Method for rapidly screening simulated material solution for freeze-drying process development and characterization

Abstract

The invention aims to quickly and accurately screen a suitable simulated material solution by using mass transfer resistance, namely measure the water loss within a certain time in a freeze-drying process in a laboratory, and select a simulated material solution with the same or similar water loss as the stock solution as a final selected simulated material solution for freeze-drying process development and engineering batches.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, in particular the field of freeze-drying process of biological medicine, and specifically relates to the use of freeze-drying weight loss to simulate the mass transfer resistance of different types and concentrations of simulated material solutions, so as to screen the simulated material solutions suitable for filling the freeze-dryer plate layer in the test batch and engineering batch. BACKGROUND

[0002] The preparation of freeze-dried preparations of biological medicines usually includes several unit operations, such as thawing, mixing, sterilization filtration, filling, freeze-drying, capping, appearance inspection and packaging. Before production, the low-risk method of laboratory process development is to conduct full-scale research using the same stock solution and equipment as clinical or commercial production. In addition, the production department will also conduct engineering batches during the development of some drugs to better study the actual problems encountered in the production process.

[0003] However, even the smallest volume requirement in the production equipment or in the laboratory process development equipment far exceeds the volume of the stock solution that can be used. Therefore, one of the existing solutions is to use a surrogate solution that does not contain active molecules. For example, in laboratory freeze-drying process development or engineering batch production, a placebo form of the drug product (i.e. not containing active ingredients) is selected as a surrogate to fill the freeze-dryer plate layer. However, the utility of the placebo in process development is limited. During freeze-drying, if the protein can provide a larger sublimation resistance, the freeze-drying time of the placebo and the stock solution will differ greatly, i.e. the test batch or engineering batch freeze-drying end time will be much lower than the GMP actual production batch, therefore, the test batch or engineering batch cannot truly simulate the actual production situation.

[0004] In recent years, surrogate solutions have received more attention. They have similar collapse temperature (Tc), glass transition temperature (Tg'), solid content and other properties to the original solution, and thus can be used for laboratory freeze-drying process development or filling the freeze-drying machine plate layer in the engineering batch to obtain similar freeze-drying endpoints to the GMP actual production batch. However, existing studies show that when screening surrogate solutions, the collapse temperature and glass transition temperature need to be as consistent as possible (see Kevin Boksa et al., Case Study in the Design of a Surrogate Solution for Use in Biopharmaceutical, AAPS PharmSciTech (2021) 22:32). However, testing these two sets of data often requires a freeze-drying microscope and a modulated differential scanning calorimeter in the laboratory, which has the following disadvantages: (1) both instruments are expensive (more than 1 million yuan) and require regular maintenance, which will increase the operating costs of the laboratory; (2) the freeze-drying microscope often requires visual determination of the collapse temperature, which is subjective and difficult to obtain a more realistic value; the modulated differential scanning calorimeter needs to accurately separate the reversible and irreversible endothermic peaks to obtain a more accurate glass transition temperature; the operation of both instruments requires experienced laboratory personnel, which also increases the labor costs of the laboratory; (3) similar collapse temperature and glass transition temperature is a relatively effective surrogate material screening standard, but there is currently no research to prove that similar collapse temperature and glass transition temperature are equivalent to similar sublimation resistance, i.e. if the surrogate solution has similar collapse temperature and glass transition temperature, it cannot guarantee that it has the same freeze-drying endpoint as the GMP actual production batch.

[0005] Therefore, there is a need to develop a new, more economical, simple, fast and accurate method for screening surrogate solutions for freeze-drying process development and characterization. SUMMARY

[0006] The present application aims to use mass transfer resistance to quickly and accurately screen suitable surrogate solutions, i.e. to measure the amount of water loss in a certain time during the laboratory freeze-drying process, and to select a surrogate solution with the same or similar water loss as the original solution as the final selected surrogate solution for freeze-drying process development and engineering batch.

[0007] Specifically, the present application relates to a method for screening a surrogate solution of an original solution containing an active ingredient, which comprises:

[0008] (1) preparing an original solution containing an active ingredient;

[0009] (2) selecting a test surrogate material and preparing test surrogate solutions of different concentrations;

[0010] (3) under the same freeze-drying conditions, freeze-drying the bulk solution prepared in step (1) and the test mimic material solution prepared in step (2) respectively;

[0011] (4) determining the water loss of each sample after freeze-drying;

[0012] (5) determining whether the test mimic material solution of different concentrations can be used as the alternative mimic material solution of the bulk solution by statistically analyzing the water loss;

[0013] (6) if the result of step (5) is that the test mimic material solution can be used as the alternative mimic material solution of the bulk solution, selecting the test mimic material solution of the concentration closest to the water loss of the bulk solution as the mimic material solution of the bulk solution.

[0014] The method of the present application avoids the dependence on expensive instruments, simplifies the screening process, and ensures that the freeze-drying endpoint is highly matched with the GMP actual production batch, and is a more economical, simple, rapid and accurate screening method. The mimic material solution obtained by screening using the method of the present application can be used in freeze-drying process development and engineering batch, and ensures that the freeze-drying endpoint is consistent with the GMP actual production batch.

[0015] In some embodiments, the active ingredient contained in the bulk solution of the present application is a biopharmaceutical molecule. In other embodiments, the biopharmaceutical molecule is selected from one or more of the following, without limitation: a protein, a polypeptide, a nucleic acid or any derivative thereof (e.g. a fusion or conjugate), for example, selected from one or more of the following, without limitation: an antibody (e.g. a traditional monoclonal antibody or a bispecific / multispecific antibody) or an antigen-binding fragment thereof, a fusion protein, a bioconjugate (e.g. an immunoconjugate, e.g. an antibody-drug conjugate), a polypeptide, a small nucleic acid drug.

[0016] In some embodiments, the biopharmaceutical molecule is a protein. In some specific embodiments, the protein is a protein having a quaternary structure composed of more than one peptide chain. In some more specific embodiments, the protein has a post-translational modification. In some more specific embodiments, the post-translational modification is selected from the following or any combination thereof: glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, fatty chain / fatty acid chain modification, polyethylene glycol modification, proteolytic cleavage or modification with non-naturally occurring amino acids, etc. In some more specific embodiments, the protein is an immunoglobulin and / or comprises an Ig-like domain. In some more specific embodiments, the protein is an antibody or an antigen-binding fragment thereof, e.g. a monoclonal or polyclonal antibody. In some more specific embodiments, the protein is an antibody derivative, e.g. an immunoconjugate comprising a monoclonal antibody.

