulation may exhibit heterogeneous differences in size, charge, and post-translational modifications during the production process. Therefore, the presence of different antibodies increases the complexity of the co-prescription formulation development process and poses challenges to the characterization and control of product quality. Inter-antibody interactions have a significant impact on the colloidal stability of proteins in formulations and product quality and safety. For example, attractive cross-interactions between different protein drugs may lead to the formation of heterogeneous protein aggregates that are not detected in a single formulation, thereby affecting product quality. Therefore, in the development of co-prescription biologics, analyzing nonspecific attractive and repulsive protein interactions can guide the rational development of protein co-prescription formulations to achieve colloidal stability of protein drugs to ensure the quality and safety of drugs.

[0004] There are currently several analytical methods that can detect the type and strength of antibody interactions in co-formulated formulations, such as dynamic light scattering (DLS), static light scattering (SLS), differential scanning calorimetry (DSC), isothermal titration calorimetry (ITC), and circular dichroism (CD). However, these methods have some limitations and shortcomings, such as the use of ITC to detect interactions, especially at high concentrations, nonspecific and non-covalent interactions (electrostatic, ionic or hydrophobic) between two antibodies can lead to reversible binding, which may not cause significant enthalpy changes in ITC experiments; traditional SLS methods usually require tedious sample preparation and experimental work.

[0005] Composition gradient multi-angle light scattering (CG-MALS) is a technique used to study molecular interactions, especially useful for studying the self-assembly and complex formation of proteins and other biomacromolecules. This technique combines concentration gradient and multi-angle laser scattering analysis. In a composition gradient multi-angle light scattering (CG-MALS) experiment, two or more solutions (usually solutions of proteins or other biomacromolecules) pass through a special mixing device to form a concentration gradient. The sample with this concentration gradient flows through a flow cell, where the sample is illuminated by lasers at multiple different angles. By measuring the light scattering intensity at different angles, information about the size, shape and interactions of the sample molecules can be obtained. In particular, by analyzing the scattering intensity that varies with concentration, the type of interaction between molecules (such as whether complexes or aggregates are formed), as well as the strength and characteristics of these interactions, can be inferred. The composition gradient multi-angle light scattering (CG-MALS) technology can provide label-free molecular interaction data, which is very important for understanding the function and interaction mechanism of biomolecules. Therefore, we can use it to determine the weight average molecular weight (M) of protein preparations in different formulations. W ), the second virial coefficient (A2) and the equilibrium dissociation constant (Kd). The second virial coefficient A2 is an important parameter for determining whether macromolecules in a solution have attractive or repulsive interactions. It is calculated based on the Debye equation measured by static light scattering. The cross virial coefficient (A 11 ) represents the interaction between heterogeneous molecules. Positive and negative values ​​of A2 represent repulsive and attractive interactions between macromolecules in solution, respectively. In addition, by collecting and analyzing the Rayleigh ratio as a function of solute concentration and scattering angle, it can be used to detect and characterize the reversible binding of macromolecules in solution. Kd is a parameter that describes the intermolecular binding strength of a specific association. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a method for predicting the stability of protein co-prescription preparations. Through the second virial coefficient and the cross virial coefficient, the stability of protein co-prescription preparations can be well predicted, which is conducive to reducing the workload in the early stage of drug development.

[0007] In order to solve the above technical problems, the present invention provides a method for predicting the stability of a protein co-prescription preparation, wherein:

[0008] The co-prescription preparation includes n proteins with active pharmaceutical ingredients, where n is in the range of n≥2;

[0009] When the second virial coefficients of n proteins with active pharmaceutical ingredients are all positive, and the cross-virial coefficients between proteins with heterogeneous active pharmaceutical ingredients are also positive, the long-term stability of the co-prescription preparation is good;

[0010] When at least one of the second virial coefficients of n proteins with active pharmaceutical ingredients is negative, and / or at least one of the cross-virial coefficients between proteins with heterogeneous active pharmaceutical ingredients is negative, the co-prescription preparation has poor long-term stability.

