Subcutaneous administration preparation of coupled medicine as well as preparation method and application of subcutaneous administration preparation

Abstract

The invention relates to a subcutaneous administration preparation of a coupled drug as well as a preparation method and application of the subcutaneous administration preparation. The subcutaneous administration preparation comprises an antibody coupled drug and recombinant hyaluronidase. According to the invention, the hyaluronidase and the antibody coupling medicine are matched for use, so that the stability is relatively high, and the hyaluronidase can be compounded with the high-concentration antibody coupling medicine for use.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a subcutaneous drug-coupled preparation and a preparation method and application thereof. Background Art

[0002] In recent years, drug conjugates have become a hot topic in the field of new drug research and development. Due to their excellent therapeutic effects, they have become important drug molecules in tumor treatment. Antibody-drug conjugates (ADCs) are chemical immunoconjugates composed of highly active drugs and monoclonal antibodies. They have both the high activity of small molecules and the high targeting of monoclonal antibodies. Through this property, drugs can be "precisely" delivered to target cells, thereby further increasing the concentration of drugs accumulated in tumor sites and reducing the damage of drugs to other tissues and organs in the body, thereby achieving the purpose of increasing drug effects and reducing their toxic side effects. The antibody part is responsible for accurately transporting ADC drug molecules to the surface of target cells, and the connector is responsible for releasing small molecules in or on the surface of target cells, while highly active small molecules efficiently kill tumor cells. Therefore, ADCs are also called "biological missiles" or "magic bullets."

[0003] All the antibody-drug conjugates that have been marketed are administered intravenously, which requires professional and skilled medical staff to perform and is time-consuming, taking several hours. Subcutaneous injection of biotherapeutic drugs has become a valuable alternative to intravenous administration in many disease areas. Many traditional intravenous drugs have been further developed by pharmaceutical companies based on subcutaneous preparations, which have been used in rheumatoid arthritis, primary immunodeficiency disease, multiple sclerosis, and tumor drug antibody drugs.

[0004] However, the volume limitation of subcutaneous administration is the primary challenge in the development of conjugate drug formulations. High-concentration formulations are the preferred solution to this problem, but there are still challenges for some conjugate drug molecules that are difficult to concentrate at high concentrations or for which the dosage is large. Currently, there are a variety of technical types that can be used: ① Reducing drug viscosity by adding special substances, but some substances are not pharmaceutical excipients, and their toxicity is unknown, so there are risks in clinical application and project application at the current stage; ② Reducing drug viscosity by preparing protein samples into suspension fluids, but the preparation process is relatively complex, and there are currently no marketed products; ③ Subcutaneous delivery of high-viscosity drugs through drug delivery devices, which are expensive; ④ Subcutaneous drug delivery through diffusion enhancers.

[0005] Since its discovery in 1928, hyaluronidase is an enzyme that can hydrolyze the β1→4 glycosidic bonds of hyaluronic acid, which is widely present under the skin, into oligosaccharides of various lengths. It has been used as a functional excipient in antibody drugs and small molecule drugs, but there are currently no reports on its application in conjugated drugs.

[0006] Therefore, how to develop an antibody-drug conjugate preparation that has no effect on the effect of the antibody-drug conjugate and can be administered subcutaneously has become an urgent problem to be solved. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a subcutaneous drug-coupled preparation and a preparation method and application thereof. It is found in the present invention that the combined use of hyaluronidase and antibody-coupled drugs has high stability and can be compounded with high-concentration antibody-coupled drugs for use.

[0008] To achieve this object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a subcutaneous drug-conjugate preparation, wherein the subcutaneous drug-conjugate preparation comprises an antibody-conjugate drug and a recombinant hyaluronidase.

[0010] In the present invention, recombinant hyaluronidase is used in combination with an antibody-drug conjugate, wherein the hyaluronidase can degrade hyaluronic acid during subcutaneous injection to reversibly change the dermal structure, increase tissue permeability, and thus promote drug diffusion. Hyaluronic acid is a diffusion enhancer and the main mucopolysaccharide form in the dermis. It is a physical barrier to the diffusion of subcutaneously administered drugs. Hyaluronidase reversibly changes the dermal structure by degrading hyaluronic acid, increases tissue permeability, and thus promotes drug diffusion.