[0017] In some more specific embodiments, the monoclonal antibody is a mouse antibody, a rat antibody, a chimeric antibody, a humanized antibody or a fully human antibody. In some more specific embodiments, the monoclonal antibody is a multispecific antibody, such as a bispecific antibody or a trispecific antibody.

[0018] In some specific embodiments, the antibodies or antigen-binding fragments thereof of the present invention are selected from, but are not limited to, one or more of the following: rituximab, trastuzumab, bevacizumab, cetuximab, pembrolizumab, nivolumab, pertuzumab, atezolizumab, adalimumab, ustekinumab, dupilumab, secukinumab, ixekizumab, ocrelizumab, ofatumumab, alizumab, evolocumab, denosumab, Antibodies, aducanumab, alemtuzumab, atumomab, atezolizumab, anetuzumab, avelumab, bapilumab, basiliximab, betumumab, bermedizumab, besolumab, belotuzumab, vebruximab, vebruximab, bodalumab, cemiplimab, simpanumab, clituzumab, clenezumab, daclizumab, daratumumab, denosumab, dotalimumab, durvalumab, edemostat Lotumab, elotuzumab, ipavacumab, enroku, epratuzumab, edamame, gemtuzumab, gemtuximab, gonelizumab, ibritumomab tiuxetan, inelizumab, infliximab, inotuzumab, ipilimumab, isatuximab, rosopetuzumab, labetuzumab, lencanezumab, lantuzumab, moglizumab, nexitozumab, nimotuzumab, natalizumab, nasituximab, olaparib, In some preferred embodiments, the antibody or antigen-binding fragment thereof of the present invention is trastuzumab or bevacizumab, or an antigen-binding fragment thereof. In some preferred embodiments, the antibody or antigen-binding fragment thereof of the present invention is trastuzumab or bevacizumab, or an antigen-binding fragment thereof.

[0019] In some embodiments, the antibody or antigen-binding fragment thereof of the present application is non-limitingly selected from one or more of the following: belimumab, epratuzumab, trademersatumab, BVX001, motavizumab, zanolimumab, evinacumab, grofituzumab, zibovudine, catumaxomab, cadonilimab, elotuzumab, talacotuzumab, enanatumab, faricimab, bimekizumab, evolocumab. In some preferred embodiments, the antibody or antigen-binding fragment thereof of the present application is enanatumab, zanolimumab, zibovudine, evolocumab, or an antigen-binding fragment thereof.

[0020] In some embodiments, the biopharmaceutical molecule is a fusion of proteins (e.g., a fusion protein) or a conjugate. In some embodiments, the conjugate is an immunoconjugate, e.g., an antibody conjugate drug. In some embodiments, the antibody conjugate drug of the present application is non-limitingly selected from one or more of the following: an antibody-radionuclide conjugate, an antibody-immunostimulant conjugate, an antibody-degrader conjugate, an antibody-fragment conjugate, an antibody-oligonucleotide conjugate, an antibody-cell conjugate, or an antibody-biopolymer conjugate.

[0021] In some embodiments, the biopharmaceutical molecule is non-limitingly selected from one or more of the following immunoconjugates: gemtuzumab ozogamicin, vibostuzumab, emicymatuzumab (trastuzumab emtansine conjugate), emicymatuzumab biosimilar, ocaratuzumab, deruxtezumab, verpustuzumab, venetoclax, gosatuzumab, beiramatuzumab mafodotin, Akalux, vedisutuzumab, talanituzumab, sintalimus, epaxalimus SN38, ZW-49. In some preferred embodiments, the immunoconjugate of the present application is emicymatuzumab, emicymatuzumab biosimilar, deruxtezumab, verpustuzumab, ZW-49.

[0022] In some embodiments, the biopharmaceutical molecule has a concentration in the stock solution of about 1-100 mg / mL, preferably about 10-50 mg / mL, more preferably about 15-40 mg / mL, most preferably about 20-30 mg / mL, e.g., about 20 mg / mL, about 25 mg / mL, or about 30 mg / mL.

[0023] In some embodiments, the stock solution of the present application is a "lyophilized stock solution," which generally refers to a solution comprising an active ingredient (i.e., a biopharmaceutical molecule) and a buffer, a lyoprotectant, a stabilizer, an antioxidant, a surfactant, etc., configured prior to lyophilization. In some embodiments, in addition to the active ingredient, the stock solution of the present application can comprise one or more substances non-limitingly selected from the group consisting of a buffer, a lyoprotectant, an amino acid, a stabilizer, a preservative, an antioxidant, an osmotic pressure adjusting agent, an adjuvant, a chelating agent, an acid-base adjusting agent, a bulking agent, and a surfactant.

[0024] In some embodiments, the mimetic material of the present application is a substance suitable for use in a lyophilization process, which is non-limitingly selected from one or more substances selected from the group consisting of a sugar, e.g., a monosaccharide, a disaccharide, or a polysaccharide; a protein; a polymer; an organic acid; an alcohol; and an amino acid. Typically, the concentration of the mimetic material in the mimetic material solution is about 1-400 mg / mL, e.g., about 1-100 mg / mL, about 100-200 mg / mL, about 200-300 mg / mL, or about 300-400 mg / mL.

[0025] In some embodiments, the mimetic material of the present application is non-limitingly selected from one or more substances selected from the group consisting of a sugar, e.g., a monosaccharide, a disaccharide (e.g., lactose, sucrose, or trehalose), or a polysaccharide (e.g., dextran, polysucrose, sorbitol, mannitol, hydroxyethyl starch); a protein (e.g., human serum albumin, bovine serum albumin, or other albuminoid); a polymer (e.g., polyethylene glycol or polyvinylpyrrolidone (PVP)); an organic acid (e.g., citric acid); and an amino acid (e.g., histidine or glycine).