[0011] In the present invention, when the second virial coefficient is positive, it indicates that the interaction between the molecules of the active pharmaceutical ingredient protein itself is repulsive, and when it is negative, it indicates that the interaction between the molecules of the active pharmaceutical ingredient protein itself is attractive; when the cross virial coefficient is positive, it indicates that the interaction between the heterogeneous active pharmaceutical ingredient protein molecules represented by it is repulsive, and when it is negative, it indicates that the interaction between the heterogeneous active pharmaceutical ingredient protein molecules represented by it is attractive. Therefore, when the second virial coefficients of the proteins with active pharmaceutical ingredients in n are all positive, and the cross virial coefficient is also positive, it means that the homogeneous protein molecules of the active pharmaceutical ingredients and the heterogeneous protein molecules repel each other, and therefore, the co-prescription preparation has good long-term stability; on the contrary, if the second virial coefficient of any active pharmaceutical ingredient protein is negative, it means that the active pharmaceutical ingredient protein molecules are easy to agglomerate, which is not conducive to the long-term stability of the co-prescription preparation. Similarly, when the cross virial coefficient between heterogeneous active pharmaceutical ingredient protein molecules is negative, it means that the heterogeneous active pharmaceutical ingredient protein molecules are easy to agglomerate to form heterogeneous aggregates, which is also not conducive to the long-term stability of the co-prescription preparation. It should be noted that heterogeneous proteins are prone to form heterogeneous aggregates, and the composition and proportion of heterogeneous aggregates can be further simulated using the fitting stoichiometric method.

[0012] In a specific embodiment, the second virial coefficients of the n proteins having pharmaceutically active ingredients are all greater than 1×10 -5 mol·mL / g 2 , or at 5×10 -5 mol·mL / g 2 Up to 2×10 -4 mol·mL / g 2 The range is 5×10 - 5 mol·mL / g 2 Up to 2×10 -4 mol·mL / g 2 The range is the preferred value range; in this specific embodiment, the second virial coefficient cannot be too small, which means that the repulsive interaction between molecules is smaller than the repulsive effect of steric hindrance, and the repulsion between molecules is insufficient. The second virial coefficient cannot be too large either, which indicates that the repulsion between protein molecules is very strong, which will affect the uniform distribution of proteins in the solution and increase the viscosity of the solution.

[0013] In a specific embodiment, the cross-virial coefficients of the n proteins with pharmaceutical active ingredients are all greater than 1×10 -5 mol·mL / g 2 , or at 5×10 -5 mol·mL / g 2 -2×10 -4 mol·mL / g 2 The range is 5×10 - 5 mol·mL / g 2 -2×10 -4 mol·mL / g 2 The range is the preferred value range; in this specific embodiment, the cross-virial coefficient cannot be too small, which means that the repulsive interaction between molecules is smaller than the repulsive effect of steric hindrance, and the repulsion between molecules is insufficient. The cross-virial coefficient cannot be too large either, which indicates that the repulsion between protein molecules is very strong, which will affect the uniform distribution of proteins in the solution and increase the viscosity of the solution.

[0014] In a specific embodiment, the value of n is 2 or 3; understandably, generally speaking, the proteins of the active pharmaceutical ingredients contained in the current co-prescription preparations are usually two or three, more usually two.

[0015] In a specific embodiment, the protein of the active pharmaceutical ingredient includes antibodies, enzymes, fusion proteins, antibody-drug conjugates and polypeptides, preferably monoclonal antibodies, such as ipilimumab, pembrolizumab and eloumab.

[0016] In a specific embodiment, the total protein concentration of the active pharmaceutical ingredient is 1 mg / mL-1000 mg / mL, or 1 mg / mL-500 mg / mL, or 5 mg / mL-300 mg / mL or 20 mg / mL. It is understandable that the total protein concentration of the active pharmaceutical ingredient is determined by comprehensively considering various factors such as protein solubility, stability, drug formulation and safety.