[0011] Preferably, the amino acid sequence of the recombinant hyaluronidase includes the sequence shown in SEQ ID No.1 or the sequence after mutation of SEQ ID No.1.

[0012] SEQ ID No.1:

[0013] LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDW EEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTAL YPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTL AAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY.

[0014] Preferably, the mutated sites include site 12 substituted by V to E, site 58 substituted by V to K, site 83 substituted by I to V, site 140 substituted by Q to K, site 142 substituted by V to P, site 143 substituted by Q to K, site 165 substituted by L to F, site 234 substituted by Q to M, site 249 substituted by E to V, site 261 substituted by S to N, site 277 substituted by V to K, site 291 substituted by G to V, site 310 substituted by M to Q, site 368 substituted by D to R, site 392 substituted by E to Q, site 395 substituted by S to A, site 421 substituted by D to H, site 431 substituted by D to K, site 433 substituted by F to V, site 438 substituted by M to N, site 439 substituted by E to A, and site 445 substituted by I to M, any one or a combination of at least two.

[0015] Preferably, the drug connected in the antibody-drug conjugate includes any one of camptothecin derivative SN38, exitecan derivative Dxd, calicheamicin, dukamycin, anthraxin, monomethyl auristatin E, monomethyl auristatin F, maytansine DM1, maytansine DM4, Tubulysins, eribulin or α-amanitin, or a combination of at least two thereof.

[0016] The drugs connected in the antibody-drug conjugate include DNA topoisomerase inhibitors (such as camptothecin derivatives and SN38, exotecan and its derivative Dxd), DNA alkylating agents (such as calicheamicin, dukamycin and anthraxin PBD, etc.), microtubule protein disruptors (such as monomethyl auristatin E, monomethyl auristatin F, maytansine DM1, maytansine DM4, Tubulysins, Eribulin), RNApoⅢ inhibitors (such as α-amanitin), etc.

[0017] In a specific embodiment, the antibody-drug conjugate used is Trastuzumab-Dxd (trastuzumab-exitecan derivative conjugate).

[0018] Preferably, the antibody comprises any one of a bispecific antibody, a monospecific antibody or an antibody fragment.

[0019] Preferably, the targets of the antibody include any one or a combination of at least two of HER2, HER3, cMet, Trop-2, Claudin 18.2, Nectin-4, FRα, PD-1, PD-L1, EGFR, cMet, HER3 or EGFR.

[0020] Preferably, the antibody coupling ratio of the antibody-drug conjugate is 2-8, for example, it can be 2, 3, 4, 5, 6, 7 or 8.

[0021] In one embodiment of the present invention, the antibody-drug conjugate used is trastuzumab linked to an isotecan derivative with an antibody conjugation ratio of 6. It can be seen from this embodiment that adding hyaluronidase and excipients to the antibody-drug conjugate has no effect on the physical properties and drug properties of the antibody-drug conjugate. There is reason to believe that other antibody-drug conjugates can also achieve similar effects.

[0022] Preferably, the concentration of the antibody-drug conjugate is 10-150 mg / mL, for example, it can be 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL or 150 mg / mL, etc.

[0023] In the present invention, the concentration of the antibody-drug conjugate that can be used is 10-150 mg / mL, that is, the compound preparation of the present invention can be used in combination with the antibody-drug conjugate.

[0024] Preferably, the concentration of the recombinant hyaluronidase is 500-10000U / mL, for example, it can be 500U / mL, 1000U / mL, 1500U / mL, 2000U / mL, 2500U / mL, 3000U / mL, 3500U / mL, 4000U / mL, 4500U / mL, 5000U / mL, 5500U / mL, 6000U / mL, 6500U / mL, 7000U / mL, 7500U / mL, 8000U / mL, 8500U / mL, 9000U / mL, 9500U / mL or 10000U / mL, etc.

[0025] It is found in the present invention that the concentration of recombinant hyaluronidase can be compounded with the antibody-drug conjugate within the above range. According to the above, the recombinant hyaluronidase increases the subcutaneous injection volume, and a higher injection volume can be obtained by adjusting the concentration of the recombinant hyaluronidase.

