CN117797264A

CN117797264A - Surfactant for macromolecule therapeutic preparation

Info

Publication number: CN117797264A
Application number: CN202311492486.0A
Authority: CN
Inventors: 王益彤; 王亭亭; 陈全民; 郭哲明; 吴昊
Original assignee: Wuxi Biologics Shanghai Co Ltd
Current assignee: Wuxi Biologics Shanghai Co Ltd
Priority date: 2023-11-09
Filing date: 2023-11-09
Publication date: 2024-04-02

Abstract

Disclosed is a pharmaceutical formulation comprising: macromolecular drugs, buffer solutions, stabilizers, surfactants and sterile water for injection; the buffer solution is one or the combination of more than two of citrate, histidine salt or phosphate; the stabilizer is selected from sodium chloride, amino acid, polyol or any combination thereof, the amino acid is selected from arginine, aspartic acid, histidine or any combination thereof, and the polyol is selected from sucrose, sorbitol, mannitol or any combination thereof; the surfactant is selected from any one or combination of polysorbate 20 (PS 20), polysorbate 80 (PS 80), poloxamer 188 (P188), octyl-beta-D-glucopyranoside (OGP); and the pH of the pharmaceutical formulation is 4.5-7.5.

Description

Surfactant for macromolecule therapeutic preparation

(1) Technical field

The present invention relates to the field of biotechnological pharmaceutical formulations, in particular to pharmaceutical formulations of therapeutic proteins comprising a surfactant which is resistant to degradation and of controlled content.

(2) Background art

The availability of new biopharmaceuticals for patients to treat critical diseases has steadily increased in the last decade from oncology to the various therapeutic areas of metabolic diseases. Accordingly, therapeutic protein drugs, particularly monoclonal antibodies and their related drug classes, such as bispecific antibodies and antibody-conjugated drugs, have been rapidly developed. Thus, maintaining the stability of the biologic during biologic processing, pharmaceutical manufacturing, shipping, storage, and patient administration is critical. There are many mechanisms that may trigger protein aggregates (e.g., hydrophobic binding, chemical modification, and interfacial interactions of partially denatured protein molecules) to form visible and sub-visible particles outside of acceptable standards, thereby affecting product quality. Proteins typically undergo four types of "instability": chemical instability, conformational instability, colloidal instability, and interfacial instability. By reasonable formulation design to remove or mitigate these "instabilities", a protein pharmaceutical product can be obtained that is stable over a long period of time. Therefore, an important process in the development of formulation is the screening of adjuvants in order to obtain protein drugs that are stable as long as possible.

The interface is ubiquitous with proteins, whether during production, transport, storage or clinical administration. Mainly comprises an air/water interface generated during mixing of liquid preparation, an ice/water interface generated during repeated freezing and thawing and a coating material/water interface generated during transportation, wherein the existence of the interfaces can lead to unfolding or adsorption of proteins, thus causing aggregation of the proteins or insufficient actual content of the proteins in the liquid medicine, and simultaneously, other types of instability can be caused. The surfactant is used as an auxiliary material for improving the interfacial stability of protein medicines. Surfactants generally prevent proteins from aggregating by preventing them from entering the hydrophobic interface (glass bottle wall or air interface). There are mainly two possible mechanisms: interface competition mechanisms and surfactant-protein complex mechanisms. The preferential occupation of the surfactant at the interface, compared to the protein molecule, is a thermodynamically more stable state and is related to the kinetic mass. In contrast, surfactant-protein complexation is primarily through direct binding of the surfactant to the exposed hydrophobic surface of the native protein, thereby increasing protein stability. The most commonly used surfactants at present are nonionic surfactants: polysorbates (PS 20 and PS 80) and poloxamers (P188). However, in recent years, it has been shown that polysorbates degrade by various mechanisms such as autoxidation, cleavage of ethylene oxide subunits and hydrolysis of fatty acid ester bonds. The free fatty acid esters also form insoluble particles, which can cause turbidity in the formulation. These all affect the activity and stability of protein drugs. While work has shown and can mitigate degradation of polysorbates in formulations by the addition of antioxidants, the choice of surfactants in formulation formulations still presents a great challenge, as their degradation is likely to be due to the presence of lipases in host cells.