[0017] In a specific embodiment, the protein co-prescription formulation further includes pharmaceutically acceptable excipients and a buffer system, and the pH value of the system ranges from 4.0 to 8.0 or 4.5 to 7.0. In this specific embodiment, pharmaceutically acceptable excipients include excipients, diluents, adhesives, lubricants, humectants, emulsifiers, preservatives, antioxidants, buffers, antibacterial agents, disintegrants, surfactants, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners, etc., such as water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptide substances, cellulose, nanogels, or combinations thereof; buffer systems such as histidine / histidine hydrochloride buffer.

[0018] In a specific embodiment, the prediction method further comprises obtaining the second virial coefficient and the cross virial coefficient by means of a component gradient multi-angle light scattering instrument; wherein, when the weight average molecular weight determined by the component gradient multi-angle light scattering instrument differs from the theoretical molecular weight by no more than 10%, the second virial coefficient and the cross virial coefficient obtained by fitting are determined to be valid. Preferably, the weight average molecular weight, the second virial coefficient and the cross virial coefficient are determined by using the Reversible association+CVC mode of the component gradient multi-angle light scattering instrument.

[0019] The present invention predicts the compatibility stability of co-prescription preparations through the second virial coefficient and the cross virial coefficient of the active pharmaceutical ingredient protein of the co-prescription preparations, and the prediction result is accurate, which is beneficial to improving the efficiency of drug research and development; the second virial coefficient and the cross virial coefficient of the present invention are measured by a component gradient multi-angle light scattering instrument, and there is no need to label or otherwise process the samples, the sample preparation and instrument operation are simple, and the detection speed is fast. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solution of the present invention, the following briefly introduces the drawings required for use in the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 This is the stability test result in Example 3. DETAILED DESCRIPTION

[0022] The following will be combined with the accompanying drawings to clearly and completely describe the technical solutions in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Main Materials:

[0024] L-Histidine was purchased from Merck;

[0025] L-Histidine Monohydrochloride was purchased from Merck;

[0026] Polysorbate 80 was purchased from Croda;

[0027] Sucrose was purchased from Pfanstiehl;

[0028] Evolocumab, Ipilimumab and Pembrolizumab are prepared by the applicant.

[0029] Main equipment:

[0030] Centrifuge (model 5804R) was purchased from Eppendorf;

[0031] The clarity tester (model YB-2) was purchased from Tianda Tianfa Company;

[0032] UV spectrophotometer (model Nano Drop One) was purchased from Thermo Fisher Scientific;

[0033] Composition gradient multi-angle light scattering (Calypso II and DAWN TM ) was purchased from Wyatt Technologies.

[0034] Size exclusion high-performance liquid chromatography (SE-HPLC);

[0035] Imaged Capillary Isoelectric Focusing (iCIEF);

[0036] Non-reduced Caliper Sodium Dodecyl Sulfate (Caliper-SDS-NR);

[0037] The experimental methods for which specific conditions are not specified in the following examples may be based on conventional methods in the art, for example, reference to Molecular Cloning Laboratory Manual (3rd edition, Cold Spring Harbor Laboratory Press, New York, 1989) or according to the conditions recommended by the supplier.