[0026] Preferably, the subcutaneous administration preparation further comprises an auxiliary material.

[0027] Preferably, the excipients include a buffer, a surfactant and a stabilizer.

[0028] Preferably, the buffer comprises any one or a combination of at least two of histidine buffer, glutamate buffer, acetate buffer, succinate buffer, phosphate buffer, Tris buffer, 2-(N-morpholino)ethanesulfonic acid MES buffer, 3-(N-morpholino)propanesulfonic acid MOPS buffer, piperazine-N,N'-bis(2-ethanesulfonic acid) Pipes buffer).

[0029] Preferably, the pH value of the buffer is 5.0-6.5, for example, it can be 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4 or 6.5, etc.

[0030] In the present invention, it is found that within the above pH range, the appearance of hyaluronidase is clear and the enzyme activity is relatively high, and in the compound preparation, within the above range, the activity of the antibody-drug conjugate is relatively high.

[0031] Preferably, the concentration of the buffer is 10-50 mM, for example, it can be 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM or 50 mM, etc.

[0032] Preferably, the stabilizer comprises any one of sucrose, trehalose, dextran, glucose, lactose, maltose, xylose, mannitol or sorbitol, or a combination of at least two thereof.

[0033] Preferably, the concentration of the stabilizer is 1%-10%, for example, it can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%.

[0034] Preferably, the surfactant includes any one of polysorbate 80, polysorbate 20, poloxamer 188, polyethylene glycol 3350, sodium dodecyl sulfate or sodium dodecylbenzene sulfonate, or a combination of at least two thereof.

[0035] Preferably, the concentration of the surfactant is 0.01%-0.2%, for example, it can be 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19% or 0.2%, etc.

[0036] Preferably, the auxiliary material further comprises amino acids and / or chelating agents.

[0037] Preferably, the amino acids include any one or a combination of at least two of arginine, proline, methionine, cysteine, glutamine, glycine, leucine, lysine, phenylalanine, serine, tryptophan, tyrosine or valine.

[0038] Preferably, the concentration of the amino acid is 5-150 mM, for example, it can be 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM or 150 mM, etc.

[0039] Preferably, the chelating agent comprises ethylenediaminetetraacetic acid and / or diethylenetriaminepentaacetic acid.

[0040] Preferably, the concentration of the chelating agent includes 0.001%-0.04%, for example, it can be 0.001%, 0.002%, 0.0013%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03% or 0.04%, etc.

[0041] In a second aspect, the present invention provides a method for preparing a subcutaneous drug-coupled preparation according to the first aspect, the preparation method comprising mixing an antibody-coupled drug, recombinant hyaluronidase and excipients, and filtering to obtain the subcutaneous drug-coupled preparation.

[0042] Preferably, the pore size of the filtering membrane is 0.1-0.5 μm, for example, it can be 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm or 0.5 μm.

[0043] In a third aspect, the present invention provides a use of the subcutaneous drug-coupled preparation according to the first aspect in the preparation of an antibody drug.

[0044] Compared with the prior art, the present invention has at least the following beneficial effects:

[0045] The present invention finds that the combination of hyaluronidase and antibody-drug conjugate has high stability and can be used in combination with high-concentration antibody-drug conjugates. When used in combination with a variety of excipients, the stability of hyaluronidase can be significantly improved, and the quality attributes of the antibody-drug conjugate have no effect. It can also be used in combination with high-concentration antibody-drug conjugates to significantly increase the concentration of the antibody-drug conjugate. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a bar graph showing the effect of pH / buffer on the stability of hyaluronidase activity.

[0047] Figure 2 This is a bar chart showing the effect of excipients on the stability of hyaluronidase activity.

[0048] Figure 3 Bar graph showing the effect of excipients on the SEC purity stability of hyaluronidase.

[0049] Figure 4 This is a graph showing the enzyme activity stability results of the combined preparation of hyaluronidase and ADC.

[0050] Figure 5 This is a graph showing the SEC purity (main peak) stability results of the combined preparation of hyaluronidase and ADC.

[0051] Figure 6 This is a graph showing the SEC purity (polymer) stability results of the combined preparation of hyaluronidase and ADC.

[0052] Figure 7 This is a graph showing the SEC purity (oligomer) stability results of the combined preparation of hyaluronidase and ADC.