[0026] In some embodiments, the mimetic material of the present application is non-limitingly selected from one or more substances selected from the group consisting of dextran, e.g., dextran 10, dextran 20, dextran 40, dextran 70, dextran 150, or dextran 400; bovine serum albumin; human serum albumin; polysucrose, e.g., polysucrose 70, polysucrose 400; hydroxyethyl starch, e.g., hydroxyethyl starch 130 or hydroxyethyl starch 200; polyethylene glycol, e.g., polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 1000, polyethylene glycol 1500, polyethylene glycol 3350, polyethylene glycol 4000, or polyethylene glycol 6000; polyvinylpyrrolidone (PVP), e.g., PVP K30 or PVP K90; sucrose; trehalose / trehalose dihydrate; citric acid; mannitol; sorbitol; histidine; glycine; and lactose.

[0027] In some embodiments, the mimetic material of the present application is non-limitingly selected from one or more substances selected from the group consisting of dextran, bovine serum albumin, polysucrose, sucrose, trehalose / trehalose dihydrate.

[0028] In some embodiments, the mimetic material of the present application is non- limitingly selected from one or more of the following: Dextran 20, Dextran 70, Dextran 150, Bovine Serum Albumin, Polysucrose 70, Sucrose, Trehalose dihydrate.

[0029] In some embodiments, the mimetic material of the present application is a polysaccharide, such as a dextran (e.g., Dextran 10, Dextran 20, Dextran 40, Dextran 70, Dextran 150, or Dextran 400, preferably Dextran 20, Dextran 70, or Dextran 150) or a polysucrose (e.g., Polysucrose 70, Polysucrose 400, preferably Polysucrose 70). Typically, the concentration of the mimetic material in the mimetic material solution is about 1-100 mg / mL, preferably about 10-90 mg / mL, about 20-80 mg / mL, about 30-70 mg / mL, or about 40-60 mg / mL, such as about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, or about 100 mg / mL, preferably about 30 mg / mL, about 50 mg / mL, or about 70 mg / mL.

[0030] In some embodiments, the mimetic material of the present application is a disaccharide, such as lactose, sucrose, or trehalose, preferably sucrose or trehalose dihydrate. Typically, the concentration of the mimetic material in the mimetic material solution is about 100-400 mg / mL, preferably about 120-380 mg / mL, about 140-360 mg / mL, about 160-340 mg / mL, about 180-320 mg / mL, about 200-300 mg / mL, about 220-280 mg / mL, about 240-260 mg / mL, such as about 140 mg / mL, about 160 mg / mL, about 180 mg / mL, about 190 mg / mL, about 200 mg / mL, about 220 mg / mL, about 240 mg / mL, about 260 mg / mL, about 280 mg / mL, or about 300 mg / mL, preferably about 180 mg / mL, about 190 mg / mL, about 200 mg / mL, about 220 mg / mL, or about 240 mg / mL.

[0031] In some embodiments, the mimetic material of the present application is a protein, such as bovine serum albumin or human serum albumin, preferably bovine serum albumin. Typically, the concentration of the mimetic material in the mimetic material solution is about 1-100 mg / mL, preferably about 10-90 mg / mL, about 20-80 mg / mL, about 30-70 mg / mL, or about 40-60 mg / mL, such as about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, or about 100 mg / mL, preferably about 20 mg / mL, about 40 mg / mL, or about 60 mg / mL.

[0032] In some embodiments, the present application relates to a method of screening a mimetic material solution of a stock solution comprising an active ingredient, wherein the active ingredient is an immunoglobulin or a fragment or a derivative thereof, such as a monoclonal antibody or an antibody drug conjugate (ADC); and the mimetic material is selected from one or more of the following: dextran, bovine serum albumin, polysucrose, sucrose, trehalose / trehalose dihydrate.

[0033] In some more preferred embodiments, the active ingredient is selected from one or more of the following: trastuzumab, bevacizumab, inotuzumab, inebilizumab, zanolimumab, epratuzumab, enemumab, enemumab biosimilar, denintuzumab, vedotinuzumab, or ZW-49.

[0034] In some more preferred embodiments, the mimetic material is selected from one or more of the following: dextran 20, dextran 70, dextran 150, bovine serum albumin, polysucrose 70, sucrose, trehalose dihydrate.

[0035] In some preferred embodiments, the present application relates to a method of screening a mimetic material solution of a stock solution comprising an active ingredient, wherein the active ingredient is a protein having a quaternary structure and post-translational modifications, and the mimetic material is selected from one or more of the following: dextran, bovine serum albumin, polysucrose, sucrose, trehalose / trehalose dihydrate. Preferably, the mimetic material is selected from one or more of the following: dextran 20, dextran 70, dextran 150, bovine serum albumin, polysucrose 70, sucrose, trehalose dihydrate.

[0036] In some preferred embodiments, the present application relates to a method of screening a mock material solution of a stock solution comprising an active ingredient, wherein said active ingredient is an immunoglobulin, and said mock material is selected from one or more of the following: Dextran, Bovine Serum Albumin, Polysucrose, Sucrose, Trehalose / Trehalose dihydrate. Preferably, said mock material is selected from one or more of the following: Dextran 20, Dextran 70, Dextran 150, Bovine Serum Albumin, Polysucrose 70, Sucrose, Trehalose dihydrate.

[0037] In some more preferred embodiments, the present application relates to a method of screening a mock material solution of a stock solution comprising an active ingredient, wherein said active ingredient is a monoclonal antibody, and said mock material is selected from one or more of the following: Dextran, Bovine Serum Albumin, Polysucrose, Sucrose, Trehalose / Trehalose dihydrate. Preferably, said mock material is selected from one or more of the following: Dextran 20, Dextran 70, Dextran 150, Bovine Serum Albumin, Polysucrose 70, Sucrose, Trehalose dihydrate. Preferably, said antibody is selected from Trastuzumab, Bevacizumab, Enavatuzumab, Inebilizumab, Zanolimumab and Evinacumab, and antigen-binding fragments thereof.

[0038] In some more preferred embodiments, the present application relates to a method of screening a mock material solution of a stock solution comprising an active ingredient, wherein said active ingredient is an antibody drug conjugate (ADC), and said mock material is selected from one or more of the following: Dextran, Bovine Serum Albumin, Polysucrose, Sucrose, Trehalose / Trehalose dihydrate. Preferably, said mock material is selected from one or more of the following: Dextran 20, Dextran 70, Dextran 150, Bovine Serum Albumin, Polysucrose 70, Sucrose, Trehalose dihydrate. Preferably, said antibody drug conjugate is selected from Enfortumab vedotin, Enfortumab vedotin biosimilar, Deruxteumab, Vedotin, and ZW-49.