The antibody-drug conjugate (ADC) combines the high specificity of monoclonal antibody drugs with the high activity of small molecule cytotoxic drugs, so as to improve the targeting of tumor drugs and reduce the toxic and side effects. Compared to traditional fully or partially humanized antibodies or antibody fragments, ADCs are theoretically more therapeutic due to their ability to release highly active cytotoxins in tumor tissue. It has higher tolerance or lower side effects than the fusion protein. The accurate identification of the ADC drug to the target spot and the noninjugated cell are not affected, so that the drug effect is greatly improved, the toxic and side effects are reduced, and the attention of the personnel in the field of medicine research and development is rapidly developed in recent years. Due to the special composition structure of the ADC, the ADC is not completely similar to a pure protein drug when the ADC is subjected to preparation development. The corresponding monoclonal antibody (mAb) moiety in ADC is typically stored at-70 ℃ after production and purification, whereby the mAb intermediate undergoes a series of powerful conditions of bulk freeze thawing, stirring, contact with small amounts of organic solvents, etc. during conjugation with small molecule drugs, which presents a major challenge for the stability of the mAb intermediate. For mAb intermediates with poor stability, it is generally necessary to add higher concentrations of surfactant to ensure stability of the protein during this series of processes. However, the addition of conventional surfactants causes the following two problems: 1. in view of the higher molecular weight of polysorbates (PS 20 and PS 80) and poloxamers (P188), it is difficult to remove them by the subsequent UF/DF (ultrafiltration/diafiltration) step, and thus the surfactant content in the formulation is difficult to determine; 2. residual surfactant may have an effect on the coupling efficiency of the ADC. Thus, there is a need in the art to find surfactants that can stabilize mAb intermediates, do not degrade themselves, and can be removed by UF/DF steps.

(3) Summary of the invention

The invention aims to solve the problems that a nonionic surfactant in the existing preparation formula is easy to degrade and difficult to remove, and the like, and provides a novel surfactant which can provide stability of protein drugs in antibody-coupled pharmaceutical preparations without influencing the quality of finished products of the preparations and the ratio of drug to antibody (DAR).

In a first aspect the present invention provides a pharmaceutical formulation for use in the preparation of an antibody drug conjugate, the pharmaceutical formulation comprising: macromolecular drug, buffer solution, stabilizer, surfactant and sterile water for injection, wherein:

the concentration of the macromolecular medicament is 5-200mg/mL;

the buffer solution is one or more than two of citrate, histidine salt or phosphate, and the concentration of the salt is 10-20mmol/L, preferably 15-20mmol/L;

the stabilizer is selected from sodium chloride, amino acid, polyol or any combination thereof, the amino acid is selected from arginine, aspartic acid, histidine or any combination thereof, the polyol is selected from sucrose, sorbitol, mannitol or any combination thereof, the concentration of the sodium chloride is 100-200mmol/L, the concentration of the amino acid is 10-200mmol/L, and the concentration of the polyol is 1-15wt%, based on the total weight of the preparation;

the surfactant is selected from any one or combination of polysorbate 20 (PS 20), polysorbate 80 (PS 80), poloxamer 188 (P188), octyl-beta-D-glucopyranoside (OGP), and the concentration of the surfactant is 0.01-0.08 wt%, based on the total weight of the preparation;

and the pH of the pharmaceutical formulation is 4.5-7.5.

In one embodiment of this aspect, the macromolecular drug is selected from the group consisting of an antibody drug conjugate, an antibody, preferably a monoclonal antibody, more preferably an antigen binding fragment, still more preferably a Fab, F (ab) 2, fv or scFv fragment, most preferably a monoclonal antibody portion of an antibody conjugated drug.

In another embodiment of this aspect, the stabilizer is preferably one or a combination of two or more of an amino acid, a polyol. Preferably the concentration of amino acid is 20mmol/L. In another embodiment, the polyol is preferably sucrose or sorbitol, the concentration of the polyol being 4.5wt% to 8.8wt%, preferably 4.5% sorbitol, or preferably 8% sucrose, based on the total weight of the formulation.

In another embodiment, the pH of the formulation is 5.0-7.5, preferably 5.5.

In yet another preferred embodiment, the buffer salt is one of citrate, histidine or phosphate, preferably histidine; the polyalcohol is sucrose; the surfactant is octyl-beta-D-glucopyranoside (OGP); preferably, the concentration of the octyl-beta-D-glucopyranoside is 0.05wt% based on the total weight of the formulation.

In another aspect of the invention, a kit is provided comprising a formulation of the invention, and a container containing the formulation.

In one embodiment of this aspect, the kit further comprises instructions.