[0053] Figure 8 This is a graph showing the stability results of the DAR value of the combination preparation of hyaluronidase and ADC.

[0054] Fig. 9 This is a graph showing the stability results of the free drug content in the combination preparation of hyaluronidase and ADC. DETAILED DESCRIPTION

[0055] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0056] Sources of reagents used in the following examples:

[0057] Histidine buffer: 1.04352.9010, Merck;

[0058] Histidine hydrochloride: 1.04354.0500, Merck;

[0059] TCEP: C4706-2G, Sigma-Aldric;

[0060] ZnCl2: STBK6550, Sigma-Aldrich;

[0061] Dimethylacetamide: Sigma-Aldrich;

[0062] GGFG-Dxd: WuXi AppTec.

[0063] Example 1

[0064] This example explores the effect of different pH / buffer conditions on the stability of hyaluronidase

[0065] As shown in Table 1, four groups of reagents at different pH values ​​were used to explore the effects on the stability of hyaluronidase. The hyaluronidase used in this example was the sequence SEQ ID No. 2 after mutation of SEQ ID No. 1.

[0066] SEQ ID No.2:

[0067] LNFRAPPVIPNEPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYKDRLGYYPYIDSITGVTVNGGIPQKVSLQDHLDKAKKDITFYMPVDNLGMAVIDW EEWRPTWARNWKPKDVYKNRSIELVQQKNPKLSLTEATEKAKQEFEKAGKDFFVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTAL YPSIYLNTQMSPVAATLYVRNRVRVAIRVSKIPDAKNPLPVFAYTRIVFTDQKLKFLSQDELVYTFVETVALGASGIVIWGTLSIQRSMKSCLLLDNYMETILNPYIINVTL AAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPRNFAIQLEKGGKFTVRGKPTLEDLQQFAEKFYCSCYSTLSCKEKADVKDTDAVHVCIADGVCIKAVLKPPNATEEPQMFY.

[0068] The hyaluronidase solution was replaced with the corresponding buffer by centrifugal exchange. The concentrations after the replacement were 2.2 mg / mL, 1.9 mg / mL, 1.8 mg / mL and 3.9 mg / mL, respectively, and then diluted to 0.5 mg / mL with the corresponding buffer. In the biological safety cabinet, all preparations were sterilized and filtered through a 0.22 μm filter, and 0.6 mL was filled into a sterile 2 mL medium borosilicate glass tube anti-jump stopper syringe bottle, which was plugged with a chlorobutyl coated rubber stopper and rolled. The samples were placed at 35°C for 3 days, 7 days and 2 weeks to examine the appearance, pH value and enzyme activity.

[0069] Table 1

[0070]

[0071] (1) Appearance and pH value

[0072] The specific results are shown in Table 2. From the 2-week data at 35°C, it can be seen that in the histidine buffers of pH 5.5, pH 6.0 and pH 6.5 and the phosphate buffer of pH 7.0, there is no obvious change in the pH value, and particles appear in the appearance, which may be due to the lack of added surfactant.

[0073] Table 2

[0074]

[0075] (2) Enzyme activity

[0076] After being placed at 35°C for 3 days, 7 days and 2 weeks, the samples were placed at 40°C for 2 weeks and 4 weeks, and the enzyme activity results were investigated. The enzyme activity was determined by turbidity, where hyaluronidase was incubated with hyaluronic acid and then undigested hyaluronic acid was precipitated with acidified serum protein (horse serum) to determine the enzyme activity. The turbidity was measured at a wavelength of 640 nm, and the turbidity reduction of hyaluronic acid in the well plate indicated the enzyme activity, and the specific results are shown in Table 3.

[0077] Table 3

[0078]

[0079] Specific results such as Figure 1 As shown, when the sample was placed at 35°C for 2 weeks, the enzyme activity did not decrease significantly. When the sample was placed at 40°C, the F4 (phosphate buffer pH 7.0) group was the first to show a decrease in enzyme activity compared to other groups (histidine buffer). Therefore, histidine is preferred as the buffer for hyaluronidase to screen the auxiliary materials for hyaluronidase.