[0039] In some embodiments, the mock material solution of the present application, in addition to the mock material, can comprise one or more substances selected, without limitation, from the following: buffers, lyoprotectants, amino acids, stabilizers, preservatives, antioxidants, osmolality adjusting agents, adjuvants, chelating agents, pH adjusting agents, bulking agents, surfactants.

[0040] In some embodiments, the buffer in the stock or mock material solution of the present application is selected, without limitation, from one or more of the following: histidine buffer, glutamic acid buffer, acetic acid buffer, succinic acid buffer, citric acid buffer, phosphate buffer, adipic acid buffer, Tris buffer, MES buffer, MOPS buffer, PIPES buffer. Preferably, the buffer is a histidine buffer (e.g., histidine-histidine hydrochloride) or a succinic acid buffer (e.g., succinic acid-sodium succinate). Typically, the concentration of the buffer in the stock or mock material solution is about 1-100 mM, preferably about 1-50 mM, more preferably about 10-40 mM, further preferably about 20-30 mM, e.g., about 10 mM, about 20 mM, or about 30 mM.

[0041] In some embodiments, the lyoprotectant in the stock or mock material solution of the present application is selected, without limitation, from one or more of the following: sucrose, trehalose, glucose, lactose, maltose, xylose, mannitol, sorbitol, and dextran. Preferably, the lyoprotectant is sucrose or trehalose (e.g., trehalose dihydrate). Typically, the concentration of the lyoprotectant in the stock or mock material solution is about 1-20% (w / v), preferably about 1-10% (w / v), more preferably about 2-8% (w / v), further preferably about 4-6% (w / v), e.g., about 6% (w / v) or about 8% (w / v).

[0042] In some embodiments, the amino acid in the stock or mock material solution of the present application is selected, without limitation, from one or more of the following: arginine, proline, methionine, cysteine, glutamine, glycine, leucine, lysine, phenylalanine, serine, tryptophan, tyrosine, valine.

[0043] In some embodiments, the stabilizer in the stock or mock material solution of the present application is selected, without limitation, from one or more of the following: sucrose, trehalose, mannitol, sorbitol, glycine, arginine, glutamic acid, cyclodextrin, human serum albumin, glycerol, polyethylene glycol.

[0044] In some embodiments, the preservative in the stock or mock material solution of the present application is selected, without limitation, from one or more of the following: benzyl alcohol, phenol, m-cresol, parabens.

[0045] In some embodiments, the antioxidant in the stock or mock material solution of the present application is selected, without limitation, from one or more of the following: methionine, ethylenediaminetetraacetic acid, ascorbic acid, gentisic acid.

[0046] In some embodiments, the osmotic pressure adjusting agent in the stock or mock material solution of the present application is non-limitingly selected from one or more of the following: sodium chloride, glycerol, glucose.

[0047] In some embodiments, the adjuvant in the stock or mock material solution of the present application is non-limitingly selected from one or more of the following: aluminum hydroxide, aluminum phosphate, liposome / nanoparticle.

[0048] In some embodiments, the chelating agent in the stock or mock material solution of the present application is non-limitingly ethylenediaminetetraacetic acid.

[0049] In some embodiments, the acid-base adjusting agent in the stock or mock material solution of the present application is non-limitingly selected from one or more of the following: hydrochloric acid, sodium hydroxide.

[0050] In some embodiments, the bulking agent in the stock or mock material solution of the present application is non-limitingly selected from one or more of the following: lactose, microcrystalline cellulose.

[0051] In some embodiments, the surfactant in the stock or mock material solution of the present application is non-limitingly selected from one or more of the following: polysorbate 80, polysorbate 20, poloxamer 188, poloxamer 407, polyethylene glycol 3350, sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, or polyoxyethylene castor oil derivative. Preferably, the surfactant is polysorbate 80 or polysorbate 20. Typically, the concentration of the surfactant in the stock or mock material solution is about 0.001-0.1% (w / v), preferably about 0.005-0.08% (w / v), more preferably about 0.01-0.06% (w / v), further preferably about 0.02-0.04% (w / v), for example about 0.005% (w / v), about 0.01% (w / v), or about 0.02% (w / v).

[0052] In some embodiments, the mock material solution of the present application contains only the mock material and a solvent (e.g., water). In some embodiments, the mock material solution of the present application contains, in addition to the mock material and a solvent (e.g., water), other substances such as a buffer, a lyoprotectant, and a surfactant, etc., preferably in the respective categories and amounts as described above.

[0053] In some embodiments, the mimetic solution of the present application preferably comprises a polysaccharide (e.g., dextran 20, dextran 70, dextran 150, polysucrose 70), a protein (e.g., bovine serum albumin), or a disaccharide (e.g., sucrose, trehalose dihydrate) as the mimetic material; a histidine buffer (e.g., histidine-histidine hydrochloride) or succinate buffer (e.g., succinic acid-sodium succinate) as the buffer; sucrose or trehalose (e.g., trehalose dihydrate) as the lyophilization protectant; and polysorbate 80 or polysorbate 20 as the surfactant, preferably each of their respective kinds and amounts as described above.

[0054] In some embodiments, in the method of the present application, a substance with a molecular weight similar to that of the active ingredient in the stock solution is selected as the mimetic material or a component in the stock solution other than the active ingredient is selected as the mimetic material, according to the relative molecular weight of the active ingredient in the stock solution.

[0055] In some embodiments, in the method of the present application, the concentration range of the test mimetic solution is selected according to the molecular weight and concentration of the active ingredient in the formulated stock solution. In some embodiments, 2-10 concentrations of the test mimetic solution are formulated, preferably 3-8 concentrations of the test mimetic solution, more preferably 4-7 concentrations of the test mimetic solution, such as 2, 3, 4, 5, or 6 concentrations of the test mimetic solution. In some embodiments, at least 3, preferably at least 5, more preferably at least 10, of each concentration of the test mimetic solution are formulated.