In another aspect the present invention provides the use of a combination of a buffer solution, a stabiliser and a surfactant, the buffer solution being one or a combination of two or more of a citrate, a histidine salt or a phosphate salt and the salt having a concentration of 10 to 20mmol/L, preferably 15 to 20mmol/L;

the stabilizer is one or more of sodium chloride, amino acid and polyalcohol, wherein the amino acid is one or more of arginine, aspartic acid and histidine, the polyalcohol is one or more of sucrose, sorbitol and mannitol, the concentration of the sodium chloride is 100-200mmol/L, the concentration of the amino acid is 10-200mmol/L, and the concentration of the polyalcohol is 1-15wt% based on the total weight of the preparation;

the surfactant is one or more than two of polysorbate 20 (PS 20), polysorbate 80 (PS 80), poloxamer 188 (P188) and octyl-beta-D-glucopyranoside (OGP), and the concentration of the surfactant is 0.01-0.08 wt%, based on the total weight of the preparation;

and the pH of the liquid formulation is 4.5-7.5; for the preparation of a pharmaceutical formulation comprising the addition of a macromolecular drug selected from the group consisting of antibody drug conjugates, antibodies, preferably monoclonal antibodies, more preferably antigen binding fragments, still more preferably Fab, F (ab) 2, fv or scFv fragments, most preferably monoclonal antibody portions of antibody conjugated drugs, said formulation further comprising sterile water.

In another aspect of the invention there is provided the use of a pharmaceutical formulation of the invention for the preparation of an antibody drug conjugate product.

The liquid preparation of the invention can keep therapeutic protein stable, so that the protein can stably exist in the preparation formula, the quality of the product is improved, the service life of the product is prolonged, and the clinical use safety is improved. The liquid preparation has better thermal stability, and can be kept stable under the conditions of high temperature acceleration, long-term refrigeration, repeated freezing and thawing and the like.

(4) Description of the drawings

FIG. 1 shows the pyrene molecule hydrophobic factor (I) ₁ /I ₃ ) A profile of variation with surfactant OGP concentration;

FIG. 2 shows fluorescence spectra of BSA in the presence of OGP molecules at different concentrations;

FIG. 3 shows the fluorescence quenching degree of iodide ions with the concentration of iodide ions.

(5) Detailed description of the preferred embodiments

In this application, "macromolecule therapeutic formulation" refers to or consists of a composition comprising a biologic. For example, the macromolecular therapeutic preparation may be a stock solution of a macromolecular pharmaceutical product, a simulated solution containing a macromolecular pharmaceutical product, or the macromolecular pharmaceutical product itself. In this application, "macromolecular pharmaceutical product" refers to a product comprising therapeutic protein drugs and made from various biological materials. In some embodiments of the present application, the macromolecular pharmaceutical product is selected from the group consisting of cytokines, erythropoietin, plasminogen activator, plasma factors, growth hormone, growth factor, insulin, immunoglobulins, antibodies (particularly monoclonal antibodies) and vaccines (e.g., peptide, polypeptide or protein vaccines). In some embodiments, the macromolecular pharmaceutical product comprises or consists of an immunoglobulin. In some embodiments, the macromolecular pharmaceutical product comprises or consists of an antibody. In some embodiments, the biologic comprises or consists of a monoclonal antibody.

The term "antibody" includes whole antibodies and antigen-binding fragments thereof, wherein the antigen-binding fragment comprises an antigen-binding region and at least a portion of a heavy chain constant region comprising asparagine (N) 297 located in CH 2. Typically, the "variable region" contains the antigen binding region of an antibody and relates to the specificity and affinity of binding. See Fundamental Immunology, 7 th edition, paul, wolters Kluwer Health/Lippincott Williams & Wilkins (2013). Light chains are generally classified as either kappa or lambda. Heavy chains are generally classified as gamma, mu, alpha, delta or epsilon, which in turn define immunoglobulin classes IgG, igM, igA, igD and IgE, respectively.

The term "antibody" also includes bivalent or bispecific molecules, diabodies, triabodies and tetrabodies. "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. In some embodiments provided herein, the antibody or antigen binding fragment thereof is a Fab, F (ab) 2, fv, or scFv fragment. In some embodiments provided herein, the antibody is a fully human antibody. In some embodiments of the methods provided herein, the antibody or antigen binding fragment thereof is recombinantly produced.

The term "monoclonal antibody moiety" according to the present invention refers to a monoclonal antibody conjugated in an antibody conjugated drug, or a monoclonal antibody to be used as conjugated moiety in an ADC pharmaceutical formulation, as well as any antigen binding fragment or any functional form thereof.