[0080] Example 2

[0081] This example explores the effect of different excipients on the stability of hyaluronidase

[0082] In order to explore the effects of different excipients on hyaluronidase, sodium chloride (NaCl), arginine hydrochloride (Arg·HCl), methionine (Met) and ethylenediaminetetraacetic acid (EDTA) were mixed with hyaluronidase to explore their effects on its stability.

[0083] The specific groups are shown in Table 4. The hyaluronidase solution was replaced with the buffer solution by centrifugal exchange, and then different excipient solutions were added, and finally diluted to 0.5 mg / mL and 4000 U / mL with the corresponding buffer solution. In the biological safety cabinet, all preparations were sterilized and filtered through a 0.22 μm filter, and filled into sterile 2 mL medium borosilicate glass tube anti-jump stopper syringe bottles, plugged with chlorobutyl coated rubber stoppers, and capped. The pH value, osmotic pressure, protein concentration, molecular size exclusion chromatography purity and thermal denaturation temperature of the T0 sample were measured. At the same time, the sample was placed at 40°C for 4 weeks to examine the appearance, pH value, molecular size exclusion chromatography purity and enzyme activity.

[0084] Table 4

[0085]

[0086] (1) Appearance and physical parameters

[0087] The specific results are shown in Table 5. Different excipients have no effect on the appearance of hyaluronidase, and there is no significant change in pH value, osmotic pressure and hyaluronidase concentration over a long period of time, indicating that excipients have no effect on the appearance and some physical parameters of hyaluronidase.

[0088] Table 5

[0089]

[0090] (2) Thermal denaturation temperature

[0091] The thermal denaturation temperature of the sample was determined using MicroCal PEAQ DSC (Malvern, MAL1268569). The sample concentration was 0.5 mg / mL and the sample detection volume was 400 μL. The temperature range was 20-100°C, the heating rate was 200°C / h, and the thermal spectra were analyzed in the MicroCal PEAQ DSC automatic data analysis software.

[0092] Table 6

[0093]

[0094] The specific results are shown in Table 6. In the five groups of formulations, the thermal denaturation onset temperatures (Tm onset) of the recombinant hyaluronidase were all higher than 50°C, indicating that the molecule has good conformational stability.

[0095] (3) Enzyme activity

[0096] The enzyme activity was determined by turbidimetry by incubating the hyaluronidase variant with hyaluronic acid and then precipitating the undigested hyaluronic acid with acidified serum protein (horse serum). The turbidity was measured at a wavelength of 640 nm, and the decrease in turbidity of the hyaluronic acid in the well plate indicated the enzyme activity.

[0097] Table 7

[0098]

[0099]

[0100] Specific results such as Figure 2 As shown in the figure, after being placed at 40°C for 1 week, the enzyme activity of hyaluronic acid in A2 (Arg·HCl) decreased. After being placed at 40°C for 2 or 4 weeks, the decrease in the A3 / A5 (NaCl+Met) group and A4 (NaCl+EDTA) was less than that in A1 (NaCl) and A2 (Arg·HCl).

[0101] (4) Molecular size exclusion chromatography purity

[0102] By size exclusion chromatography, proteins are separated according to their size to detect high molecular weight substances and low molecular weight hydrolysis products in the sample. This method uses an ultra-high performance liquid chromatograph equipped with a SEC column (Waters Acquity BEH SEC 150×4.6mm, 1.7μm) and a UV detector (detection wavelength: 280nm). Mobile phase: 50mM phosphate buffer, 300mM sodium chloride, pH 6.8. Flow rate: 0.4mL / min. The sample load was 10μg. The specific results are shown in Table 8.

[0103] Table 8

[0104]

[0105] Specific results such as Figure 3 As shown, the A3 (sodium chloride + methionine) group and the A4 (sodium chloride + EDTA) group can effectively maintain the thermal stability and antioxidant properties of hyaluronidase. Therefore, in the feasibility study of the combination of hyaluronidase and antibody-drug conjugates, the antioxidant methionine was added to improve stability.

[0106] In this experiment, it was found that the use of methionine can significantly improve stability, and it also has a good effect in A5. A5 will be used in subsequent experiments.