[0056] In some embodiments, in the screening method of the present application, the amount of water loss of the mimetic solution of the selected stock solution is 80%-120%, preferably 90%-110%, of the amount of water loss of the stock solution.

[0057] In some embodiments, the ratio of the time to primary dry end point of the mimetic solution of the selected stock solution to that of the stock solution is 0.8-1.2, preferably 0.9-1.1.

[0058] In some embodiments, the stock solution and the test mimetic solution of the present application are formulated in a container, such as a vial, preferably the vial is of a size of 2R, 6R, 10R, 20R, 30R, 50R, preferably 2R, 6R, 10R, more preferably 2R. In some embodiments, the stock solution and the test mimetic solution of the present application are filled in the vial to an amount of about 1 / 3 to about 1 / 2 of the size of the vial.

[0059] In some embodiments, in the screening method of the present application, the lyophilization is stopped when about 30%-about 80%, preferably about 40%-about 60%, of the primary drying is completed.

[0060] In some embodiments, the statistical analysis in the screening method of the present application comprises:

[0061] (a) calculating a group within sample mean of the amount of water loss of the samples of the test simulant solution at each concentration

[0062]

[0063] where Δm i,j is the amount of water loss of the jth sample of the ith concentration of the test simulant solution, n is the number of samples of the ith concentration of the test simulant solution, and 1 < j < n, n > 2, k is the number of concentrations, and 1 < i < k, k > 2;

[0064] (b) calculating a grand mean of the amount of water loss of the samples of the test simulant solution at all concentrations

[0065]

[0066] (c) calculating a group within sum of squares and a group between sum of squares of the samples of the test simulant solution, wherein:

[0067] the group within sum of squares SSE is calculated as

[0068]

[0069] the group between sum of squares SSA is calculated as

[0070]

[0071] (d) calculating a statistic F,

[0072]

[0073] (e) when F is greater than a critical value F α corresponding to the numerator degrees of freedom k-1 and the denominator degrees of freedom (n-1) x k in an F-distribution table, wherein the significance level a corresponding to the F-distribution table is 0.05, preferably 0.025, the test simulant solution at the different concentrations is determined to be a candidate simulant solution for the stock solution.

[0074] In the above embodiments, if F > F α , it indicates that the concentration of the simulant solution has an effect on the amount of water loss, i.e. on the mass transfer resistance, and the concentration of the simulant solution can be screened using the group of data.

[0075] In some embodiments, the statistical analysis in the screening method of the present application further comprises:

[0076] (f) calculating the absolute value of the difference between the group mean of the sample of the amount of water loss of the sample of the test analog solution at the i-th concentration and the group mean of the sample of the amount of water loss of the sample of the test analog solution at the j-th concentration

[0077]

[0078] (g) calculating the least significant difference, LSD, of the sample of the analog solution:

[0079]

[0080] wherein t α / 2 is the critical value corresponding to the degrees of freedom (n-1) x k in the t-distribution table, wherein the significance level a corresponding to the t-distribution table is 0.05, preferably 0.025,

[0081] When is greater than LSD, the test analog solution at the i-th concentration and the j-th concentration is further selected from the candidate analog solution determined in step (e) as the candidate analog solution of the stock solution.

[0082] In the above embodiments, if then it is proved that the amount of water loss of the analog solution at the i-th concentration and the amount of water loss of the analog solution at the j-th concentration have a significant difference, i.e. the mass transfer resistance caused by the concentration of the two groups of analog solutions has a significant difference, and the concentration of the analog solution can be screened using the data of the group.

[0083] In some embodiments, the step of selecting the test analog solution of the concentration closest to the amount of water loss of the stock solution containing the active ingredient as the analog solution of the stock solution containing the active ingredient of the present application comprises selecting from the k test analog concentrations or selecting by fitting a linear equation.

[0084] In some embodiments, the lyophilization of the present application is performed in a lyophilizer. The lyophilizer of the present application can be any lyophilizer, such as a small benchtop lyophilizer (e.g. Labconco FreeZone, Christ Alpha 1-2LDplus, Telstar LyoBeta Mini, etc.), a medium floor model lyophilizer (e.g. GEA Lyophil LyoCapsule TM , Millrock REVO TM , Dongfeng Lyo-0.5, SP Hull Lyostar 4.0, etc.), a large pilot / small production scale lyophilizer (e.g. SP Scientific VirTis Genesis TMHOF Série 40, etc.). The process of the present application, in particular the freeze-drying process, is not limited to a freeze-dryer.

[0085] In some embodiments, the freeze-drying process of the present application comprises the following steps:

[0086] (1) loading, at a loading temperature of 0 to about 30°C, preferably about 25°C;

[0087] (2) pre-freezing at a pre-freezing temperature of about 10°C below the glass transition temperature (Tg') of the bulk solution and the mimic material solution; the pre-freezing time is about 150 minutes or more, and the cooling rate is about 0.25 to about 2.00°C;

[0088] (3) optional annealing, wherein the annealing temperature is above the glass transition temperature (Tg') or the collapse temperature (Tc), and the annealing time is 150 minutes or more; the annealing ramping rate is about 0.25 to about 2.00°C;

[0089] (4) primary drying, wherein the primary drying ramping rate is about 0.25 to about 2.00°C, and the primary drying temperature is below the collapse temperature of the bulk solution and the mimic material solution, and the primary drying time is about 30% to about 80%, preferably about 40% to about 60% of the primary drying completion, without secondary drying.

[0090] In some embodiments, the present application also relates to the mimic material solution screened according to the process of the present application.

[0091] In some embodiments, the present application also relates to the use of the process of the present application in the development or engineering of a freeze-drying process.

[0092] In some embodiments, the present application also relates to the use of the mimic material solution screened by the process of the present application in the development or engineering of a freeze-drying process, in particular a freeze-drying process of a biopharmaceutical molecule. BRIEF DESCRIPTION OF DRAWINGS

[0093] Figure 1 . Figure of the relationship between trehalose dihydrate concentration and loss on drying.

[0094] Definitions

[0095] It is to be understood that the terminology used herein is for the purpose of describing embodiments of the present application only and is not intended to be limiting of the scope of the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms "comprises", "comprising", "including", and "having" are intended to be inclusive and mean that there can be additional

[0096] The terms "a," "an," "the," and similar referents used herein are to be construed to be inclusive, unless otherwise indicated or clearly contradicted by context.