[0038] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0039] Example 1 Prediction of the stability of co-prescription preparations of ipilimumab and evolocumab

[0040] In this embodiment, there are four prescriptions in total. Ipilimumab and evolocumab were subjected to ultrafiltration centrifugation, liquid exchange and excipient addition treatment according to the target prescription information in Table 1, and tested by component gradient multi-angle light scattering (CG-MALS). The prescription information of the two antibodies is 20mM histidine / histidine hydrochloride buffer (pH 6.0, prescription number F1), 20mM acetic acid / sodium acetate buffer (pH 5.0, prescription number F2), 20mM phosphate buffer (pH 7.0, prescription number F3), 20mM histidine / histidine hydrochloride buffer, 140mM sodium chloride (pH 6.0, prescription number F4). The concentration of both monoclonal antibodies is 5mg / mL. When testing on the machine, the F1 prescription of ipilimumab is tested simultaneously with the F1 prescription of eloumab, the F2 prescription of ipilimumab is tested simultaneously with the F2 prescription of eloumab, the F3 prescription of ipilimumab is tested simultaneously with the F3 prescription of eloumab, and the F3 prescription of ipilimumab is tested simultaneously with the F3 prescription of eloumab. During the test, the test equipment will mix the samples of the two monoclonal antibodies to obtain the second virial coefficient (A2) and the cross virial coefficient (A 11 ). The prescription information is shown in Table 1:

[0041] Table 1

[0042]

[0043] Sample preparation before the machine: Use two 0.22μm polyvinylidene fluoride filters (purchased from Merck) in series to filter 8 samples and the corresponding 4 prescription solutions. There is no need to prepare multiple concentration gradient samples by stepwise dilution or additional labeling of samples.

[0044] The samples were directly measured using a component gradient multi-angle light scattering instrument. The component gradient multi-angle light scattering instrument contains a total of three pumps, two of which are connected to two samples to be tested, and the other pump is connected to the corresponding prescription solution. A total of 6 concentration gradients were set during the entire test process, with a flow rate of 0.75 mL / min, and the wavelength of the light scattering instrument was 658 nm. The test results are shown in Table 2. The weight-average molecular weight of ipilimumab measured under the conditions of the four prescription solutions is concentrated around 147-149 kDa, which is no more than 3% different from its theoretical molecular weight of 145.0 kDa; the weight-average molecular weight of eloumab measured under the conditions of the four prescription solutions is concentrated between 146-151 kDa, which is no more than 6.9% different from its theoretical molecular weight of 141.8 kDa. Therefore, the second virial coefficients of the two antibodies and the cross-virial coefficients between the two antibodies obtained under this condition are valid.

[0045] Table 2

[0046]

[0047] From the data in Table 2, it can be predicted that the two antibodies have good compatibility in prescriptions F1 and F2, and the co-prescription preparations have good long-term stability; while the compatibility in prescriptions F3 and F4 is poor, and the co-prescription preparations have poor long-term stability.

[0048] Example 2 Prediction of the stability of co-prescription formulations of pembrolizumab and ipilimumab

[0049] There are two prescriptions in this embodiment. Ipilimumab and pembrolizumab are subjected to ultrafiltration centrifugation, liquid exchange and excipient addition treatment according to the target prescription information in Table 3, and tested with a component gradient multi-angle light scattering instrument (CG-MALS). The prescription information of the two antibodies is 20mM histidine / histidine hydrochloride buffer, 8% sucrose and 0.02% polysorbate 80 (pH 6.0, prescription number F5), 20mM histidine / histidine hydrochloride buffer, 140mM sodium chloride (pH 6.0, prescription number F4). The concentrations of both monoclonal antibodies are 5mg / mL. When the machine is tested, the F4 prescription of pembrolizumab is tested simultaneously with the F4 prescription of ipilimumab, and the F5 prescription of pembrolizumab is tested simultaneously with the F5 prescription of ipilimumab. During the test, the test equipment will mix the samples of the two monoclonal antibodies to obtain the second virial coefficient (A2) and the cross virial coefficient (A 11 ). The prescription information is shown in Table 3:

[0050] Table 3

[0051]

[0052] Sample processing: After two 0.22 μm polyvinylidene fluoride filter membranes (purchased from Merck) are connected in series, each prescription preparation can be filtered without stepwise dilution to prepare multiple concentration gradient samples or labeling the samples.