In some embodiments herein, the antibody or antigen binding fragment is linked to the drug unit by a linker. The number of the drug units may be 1-10. In some embodiments provided herein, the linker is an enzymatically cleavable linker, and wherein the linker forms a bond with the sulfur atom of the antibody or antigen binding fragment thereof.

In this application, the stability index of the macromolecule therapeutic formulation may be selected from one or more of the following: sample appearance, concentration, pH, purity, chromatography (e.g., molecular size gel exclusion chromatography), drug/antibody ratio, insoluble particle count.

Macromolecular therapeutic preparation:

the macromolecule therapeutic formulation of the invention comprises: the monoclonal antibody portion of the antibody-conjugated drug, a buffer solution, a stabilizer, a surfactant, and sterile injection water.

In another preferred embodiment, the macromolecule therapeutic formulation comprises: monoclonal antibodies, buffer solutions and sterile injection water.

In another preferred embodiment, the macromolecule therapeutic formulation comprises: monoclonal antibodies, buffer solutions, polyols and sterile injection water.

In another preferred embodiment, the macromolecule therapeutic formulation comprises: monoclonal antibodies, buffer solutions, amino acids, surfactants, and sterile injection water.

In another preferred embodiment, the macromolecule therapeutic formulation comprises: monoclonal antibodies, buffer solutions, polyols, surfactants and sterile injection water.

The therapeutic protein concentration is between 5-150mg/mL, preferably 10-60mg/mL.

The buffer solution is one of citrate, histidine salt, acetate and phosphate, and the concentration of the buffer is 5-50mM, preferably 5-20mM.

The stabilizer is one or more of amino acid and polyalcohol.

The amino acid is one of arginine, aspartic acid and histidine. The amino acid concentration is 10-200mM, preferably 20-150mM.

The polyalcohol is one or more than two of sucrose, mannitol, trehalose and sorbitol. The concentration is 1-15wt%, preferably 5-10wt%, based on the total weight of the formulation.

The surfactant is polyoxyethylene sorbitan fatty acid ester, polyoxyethylene hydrogenated castor oil, glyceride and fatty acid ester, preferably polysorbate 20 (PS 20), polysorbate 80 (PS 80), poloxamer 188 (P188) and octyl-beta-D-glucopyranoside (OGP), and the content is 0.05-0.8 wt% based on the total weight of the preparation.

General analytical test according to the invention:

appearance: the appearance of the samples, including color, clarity and visible foreign matter, was separately examined in front of the black and white background of the YB-2 clarity detector. The illumination intensity of the clarity detector was set to 2000 to 3750 lux.

Concentration: detection of protein concentration was performed using a ultraviolet spectrophotometer of the zemoer's flight. The extinction coefficient of the exact input molecule during the study was. The sample was applied 2.5. Mu.L each time, and the average value was reported after repeated detection twice. Pure water was used as a blank prior to testing of each sample.

pH: the pH was measured using a multiparameter tester with a glass electrode. Before use, the pH calibration solution (pH 4.01,7.00,9.21) is adopted for calibration, and the calibration slope is in the range of 95.0-105%. Each sample was tested 2 times and the average was calculated.

Osmotic pressure: osmolarity testing was performed using an Advanced 2020Multi-Sample osmolarity meter. In the detection, a standard product Clinitrol 290mOsm/kg is added before and after the sample, the adding volume of each sample is 20mu L, and each sample is detected once.

Size exclusion high performance liquid chromatography (SEC-HPLC): SEC-HPLC is a method of purity analysis that separates according to the size of molecules in solution. The method is run on an Agilent high performance liquid chromatography system using SEC columns. The sample temperature was set at 5℃and the column temperature was set at 25 ℃. The mobile phase composition was 50mM PB,300mM NaCl,pH 6.8 and the flow rate was set to 1.0 mL/min. The sample was diluted to a concentration of 10mg/mL using the mobile phase, 100 μg of the sample was injected into the system and run for 20 minutes, the detection wavelength was set at 280nm. Data analysis was performed using agilent CDS software.

Insoluble particle count (MFI): the MFI was used for detection of insoluble particles in the sample. 1.5mL of the sample was transferred to a MFI-specific 96-well plate using a pipette in a biosafety cabinet and placed on an MFI instrument for detection. Data analysis was performed using software matched to the MFI and reported for ECD at 2-5 μm, 5-10 μm, 10-25 μm and insoluble particle counts greater than 25 μm.