[0097] The term "about" used in connection with a numerical value means covering numbers that are within a range having a lower limit that is 5%, 4%, 3%, 2%, or 1% less than the specified numerical value and an upper limit that is 5%, 4%, 3%, 2%, or 1% greater than the specified numerical value.

[0098] As used herein, the terms "comprising" or "including," or "having" mean including but not limited to, and are not intended to exclude, for example, additional conjointly recited elements, integers, steps, or groups thereof. In this context, "comprising" means that other elements, integers, steps, or groups can also be present, in addition to those recited. For example, when reference is made to an antibody variable region "comprising" a particular sequence, it is intended to also cover an antibody variable region consisting of that particular sequence.

[0099] As used herein, the term "biopharmaceutical molecule" refers to any biological macromolecule that can have prophylactic and / or therapeutic significance. A biopharmaceutical molecule can exist and / or function within a living organism, and typically has significant activity without requiring excessively high dosages in vivo. Like general biological macromolecules, a biopharmaceutical molecule has a molecular weight that exceeds 1 kDa. In some embodiments, a biopharmaceutical molecule has a molecular weight that exceeds 50 kDa, 75 kDa, 100 kDa, 125 kDa, or 150 kDa. In some embodiments, a biopharmaceutical molecule is a polymer, e.g., a biological polymer such as a nucleic acid (a polymer of nucleotides, such as DNA, RNA), a polypeptide (a polymer of amino acids, such as a protein), a carbohydrate, and a lipid, or a combination or complex thereof. In some embodiments, a biopharmaceutical molecule is a naturally occurring biological macromolecule. In some embodiments, a biopharmaceutical molecule has structural modifications relative to its natural parent molecule, if any, from which it is derived. In some embodiments, a biopharmaceutical molecule is isolated from natural sources. In some embodiments, a biopharmaceutical molecule is produced artificially, e.g., recombinantly and / or chemically.

[0100] In some embodiments, the biopharmaceutical molecule is a protein. In some embodiments, the biopharmaceutical molecule is a recombinant protein or a fusion protein. In some embodiments, the protein has post-translational modifications such as glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, fatty chain / fatty acid chain modification, polyethylene glycol modification, proteolytic cleavage, or modification with non-naturally occurring amino acids, etc. In some embodiments, the protein is soluble. In some embodiments, the soluble protein is a free receptor. In some embodiments, the biopharmaceutical molecule is an antibody or an antigen-binding fragment thereof. Such as a monoclonal antibody or a polyclonal antibody. In some embodiments, the antibody is a multispecific antibody, such as a bispecific antibody.

[0101] In some embodiments, the biopharmaceutical molecule of the present application is selected, without limitation, from one or more of the following: cyclic peptides such as syfovre, GLP-1 RAs such as liraglutide, semaglutide, exenatide, etc., hormones such as growth hormone, insulin, estrogen, etc., cytokines such as interferons, interleukins, etc., blood products such as immunoglobulins, albumin, etc.

[0102] In some embodiments, the antibody or antigen-binding fragment thereof of the present application is non-limitingly selected from one or more of the following: rituximab, trastuzumab, bevacizumab, cetuximab, pembrolizumab, nivolumab, pertuzumab, atezolizumab, adalimumab, ustekinumab, dupilumab, siltuximab, eculizumab, ocrelizumab, ofatumumab, atlizumab, elotuzumab, denosumab, aducanumab, alemtuzumab, altumomab, atezolizumab, avelumab, bapineuzumab, basiliximab, bebtuximab, bemarituzumab, bivatuzumab, belotumab, bavituximab, bortezumab, cemiplimab, cintirestat, clivatuzumab, crizanlizumab, dalotuzumab, daratumumab, dinutuximab, dotarizumab, devolizumab, edrecolomab, elotuzumab, epacadostat, enoblituzumab, epratuzumab, erlizumab, eculizumab, gemtuzumab, gemtuzumab, gantenerumab, inebilizumab, infliximab, inebilizumab, ipilimumab, isatuximab, losooftumab, labetuzumab, leronlimab, lintuzumab, margetuximab, mogamulizumab, nacolomab, nimotuzumab, natalizumab, necitumumab, necatorlimab, nivolumab, oregovomab, panitumumab, polatuzumab, vedotin, ponesimod, ramucirumab, ramucirumab, secolizumab, sasanlimab, sonepcizumab, siltuximab, solanezumab, tanibirumab, tisotumab, tositumomab, tralokinumab, tocilizumab, vedolizumab, volociximab, zanolimumab, zanolimumab, or antigen-binding fragments of the aforementioned antibodies.

[0103] In some embodiments, the antibody or antigen-binding fragment thereof of the present application is non-limitingly selected from one or more of the following: belinostat, emicizumab, tediprotuzumab, BVX001, motavizumab, evinacumab, grofituzumab, catumaxomab, cadonilimab, emactuzumab, taquizumab, enanatumab, faricimab, bimekizumab.

[0104] In some embodiments, the biopharmaceutical molecule is a fusion of proteins (e.g., a fusion protein) or a conjugate. In some embodiments, the conjugate is an immunoconjugate, e.g., an antibody conjugate drug. In some embodiments, the antibody conjugate drug of the present application is non-limitingly selected from one or more of the following: an antibody-radionuclide conjugate, an antibody-immunostimulant conjugate, an antibody-degrader conjugate, an antibody-fragment conjugate, an antibody-oligonucleotide conjugate, an antibody-cell conjugate, or an antibody-biopolymer conjugate.

[0105] In some embodiments, the biopharmaceutical molecule is non-limitingly selected from one or more of the following: gemtuzumab ozogamicin, vibostuzumab, inotuzumab, inotuzumab (biosimilar), ocaratuzumab, denintuzumab, vecabrutinib, venetoclax, gantenerumab, belantamab mafodotin, Akalux, vedolizumab, talmapimab, tisotumab, solitomab, epacadostat SN38.