[0053] The samples of the two antibodies treated by filtration were directly measured with a component gradient multi-angle light scattering instrument. A total of 6 concentration gradients were set in the whole test process, the flow rate was 0.75 mL / min, and the wavelength of the light scattering instrument was 658 nm. The test results are shown in Table 4, wherein the measured weight average molecular weights of Ipilimumab were 153.1 kDa and 132.5 kDa, respectively, which differed by 5.5% and 8.6% from its theoretical molecular weight of 145 kDa, respectively, and the measured weight average molecular weights of Pembrolizumab were 137.4 kDa and 148.9 kDa, respectively, which differed by 5.9% and 2.0% from its theoretical molecular weight of 146 kDa, respectively, so the second virial coefficients of the two antibodies and the cross virial coefficients between the two antibodies were fitted under this condition.

[0054] Table 4

[0055]

[0056] From the data in Table 4, it can be predicted that the two antibodies have good compatibility in the formulation F5 and are prepared into a co-prescription preparation with good long-term stability.

[0057] Example 3 Stability Verification Example

[0058] 1. Sample preparation

[0059] The preparation method is the same as the prescription F5 in Example 2, except that the concentrations of the two monoclonal antibodies are adjusted to 20 mg / mL to obtain a pembrolizumab preparation and an ipilimumab preparation, which are then mixed in a volume ratio of 1:1 to obtain a co-prescription preparation of pembrolizumab and ipilimumab. In a biological safety cabinet, the pembrolizumab preparation, the ipilimumab preparation and the co-prescription preparation are filtered separately with a 0.22 μm polyvinylidene fluoride filter membrane, and filled into 2 mL vials (1 mL / bottle), then immediately stoppered, capped and labeled, and then the sample is placed in a 40°C stability box for incubation for 4 weeks.

[0060] 2. Test methods and results

[0061] The three preparations were tested before and after incubation using SE-HPLC, iCIEF, and Caliper-SDS (NR). The test results are detailed in Figure 1 ,in Figure 1 Figure A is the SE-HPLC test results before and after incubation. Figure 1 Figure B shows the iCIEF test results before and after incubation. Figure 1 Figure C shows the Caliper-SDS (NR) test results before and after incubation. Figure 1 In the figure, T0 is the data before incubation, and 40°C-4 weeks is the result after incubation at 40°C for 4 weeks.

[0062] SE-HPLC is a purity analysis method that separates molecules in solution according to their size. ) on an Agilent HPLC system and data analysis was performed using Agilent CDS software.

[0063] Caliper-SDS (NR) was detected by HT Protein Express LabChip using PerkinElmer's LabChip GXII Touch HT instrument, and the data were analyzed using Empower software.

[0064] iCIEF is an analytical method that uses the pH gradient formed by amphoteric electrolytes under the action of an electric field to separate samples according to the difference in the isoelectric point of proteins. The isoelectric point is a property of proteins. A mixture of proteins with different isoelectric points is added to a pH gradient medium. After a certain period of focusing in the electric field, the different components focus on the pH position corresponding to their respective isoelectric points, forming a separated protein focused band. The focused band is then detected at 280nm to obtain the distribution map and pI of the sample charge heterogeneity. The pI value and relative abundance of the separated peaks are quantified using chromatography software (Empower 3).

[0065] Before incubation at 40°C: the main peak contents of pembrolizumab preparation, ipilimumab preparation and the co-prescription preparation of the two were 98.3%, 99.1% and 98.7% by SE-HPLC, respectively; the purity detected by Caliper-SDS (NR) was 97.4%, 97.2% and 97.3%, respectively; the main peaks of pembrolizumab preparation and ipilimumab preparation detected by iCIEF were 67.0% and 70.8%, respectively. In the iCIEF spectrum, the two antibodies of the co-prescription preparation could be completely separated, and the main peak of each antibody accounted for 67.7% and 65.9% of the full peak of each antibody, respectively.