Hydrophobic interaction chromatography-drug antibody ratio (HIC-DAR): detection of drug antibody coupling ratio was performed using hydrophobic interaction chromatography. The method is run on an Agilent high performance liquid chromatography system using an HIA column. Mobile phase a was 1.5M (NH ₄ ) ₂ SO ₄ And 50mM K ₂ HPO ₄ ·3H ₂ O, pH 7.0.+ -. 0.1, mobile phase B50 mM K2 HPO ₄ ·2H ₂ O2-propanol=3:1, ph 7.0±0.1. The concentration of the sample was adjusted to 2.5-20mg/mL. mu.L of sample was injected into the column and eluted with a gradient of 16 minutes at a flow rate of 1.0 mL/min. The detection wavelength was 280nm.

The invention has the technical effects that:

the invention researches the single and double specific antibodies and antibody coupling medicines with poor stability, adopts octyl-beta-D-glucopyranoside (OGP) as a surfactant and a stabilizer and a buffer system to stabilize protein medicines on the premise of not influencing the quality of finished products of preparations and not influencing the antibody ratio (DAR), can keep the stability of the medicines under the conditions of high-temperature acceleration, long-term refrigeration, repeated freezing and thawing and the like, improves the clinical use safety and prolongs the storage time. The surfactant can be effectively removed by the UF/DF process without affecting the finished product or quality of the formulation and the drug-to-antibody ratio (DAR).

The present invention is further illustrated by the following specific examples, which are intended to be illustrative only and not limiting in scope.

Example 1 comparison of the stability of different buffer systems

The protein used in the following examples is a monoclonal antibody, a commercial RANKL (nuclear factor kappa-B receptor activator) specific monoclonal IgG1 antibody Denosumab (denomab) available from the pharmaceutical biotechnology company, shanghai, china, catalog No. MC018-B04W, molecular weight 144.9kDa (monomer).

TABLE 1 buffer salt System to be screened

X represents the test item to be performed

X = appearance, pH, protein concentration, SEC-HPLC

Table 2 buffer System screening appearance test results summary

TABLE 3 summary of buffer System screening concentration and pH detection results

Table 4 summary of the buffer System screening SEC-HPLC detection results

The appearance results showed that all of the formulation solutions were colorless, micro-opalescent solutions. It is worth mentioning that all formulations showed visible particles at T0, possibly due to aggregation of the proteins under strong conditions of blowing, filtering, etc., which also show some anomalies in the protein concentration at T0, due to the presence of visible particles affecting the measurement of the concentration. After standing at 25 ℃ for four weeks, the production of a large number of visible particles was also observed. Protein concentration and pH showed significant changes after four weeks of standing at T0 and 25 ℃. The SEC results show that the phosphate buffer system and high H (more than or equal to 6.0) can cause strong aggregation of the protein, and the citrate system and the histidine system can better perform, and the stability of the protein in the histidine salt (pH 5.5) buffer system is considered to be optimal according to the condition that the SEC main peak is reduced after the SEC buffer system is placed at 25 ℃ for four weeks.

EXAMPLE 2 screening of different stabilizers

According to the screening result of the buffer salt system, 20mM histidine hydrochloride/histidine and pH 5.5 are selected for auxiliary material screening. The auxiliary materials are selected from common several kinds of auxiliary materials including salts (L-arginine hydrochloric acid), sugar alcohols (sucrose and sorbitol) and surfactants (PS 80, PS20 and P188. Five formulas are designed for the auxiliary materials and the type of the surfactants, the auxiliary materials are placed at 300rpm and 25 ℃ in a shaking way, samples are taken at different time points for each detection, the specific formula design and the investigation scheme are shown in a table 5, wherein the protein concentration is 10mg/mL. The detection items comprise appearance observation, pH measurement, concentration measurement, SEC-HPLC and MFI, and the detection results are shown in tables 5 to 9.

TABLE 5 buffer salt/adjuvant System to be screened

Y represents the test item to be performed

Y = appearance, pH, protein concentration, SEC-HPLC, osmolarity, MFI

TABLE 6 buffer salt/adjuvant System screening appearance test results summary

TABLE 7 concentration and pH detection summary of buffer salt/adjuvant System Screen

Table 8 buffer salt/adjuvant System screening SEC-HPLC detection results summarize

TABLE 9 buffer salt/adjuvant System screening MFI detection results summary

The appearance results showed that all of the formulation solutions were colorless, micro-opalescent solutions. A large amount of visible particles appeared in the formulation without the addition of surfactant after T0 and three days of shaking, but no visible particles were observed in the formulation with the addition of surfactant. The proteins of all formulations showed no significant change in concentration, pH, and SEC results after shaking for three days. The MFI results are consistent with the appearance observations, and the particles are significantly reduced compared to the surfactant-added formulation. The above results demonstrate that surfactants play a critical role in the stabilization of the protein.