[0106] In some embodiments, the fusion protein is a fusion protein comprising an antigen binding fragment (e.g., a multispecific antibody). In some embodiments, the fusion protein is a fusion protein comprising an immunoglobulin Fc domain, i.e., is an Fc-fusion protein. In some embodiments, the non-Ig portion therein can be a therapeutic protein, e.g., a therapeutic protein derived from erythropoietin (EPO), thrombopoietin (THPO) such as a THPO binding peptide, growth hormone, interferon (IFN) such as IFN alpha, IFN beta, or IFN gamma, platelet-derived growth factor (PDGF), interleukin (IL) such as IL1 alpha or IL1 beta, transforming growth factor (TGF) such as TGF alpha or TGF beta, or tumor necrosis factor (TNF) such as TNF alpha or TNF beta, or a ligand binding fragment derived from a receptor, particularly a ligand binding fragment derived from the extracellular domain of a receptor, e.g., a therapeutic protein derived from cluster of differentiation 2 (CD2), CD4, CD8, CD11, CD14, CD18, CD20, CD22, CD23, CD25, CD33, CD40, CD44, CD52, CD58 (LFA3), CD80, CD86, CD147, CD164, IL2 receptor, IL4 receptor, IL6 receptor, IL12 receptor, epidermal growth factor (EGF) receptor, vascular endothelial growth factor (VEGF) receptor, epithelial cell adhesion molecule (EpCAM), or cytotoxic T-lymphocyte-associated protein 4 (CTLA4). In some embodiments, the fusion protein is non-limitingly selected from one or more of the following: aflibercept, eteplirsen, brolucizumab, romosozumab, abagovomab, afutuzumab, etanercept, dulaglutide, and the like.

[0107] In some embodiments, the fusion protein is tisotumab.

[0108] In some embodiments, the biopharmaceutical molecule is a nucleic acid molecule. In some embodiments, the nucleic acid is a linear nucleic acid molecule or a circular nucleic acid molecule. In some embodiments, the nucleic acid is a single-stranded nucleic acid molecule or a partially or fully double-stranded nucleic acid molecule. In some embodiments, the nucleic acid is a chemically modified nucleic acid molecule, including naturally occurring modified nucleic acid molecules, such as glycosylated or methylated nucleic acid molecules, or artificially modified derivatives, such as biotinylated p nucleic acid molecules, locked nucleic acids, peptide nucleic acids, and the like.

[0109] In some embodiments, the biopharmaceutical molecule is a liposome. In some embodiments, the liposome encapsulates other kinds of molecules, such as proteins and / or nucleic acids.

[0110] In some embodiments, the biopharmaceutical molecule is an antigen or antigenic fragment. In some embodiments, the antigen is a nucleic acid, a protein, or a polysaccharide, or any complex thereof. In some embodiments, the antigen or antigenic fragment is coupled to a carrier protein.

[0111] The term "antibody" broadly refers to any immunoglobulin (Ig) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any antigen binding fragment, mutant, variant, or derivative of such an Ig molecule that retains the essential epitope binding features of the Ig molecule. Such mutant, variant, or derivative antibody forms are known in the art, non-limiting embodiments of which are discussed below. The term "antibody" herein can refer to any form of antibody having the desired biological activity. Thus, it is used in the broadest sense consistent with the disclosure, specifically encompassing but not necessarily limited to monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, CrossMab antibodies, or camelized single domain antibodies.

[0112] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains (IgM antibodies comprise 5 of the basic heterotetramer units along with an additional polypeptide called J chain, and thus contain 10 antigen binding sites, while secreted IgA antibodies can multimerize to form polyvalent assemblages of 2-5 of the basic 4-chain units along with J chain). In the case of IgGs, the 4-chain unit is typically about 150,000 Daltons. Each L chain is linked to a H chain by one covalent disulfide bond, while each of the H chains is linked to another H chain by one or more disulfide bonds depending on the H chain isotype. Each H chain and L chain also has regularly spaced intrachain disulfide bridges. At the C-terminus of each H chain, there are number of domains (about 3 in the case of IgG1 and IgG3, 4 in the case of IgG2 and IgG4). The N-terminus of each H chain is followed by a variable domain (VH) initially followed by three constant domains (CH1, CH2, and CH3) in the case of IgG1 and IgG3, or four constant domains (CH1, CH2, CH3, and CH4) in the case of IgG2 and IgG4. The N-terminus of each L chain is followed by a variable domain (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VHand the CL is aligned with the first constant domain of the heavy chain (CH1). Particular amino acid residues of combinations thereof are identified as being responsible for the formation of loops (or "complementarity-determining regions" (CDRs)) of the binding site. The VHand VLdomains can further be subdivided into regions of even greater specificity (hypervariable loops, also called complementarity-determining regions, CDRs). See, e.g., Kindt et al., Kuby Immunology, 6th Ed., W.H. Freeman and Co.: 2007, p. 97 for more explanation on the structure and regions of an antibody.

[0113] The terms "whole antibody", "full-length antibody", and "intact antibody" are used interchangeably herein to refer to an antibody that contains at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of even greater specificity (hypervariable loops, also called complementarity-determining regions, CDRs), which are interposed between more conserved regions (framework regions, FRs). The constant regions of the antibodies may

[0114] The light chain of an antibody can be assigned to one of two types, called kappa (K) and lambda (l), based on the amino acid sequence of its constant domain. The heavy chain of an antibody can be assigned to one of five different types, IgA, IgD, IgE, IgG, and IgM, based on the amino acid sequence of its constant region, and several of these can be further divided into subclasses or isotypes, e.g., IgGl, IgG2, IgG3, and IgG4, IgAl, and IgA2.

[0115] An "IgG form of an antibody" refers to an IgG form to which the constant region of the heavy chain of the antibody belongs. For example, an IgG2 form of an antibody refers to an antibody whose heavy chain constant region is from IgG2.