[0107] Example 3

[0108] This example is used to prepare antibody-drug conjugates

[0109] The antibody used in this example is trastuzumab, wherein the antibody sequence is as follows: The light chain sequence of trastuzumab is SEQ ID No.3, and the heavy chain sequence is SEQ ID No.4.

[0110] SEQ ID No.3:

[0111] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0112] SEQ ID No.4:

[0113] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0114] 0.30 mM TCEP and 0.15 mM ZnCl2 were mixed and added to a 0.038 mM trastuzumab solution (solution system pH 7, 40 mM phosphate buffer) and the reaction mixture was allowed to stand overnight at 12°C. 5 mM EDTA was added to capture Zn 2+ , 0.52 mM tetrapeptide-coupled topoisomerase-1 inhibitor exotecan derivative (GGFG-Dxd) dissolved in dimethylacetamide was added, and the reaction was continued at 12°C for 1 hour. The product buffer was replaced with a 20 mM histidine buffer at pH 5.5 using an ultrafiltration tube (30K, UFC803024, Thermo Fisher) to remove excess small molecule impurities in the reaction system. After the preparation was completed, the purified ADC was characterized, and the results are shown in Table 9.

[0115] Table 9

[0116]

[0117] From the above results, it can be seen that this example successfully prepared an antibody-drug conjugate with a DAR of 6.

[0118] Example 4

[0119] This example explores the stability of the combined preparation of hyaluronidase and antibody-drug conjugates

[0120] The antibody-drug conjugate prepared in Example 3 was replaced with ultrafiltration to a 20 mM histidine buffer system, and then excipients and hyaluronidase (rHuPH20) were added to the target concentration according to Table 10. All preparations were sterile filtered through a 0.22 μm filter in a biological safety cabinet, and filled into sterile 2 mL borosilicate glass tube anti-jump stopper syringe bottles, plugged with chlorobutyl coated rubber stoppers, and capped. The samples were placed at 40°C for 1 week and 2 weeks.

[0121] Table 10

[0122]

[0123]

[0124] (1) Appearance and pH value

[0125] The specific results are shown in Table 11. The concentrations of different antibody-drug conjugates and pH values ​​have no effect on the appearance of the composite preparation. After being placed, the appearance is highly stable.

[0126] Table 11

[0127]

[0128] (2) Enzyme activity

[0129] The enzyme activity was determined by turbidimetry, where the hyaluronidase variant was incubated with hyaluronic acid and then the undigested hyaluronic acid was precipitated with acidified serum albumin (horse serum) to determine the enzyme activity. Turbidity was measured at a wavelength of 640 nm, and the turbidity of the hyaluronic acid in the well plate was reduced to indicate enzyme activity. The specific results are shown in Table 12.

[0130] Table 12

[0131]

[0132]

[0133] pass Figure 4 The results show that after being placed for 2 weeks, the enzyme activity of the experimental groups B2-B6 did not change much, indicating that the addition of antibody-drug conjugates had no significant effect on the activity of hyaluronidase.

[0134] Example 5

[0135] This example explores the effect of hyaluronidase combined with antibody-drug conjugates on antibody-drug conjugates

[0136] (1) Protein concentration

[0137] The specific results can be seen from Table 13. After being placed, the protein concentration decreased to a small extent, but the overall decrease was not large, indicating that hyaluronidase had no obvious effect on the antibody-drug conjugate.

[0138] Table 13

[0139]

[0140] (2) Molecular size exclusion chromatography purity

[0141] The above samples B1-B6 were detected on an Agilent 1260HPLC system equipped with a TSKgelG3000SWXL column (7.8×300mm, 2.5μm particle size) and a UV detector (detection wavelength: 280nm). The sampler temperature was set to 123°C and the column oven temperature was set to 25°C. The mobile phase was 50mM phosphate buffer (PB) and 300mM sodium chloride, the pH value was 6.8, and the flow rate was set to 1.0mL / min. 100μg of each sample was injected into an HPLC vial. The detection wavelength was set to 280nm and the running time was 18 minutes. Finally, all raw data were processed using chromatographic data analysis software (Agilent Openlab, Agilent Corporation). The specific results are shown in Table 14.