Example 3 comparison of the protective Effect of different surfactants

For some mAb intermediates, surfactant residues may make the surfactant content of the formulation difficult to determine and may have an impact on the coupling efficiency of the ADC. Therefore we have used a surfactant of lower molecular weight to investigate whether it has the effect of protecting the protein. The protective effect of the screened surfactants including polysorbate 20 (PS 20), polysorbate 80 (PS 80), poloxamer 188 (P188) and various concentrations of octyl- β -D-glucopyranoside (OGP) in a buffer system containing 8% sucrose, 20mM histidine/histidine hydrochloride, pH 5.5, was compared, and the detection items including appearance, pH measurement, concentration measurement, SEC-HPLC, and MFI were shown in table 10.

Table 10 summary of surfactant screening MFI assay results

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As can be seen from the MFI results of the different surfactant stabilized proteins, the novel surfactant OGP has a comparable protective effect on proteins compared to the surfactants PS80, PS20 and P188 commonly used in formulation development. Thus, the appropriate concentration of the novel surfactant OGP is explored next.

Four formulations were designed together for surfactant concentration screening experiments. The samples were taken at various time points for each test at 300rpm,25℃shaking-placed, -70℃C/room temperature freeze-thaw test, the specific formulation design and test protocol are shown in Table 11, with protein concentration of 10mg/mL. The test items include appearance observation, pH measurement, concentration measurement, SEC-HPLC, MFI, and the test results are shown in tables 12 to 15.

TABLE 11 surfactant OGP concentration System to be screened

Z, Q represents the test item to be performed

Z = appearance, pH, protein concentration, SEC-HPLC, osmolarity

Q＝MFI

TABLE 12 appearance test results of surfactant OGP concentration screening System

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TABLE 13 concentration of surfactant OGP concentration screening System concentration and pH detection results are summarized

TABLE 14 summary of SEC detection results for surfactant OGP concentration screening System

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Table 15 buffer system screening MFI test results summary

Proteins of all formulations showed no significant change in concentration, pH, and SEC results after five rounds of freeze thawing and three days of shaking. The appearance results showed that all of the formulation solutions were colorless, micro-opalescent solutions. The appearance results were consistent with the MFI results, and an interesting phenomenon was observed, the protein stabilizing effect did not increase with increasing OGP concentration. Formulations containing 0.8% ogp produced a large amount of visible particles after five rounds of freeze thawing and three days of shaking, whereas formulations containing 0.05% ogp did not observe visible particle production. The above results demonstrate that the more surfactant content is not effective in stabilizing the protein and that the lower the level the more effective the protein is.

Example 4 comparison of effects of different surfactants on drug to antibody ratio (DAR)

The extent of effect of the surfactant octyl-beta-D-glucopyranoside (OGP) on the drug/antibody ratio (DAR) was investigated by comparison in a buffer system comprising 8% sucrose, 20mM histidine/histidine hydrochloride, pH 5.5, and the detection term included hydrophobic interaction chromatography-DAR (HIC-DAR). The results show that under the buffer system, the average DAR value is 1.62, which is close to the target DAR, and the effect of octyl-beta-D-glucopyranoside (OGP) on the coupling efficiency of the drug is small.

Example 5 comparison of the removal rates of different surfactants via UF/DF step

The removal rates of the different surfactants via the UF/DF steps were compared in a buffer system containing 8% sucrose, 20mM histidine/histidine hydrochloride, pH 5.5, and the screened surfactants included polysorbate 20 (PS 20), polysorbate 80 (PS 80), poloxamer 188 (P188), octyl- β -D-glucopyranoside (OGP), the test items included PS80 assay, P188 assay, and octyl- β -D-glucopyranoside (OGP) assay, and the test results are shown in table 16. Wherein the cleaning efficiency is the ratio of the concentration of the surfactant after UF/DF to the concentration of the surfactant T0.

The results show that octyl-beta-D-glucopyranoside (OGP) can be removed through UF/DF, so that the coupling efficiency of the subsequent ADC drugs is avoided, but polysorbate 20 (PS 20), polysorbate 80 (PS 80) and poloxamer 188 (P188) cannot be removed well through the UF/DF steps.