[0066] After incubation at 40°C for 4 weeks, the main peak contents of pembrolizumab preparation, ipilimumab preparation and their co-prescription preparation were 97.6%, 98.8% and 98.0% by SE-HPLC, respectively; the purity detected by Caliper-SDS (NR) was 95.0%, 93.5% and 94.4%, respectively; the main peaks of pembrolizumab preparation and ipilimumab preparation detected by iCIEF were 52.1% and 49.2%, respectively. In the iCIEF spectrum, the two antibodies of the co-prescription preparation could still be completely separated, and the main peak of each antibody accounted for 52.8% and 45.6% of the full peak of each antibody, respectively.

[0067] The test results showed that after incubation at 40°C for 4 weeks, the main peaks of SE-HPLC, Caliper-SDS (NR&R) and iCIEF of the three preparations decreased within an acceptable range, and the results of the co-prescription preparations were consistent with the performance of the single antibody preparations. The test results show that in the formulation (20mM histidine / histidine hydrochloride buffer, 8% sucrose, 0.02% polysorbate 80, pH 6.0), the stability of the pembrolizumab preparation, ipilimumab preparation and the co-prescription preparation of the two is good, which is consistent with the previous prediction results.

[0068] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for predicting the stability of a protein co-prescription preparation, characterized in that: The co-prescription preparation includes n proteins with active pharmaceutical ingredients, where n is in the range of n≥2; When the second virial coefficients of n proteins with active pharmaceutical ingredients are all positive, and the cross-virial coefficients between proteins with heterogeneous active pharmaceutical ingredients are also positive, the long-term stability of the co-prescription preparation is good; When at least one of the second virial coefficients of n proteins with active pharmaceutical ingredients is negative, and / or at least one of the cross-virial coefficients between proteins with heterogeneous active pharmaceutical ingredients is negative, the co-prescription preparation has poor long-term stability.

2. The prediction method according to claim 1, characterized in that: The second virial coefficients of n proteins with active pharmaceutical ingredients are all greater than 1×10 -5 mol·mL / g 2 , or at 5×10 -5 mol·mL / g 2 -2×10 -4 mol·mL / g 2 within the range.

3. The prediction method according to claim 1, characterized in that: The cross-virial coefficients of n proteins with active pharmaceutical ingredients are all greater than 1×10 -5 mol·mL / g 2 , or at 5×10 -5 mol·mL / g 2 -2×10 -4 mol·mL / g 2 within the range.

4. The prediction method according to claim 1, characterized in that: The value of n is 2 or 3.

5. The prediction method according to claim 1, characterized in that: Proteins that are active pharmaceutical ingredients include antibodies, enzymes, fusion proteins, antibody-drug conjugates, and peptides.

6. The prediction method according to claim 5, characterized in that: The protein active ingredient of the drug is a monoclonal antibody.

7. The prediction method according to claim 5, characterized in that: The total protein concentration of the active pharmaceutical ingredient is 1 mg / mL-1000 mg / mL, or 1 mg / mL-500 mg / mL, or 5 mg / mL-300 mg / mL or 20 mg / mL.

8. The prediction method according to claim 1, characterized in that: The protein co-prescription preparation also includes pharmaceutically acceptable excipients, buffer systems and surfactants; The pH value range of the system is 4.0-8.0 or 4.5-7.

0.

9. The prediction method according to claim 1, characterized in that: The prediction method also includes obtaining the second virial coefficient and the cross virial coefficient through component gradient multi-angle light scattering instrument; When the weight average molecular weight measured by the component gradient multi-angle light scattering instrument differs from the theoretical molecular weight by no more than 10%, the second virial coefficient and the cross virial coefficient obtained by the fitting are determined to be valid.

10. The prediction method according to claim 1, characterized in that: The weight-average molecular weight, second virial coefficient and cross virial coefficient were determined using a composition gradient multi-angle light scattering instrument in reversible association + CVC mode.

Images