[0142] Table 14

[0143]

[0144] By result By Figure 5-7 It can be seen that the molecular size exclusion chromatography purity of the combined preparation of hyaluronidase and antibody-drug conjugates was slightly reduced (reduced by 0.5-1.3%) compared with T0 when placed at 40°C for 2 weeks, indicating good stability and no obvious effect of hyaluronidase on the antibody-drug conjugates.

[0145] (3)DAR value

[0146] The DAR value was determined by liquid chromatography-mass spectrometry. 50 μg of protein and 10 μL of 50 mM TCEP solution were added to a 1.5 mL centrifuge tube, mixed, and incubated at 37°C for 30 min. 8μm column, 2.1×50mm) and an Agilent HPLC system equipped with a TOF mass spectrometer detector. The DAR value was calculated as follows. The specific results are shown in Table 15 and Figure 8 shown.

[0147] Light chain drug loading distribution, LP(i) = (deconvoluted mass spectrum intensity of drug-loaded light chain, LP(i) component) / (total deconvoluted mass spectrum intensity of all light chains) × 100%

[0148] Heavy chain drug loading distribution, LP(j) = (deconvoluted mass spectrum intensity of drug-loaded heavy chain, LP(j) component) / (total deconvoluted mass spectrum intensity of all heavy chains) × 100%

[0149] Light chain DAR value = ∑_i × [light chain drug loading distribution, LP(i)]

[0150] Heavy chain DAR value = ∑_j × [heavy chain drug loading distribution, LP(j)]

[0151] DAR value = 2 × light chain DAR value + 2 × heavy chain DAR value

[0152] Table 15

[0153]

[0154] (4) Free drugs

[0155] The free small molecule content was determined by the difference in binding force between the free small molecule and the column surface. The free small molecule was quantified by the standard curve of the free small molecule. The free small molecule in the trastuzumab-Dxd conjugate sample was determined by reversed-phase high performance liquid chromatography. The test conditions were as follows: the chromatographic column was Agilent Poroshell 120SB-C18, 4.6×100mm, 2.7μm, the column temperature was set to 50±2℃, the sample temperature was 12±3℃, the mobile phase A (75 / 25H2O / ACN, 0.1% TFA), the mobile phase B (30 / 70H2O / ACN, 0.1% TFA), the spectrum was collected at a wavelength of 370nm, and the running time was 12 minutes. All raw data were processed using chromatographic data analysis software (Agilent Openlab, Agilent Corporation). The results of a series of Dxd solutions with different concentrations (0.5, 1.0, 2.0, 5.0, 10.0, 20.0 μg / mL) at a wavelength of 370 nm were linearly fitted to obtain a standard curve for quantification. The specific results are shown in Table 16.

[0156] Table 16

[0157]

[0158] pass Fig. 9 As shown, when hyaluronidase is added and used in combination with antigen-coupled drugs, the free drug increases slightly compared with T0 (increase of 0.48-0.61%), indicating that it has no effect on the stability of the antibody-coupled drug.

[0159] According to the results of appearance, protein concentration, SEC purity, DAR value, and free drug of each experimental group at T0, 40℃1W, and 40℃2W, it is shown that the addition of hyaluronidase has no significant effect on the quality attributes of ADC. According to the enzyme activity results of each prescription at T0, 40℃1W, and 40℃2W, it is shown that the addition of ADC has no significant effect on the activity of hyaluronidase. The liquid prescriptions of B2, B3, B4, and B6 were placed at 40℃ for 2 weeks, and the appearance, protein concentration, DAR value, and enzyme activity did not change significantly compared with T0, the SEC purity was slightly reduced compared with T0 (reduced by 0.5-1.3%), and the free drug was slightly increased compared with T0 (increased by 0.48-0.61%).

[0160] In summary, hyaluronidase has good compatibility with ADC, and the compound preparation has good stability, which shows that the combination of hyaluronidase and ADC is feasible from the perspective of formulation development.

[0161] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A subcutaneous drug-coupled preparation, characterized in that: The subcutaneous administration preparation comprises an antibody-drug conjugate and recombinant hyaluronidase.