Example 6 mechanism exploration of OGP surfactant stabilization of therapeutic proteins

In example 3, it was found that the protective effect of OGP on the protein was not positively correlated with the concentration of OGP, which led the inventors to study the mechanism of OGP stabilization of the protein. Here, bovine Serum Albumin (BSA) was used as a model protein. In the field of colloidal chemistry, pyrene probes are commonly used to characterize the overall process of micelle formation of surfactant molecules ^[1] (Ba-Salem AO, duhamel J.Synthesis and Characterization of a Pyrene-Labeled Gemini Surfactant Sensitive to the Polarity of Its environmental.Langmuir.2021 Nov 30;37 (47): 13824-13837.doi:10.1021/acs.langmuir.1c01759.Epub 2021 Nov 18.). When the concentration of pyrene molecules in the solution is lower than 10 ^-5 M, excited state fluorescence having a fine structure is observed, and pyrene molecules have a plurality of fluorescence emission peaks. Wherein the ratio of fluorescence intensities of the first peak (373 nm) and the third peak (384 nm) is very sensitive to the polarity of the microenvironment in which the pyrene molecule is located, commonly referred to as the hydrophobic factor (I) ₁ /I ₃ ). The smaller the hydrophobic factor, the more hydrophobic the microenvironment in which the pyrene molecule is located. Pyrene probes were intended to assess the interaction of BSA with OGP.

FIG. 1 shows the hydrophobic factor (I ₁ /I ₃ ) Curves as a function of the concentration of surfactant OGP. As shown in FIG. 1, the black squares indicate a simple OGP solution, I when no OGP molecules are present in the solution ₁ /I ₃ The ratio is 1.8, I is the beginning ₁ /I ₃ The slow decrease with increasing OGP concentration indicates that surfactant molecules begin to adsorb at the interface in the solution. With increasing OGP monomer and when the concentration reaches a certain value, I ₁ /I ₃ The decrease was rapid, indicating a gradual increase in the surfactant number at the interface. Then gradually reach the platform, indicating that the OGP molecules form micelles. This process is a process that reflects the overall process of forming micelles by surfactant molecules using the gradual transfer of pyrene molecules from bulk solution to the micelle core. While the red dots are shown in the tableIt is clear that the OGP solution with BSA added, I ₁ /I ₃ The profile as a function of OGP concentration is different from that of a single surfactant solution. When the OGP concentration is low, the addition of BSA results in I ₁ /I ₃ The ratio is very low, which is a result of the BSA. When the concentration reaches a certain value I ₁ /I ₃ Increasing the concentration of OGP corresponding to the concentration of the protein and I in a pure OGP system ₁ /I ₃ The concentration at the beginning of the decrease was substantially the same. This suggests that the surfactant molecules associate around the protein by hydrophobic interactions, squeezing pyrene molecules out of the hydrophobic domains of the protein, thus I ₁ /I ₃ The ratio increases. When the combination of the surfactant molecules on the macromolecules reaches saturation, as the concentration continues to increase, the surfactant molecules start to form normal micelles, and the pyrene molecules are solubilized again in the hydrophobic domains of the pre-micelles, thus I ₁ /I ₃ The ratio again begins to decrease. Finally form micelle I ₁ /I ₃ The ratio no longer changes. This result suggests that the mechanism by which OGP stabilizes proteins may be through binding to proteins.

Fig. 2 shows BSA fluorescence spectra in the presence of different concentrations of OGP molecules. From FIG. 2, it can be seen that the fluorescence intensity of BSA decreases with increasing OGP concentration, while the peak position shifts blue (peak position shifts blue from 347nm to 335 nm). Literature ^[2] (Wen L, lyu M, xiao H, lan H, zuo Z, yin Z.J.Protein Aggregation and Performance Optimization Based on Microconformational Changes of Aromatic Hydrophobic regions.mol pharm.2018;15 (6): 2257-2267.Doi:10.1021/acs.mol pharmacea.8 b 00115) shows that the blue shift of the fluorescence peak indicates that the protein is turning into a more hydrophobic environment. Based on structural and sequence analysis of BSA, OGP molecules may be bound near tryptophan residues in BSA, resulting in increased hydrophobicity of the environment surrounding the tryptophan residues. As the OGP concentration further increases to approach the critical micelle concentration, at this point the binding of the OGP molecules to BSA becomes saturated and no more OGP molecules bind, and thus the fluorescence intensity no longer changes and the peak position no longer blue shifts.

To further verify the conclusion, an iodide quenching experiment was performed. The principle is that iodide ions can only react with tryptophan residues located on the surface of the protein as a quencher, and cannot quench tryptophan residues inside the protein. According to the Stern-Volmer quenching equation:

F ₀ /(F-F ₀ )＝1/(f ₀ K[I ^- ])+1/f ₀

wherein K is a Stern-Volmer constant; f is the fluorescence intensity in the presence of iodide ions; f (F) ₀ Fluorescence intensity in the absence of iodide ions; f (f) ₀ Tryptophan residues which are quenched by iodide ions, in proportion to the total residues; [ I ]]Iodine ion concentration.

FIG. 3 shows the degree of fluorescence quenching of iodide ions as a function of iodide ion concentration. Analysis found that the slope of the change in iodide fluorescence plotted against iodide concentration indicates the proportion of tryptophan residues quenched by iodide to total residues. FIG. 3 shows the effect of iodide on BSA fluorescence intensity with and without OGP. The slope of the red dot curve is higher than the slope of the black square curve indicating that the addition of OGP increases the proportion of tryptophan residues on the BSA surface.

Since there is a large hydrophobic cavity in the BSA molecule, almost all of the hydrophobic residues are located inside the hydrophobic cavity, and tryptophan in the BSA molecule is located just inside the hydrophobic cavity, it is highly likely that the OGP molecule enters the hydrophobic cavity inside the BSA molecule and interacts with the hydrophobic residues inside the cavity by hydrophobic forces, resulting in a decrease in the number of internal residues and thus an increase in the relative number of tryptophan residues on the surface. It was concluded that the binding sites of OGP molecules are located within the hydrophobic cavity of BSA, which is similar to the binding behavior of many nonpolar small molecule ligands. Thus we believe that the interaction between the OGP molecule and the protein may be similar to the interaction between P188 and the protein, both interacting with the protein, inhibiting aggregation while reducing adsorption at the interface.

The research results show that the liquid preparation prepared from the surfactant OGP, the buffer solution and the stabilizer and the therapeutic protein, particularly the monospecific antibody, the bispecific antibody and the antibody coupling drug with poor stability, has better stability. The surfactant OGP can be removed in the UF/DF step, and the protein medicine is stabilized on the premise of not influencing the quality of finished products of the preparation and the antibody ratio (DAR), and the stability of the protein medicine can be maintained under the conditions of high-temperature acceleration, long-term refrigeration, repeated freezing and thawing and the like, so that the clinical use safety is improved, and the storage time is prolonged.

Claims

1. A pharmaceutical formulation for use in preparing an antibody conjugate, the pharmaceutical formulation comprising: macromolecular drug, buffer solution, stabilizer, surfactant and sterile water for injection, wherein:

the concentration of the macromolecular medicament is 5-200mg/mL;

and the pH of the pharmaceutical formulation is 4.5-7.5.

2. The formulation of claim 1, wherein the macromolecular drug is selected from the group consisting of antibody drug conjugates, antibodies, preferably monoclonal antibodies, more preferably antigen binding fragments, still more preferably Fab, F (ab) 2, fv or scFv fragments, most preferably monoclonal antibody portions of antibody conjugated drugs.

3. The formulation of claim 1, wherein the stabilizer is one or a combination of two or more of an amino acid, a polyol.

4. The formulation of claim 1, wherein the amino acid has a concentration of 20mmol/L.

5. The formulation of claim 1, the polyol preferably being sucrose or sorbitol, the concentration of the polyol being 4.5wt% to 8.8wt%, based on the total weight of the formulation.

6. The formulation according to claim 1, having a pH of 5.0-7.5, preferably 5.5.

7. The formulation of claim 1, wherein the buffer salt is one of citrate, histidine or phosphate, preferably histidine; the polyalcohol is sucrose; the surfactant is octyl-beta-D-glucopyranoside (OGP); preferably, the concentration of the octyl-beta-D-glucopyranoside is 0.05wt% based on the total weight of the formulation.

8. A kit comprising the formulation of claim 1, and a container containing the formulation.

9. The kit of claim 8, further comprising instructions.

10. Use of a combination of a buffer solution, a stabilizer and a surfactant, wherein the buffer solution is one or a combination of more than two of citrate, histidine salt or phosphate, and the concentration of the salt is 10-20mmol/L, preferably 15-20mmol/L;

and the pH of the liquid formulation is 4.5-7.5; for the preparation of a pharmaceutical formulation comprising the addition of a macromolecular drug selected from the group consisting of antibody drug conjugates, antibodies, preferably monoclonal antibodies, more preferably antigen binding fragments, still more preferably Fab, F (ab) 2, fv or scFv fragments, most preferably monoclonal antibody portions, said formulation further comprising sterile water.