2. The subcutaneous preparation according to claim 1, characterized in that The amino acid sequence of the recombinant hyaluronidase includes the sequence shown in SEQ ID No.1 or the sequence after mutation of SEQ ID No.1; Preferably, the mutated sites include site 12 substituted by V to E, site 58 substituted by V to K, site 83 substituted by I to V, site 140 substituted by Q to K, site 142 substituted by V to P, site 143 substituted by Q to K, site 165 substituted by L to F, site 234 substituted by Q to M, site 249 substituted by E to V, site 261 substituted by S to N, site 277 substituted by V to K, site 291 substituted by G to V, site 310 substituted by M to Q, site 368 substituted by D to R, site 392 substituted by E to Q, site 395 substituted by S to A, site 421 substituted by D to H, site 431 substituted by D to K, site 433 substituted by F to V, site 438 substituted by M to N, site 439 substituted by E to A, and site 445 substituted by I to M, any one or a combination of at least two.

3. The subcutaneous preparation according to claim 1 or 2, characterized in that: The drug connected in the antibody-drug conjugate includes any one or a combination of at least two of the following: camptothecin derivative SN38, exitecan derivative Dxd, calicheamicin, dukamycin, anthraxin, monomethyl auristatin E, monomethyl auristatin F, maytansine DM1, maytansine DM4, Tubulysins, eribulin or α-amanitin; Preferably, the antibody comprises any one of a bispecific antibody, a monospecific antibody or an antibody fragment; Preferably, the targets of the antibody include any one or a combination of at least two of HER2, HER3, cMet, Trop-2, Claudin 18.2, Nectin-4, FRα, PD-1, PD-L1, EGFR, cMet, HER3 or EGFR; Preferably, the antibody coupling ratio of the antibody-drug conjugate is 2-8.

4. The subcutaneous preparation according to any one of claims 1 to 3, characterized in that The concentration of the antibody-drug conjugate is 10-150 mg / mL; Preferably, the concentration of the recombinant hyaluronidase is 500-10000 U / mL.

5. The subcutaneous preparation according to any one of claims 1 to 4, characterized in that The subcutaneous administration preparation further includes excipients; Preferably, the excipients include a buffer, a surfactant and a stabilizer; Preferably, the buffer comprises any one or a combination of at least two of histidine buffer, glutamate buffer, acetate buffer, succinate buffer, phosphate buffer, Tris buffer, 2-(N-morpholino)ethanesulfonic acid MES buffer, 3-(N-morpholino)propanesulfonic acid MOPS buffer, piperazine-N,N'-bis(2-ethanesulfonic acid) Pipes buffer); Preferably, the pH value of the buffer is 5.0-6.5; Preferably, the concentration of the buffer is 10-50 mM.

6. The subcutaneous administration preparation according to claim 5, characterized in that: The stabilizer includes any one or a combination of at least two of sucrose, trehalose, dextran, glucose, lactose, maltose, xylose, mannitol or sorbitol; Preferably, the concentration of the stabilizer is 1%-10%; Preferably, the surfactant includes any one or a combination of at least two of polysorbate 80, polysorbate 20, poloxamer 188, polyethylene glycol 3350, sodium dodecyl sulfate or sodium dodecylbenzene sulfonate; Preferably, the concentration of the surfactant is 0.01%-0.2%.

7. The subcutaneous preparation according to claim 5 or 6, characterized in that: The auxiliary materials also include amino acids and / or chelating agents; Preferably, the amino acids include any one or a combination of at least two of arginine, proline, methionine, cysteine, glutamine, glycine, leucine, lysine, phenylalanine, serine, tryptophan, tyrosine or valine; Preferably, the concentration of the amino acid is 5-150 mM; Preferably, the chelating agent comprises ethylenediaminetetraacetic acid and / or diethylenetriaminepentaacetic acid; Preferably, the concentration of the chelating agent is comprised between 0.001% and 0.04%.

8. A method for preparing a subcutaneous drug-coupled preparation according to any one of claims 1 to 7, characterized in that: The preparation method comprises mixing the antibody-coupled drug, recombinant hyaluronidase and auxiliary materials, and filtering to obtain the subcutaneous administration preparation.

9. The method for preparing the subcutaneous drug-coupled preparation according to claim 8, characterized in that: The pore size of the filtering membrane is 0.1-0.5 μm.

10. Use of a subcutaneous drug preparation of any one of claims 1 to 7 in the preparation of an antibody drug.

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