[0116] The term "antigen-binding fragment" of an antibody refers to a molecule other than a full-length antibody that comprises a portion of a full-length antibody that is capable of binding to an antigen for which the full-length antibody is specific, or competes with the full-length antibody (i.e., with the full-length antibody from which the antigen-binding fragment is derived) for binding to the antigen. Antigen-binding fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, dAb (domain antibody), linear antibodies, single-chain antibodies (e.g., scFv); single-domain antibodies such as VHH, diabodies or fragments thereof, or camelid antibodies, diabodies, single-domain antibodies (sdAb), nanobodies. For example, Fab fragments can be obtained by papain digestion of a full-length antibody. In addition, pepsin digestion of a full-length antibody under denaturing conditions generates F(ab')2, which is a dimer of two Fab' fragments linked by their hinge regions. F(ab')2 can be reduced under denaturing conditions to break the disulfide bridges and convert the F(ab')2 dimer into a Fab' monomer. Fab' monomers are essentially Fab fragments with the hinge region. Fv fragments are composed of the VL and VH domains of a single arm of an antibody. The two domains of the Fv fragment, VL and VH, can be coded for by separate genes, but they can also be made as a single protein chain using recombinant methods, with a synthetic linker between the two domains that enables them to pair up and form a monovalent molecule by pairing of the VL and VH regions of a single protein chain. Binding fragments or derivatives generally retain at least 10% of the antigen-binding activity of the antibody from which they are derived, when the antigen-binding activity is expressed in molar concentrations. Preferably, binding fragments or derivatives retain at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the antigen-binding activity of the antibody from which they are derived.

[0117] The term "single chain antibody (scAb)" is used herein in the broadest sense, and specifically covers antibodies having mono- or multi- specificity (e.g., bispecificity) that are produced initially as a single, continuous polypeptide chain. Such single chain antibodies include, but are not limited to, those having two linked VL and VH regions. In one embodiment, the single chain antibody is a scFv.

[0118] A "diabody" is a bivalent small antibody constructed by gene fusion, e.g., it is a dimer of two polypeptide chains. The VL and VH domains of each polypeptide chain of a diabody are joined by a linker, such that the VL and VH encoded in the same polypeptide chain form a dimer with different single chain variable fragments. Diabodies generally have two antigen binding sites.

[0119] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of antibodies directed against a variety of epitopes (or having specificity for different epitopes). 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.

[0120] The term "chimeric antibody" as used herein is an antibody having the variable domains of a first antibody and the constant domains of a second antibody, wherein the first antibody and the second antibody are from different species. Typically the variable domains are obtained from an antibody of an experimental animal such as a rodent, while the constant domain sequences are obtained from a human antibody, such that the resulting chimeric antibody is less likely to induce an adverse immune response in a human subject compared to the experimental animal antibody.

[0121] The term "humanized antibody" as used herein refers to forms of antibodies that contain sequences from human and non-human (e.g., murine, rat) antibodies. Generally, a humanized antibody will comprise at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops are comparable to those of a non-human immunoglobulin, while all or substantially all of the framework (FR) regions are those of a human immunoglobulin. A humanized antibody optionally can comprise at least a portion of a human immunoglobulin constant region (Fc). In some instances, as is known to those skilled in the art, amino acid mutations can be introduced into a humanized antibody (e.g., variable domains, framework regions, and / or constant regions, if present), e.g., to improve certain properties of the antibody; such antibody forms are still within the scope of "humanized antibodies" of the present application.

[0122] As is appreciated by those skilled in the art, an antibody can have the glycosylation pattern of the cell in which the antibody is produced. For example, an antibody can contain mouse glycosylation when produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Alternatively, if produced in a rat, in a rat cell, or in a hybridoma derived from a rat cell, the antibody can contain rat glycosylation. In some embodiments, the antibodies described herein are modified to increase or decrease the extent of glycosylation of the antibody. The increase or decrease of glycosylation sites in an antibody can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0123] The term "multivalent antibody" denotes an antibody comprising two or more antigen binding sites. Multivalent antibodies are in some instances engineered to have three or more antigen binding sites, and are generally not naturally occurring antibodies.

[0124] The term "multispecific antibody" refers to an antibody that has at least two antigen binding sites, each of which binds to a different epitope of the same antigen or to different epitopes of different antigens. A multispecific antibody is an antibody that has binding specificities for at least two different antigenic epitopes. In some embodiments, the present application relates to a starting active ingredient that is a multispecific antibody, which has binding specificities for a first and a second antigen, also referred to as a "bispecific antibody." In some embodiments, the bispecific antibody comprises four antigen binding sites, and is typically a tetravalent binding protein. In some embodiments, the bispecific antibody comprises six antigen binding sites, and is typically a hexavalent binding protein.

[0125] The term "immunoconjugate" refers to a recombinant compound molecule in which an antibody is coupled (linked) to a second unit (payload). Typically, immunoconjugates comprise or consist of two to three independent components: an antibody that binds with high specificity to an antigen, a payload (effector unit) that has high biological activity at the target site, and a linker that ensures that the payload does not dissociate from the antibody during transport and reliably releases the payload at the target site or does not release the payload.

[0126] The term "payload" refers to an active moiety coupled to an antibody or antibody fragment of the present application, and can include any moiety used to attach the antibody or antibody fragment. In some embodiments, the payload can be a drug, such as a small molecule drug, a radionuclide, DNA, RNA, an enzyme, or a polypeptide, etc. In some embodiments, immunoconjugates encompass antibody drug conjugates (ADCs), antibody immunostimulant (agonist) conjugates (ISACs), antibody oligonucleotide conjugates (AOCs), antibody polypeptide conjugates (APCs), antibody polypeptide epitope conjugates (APECs), antibody radionuclide conjugates (RDCs), or antibody degrading conjugates (ADeCs), etc. In particular, when the payload is a cytotoxic agent (such as a chemotherapeutic drug), a growth inhibitory agent, a toxin such as an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof, or a radionuclide (i.e., a radioconjugate), the immunoconjugate can also be referred to as an "antibody-drug conjugate" (ADC). Suitable payloads or active moieties for attachment to an antibody include, for example, cytotoxic agents, chemotherapeutic agents, innate immune agonists (e.g., Toll-like receptor agonists (TLRs) such as ISAC drugs SBT6050, SBT6290, BDC-1001; STING agonists ISAC drugs XMT-2056, Treg cell modulating ISAC drugs ADCT-301, etc.), immunomodulatory agents, therapeutic oligonucleotides (siRNA, PMO, etc.), or radionuclides, etc. Examples

[0127] The following examples are intended to illustrate the present application and are not intended to limit the scope of the present application.

[0128] Example 1

[0129] This example used trastuzumab emtansine (T-DM1) as a test article to screen for a mock material solution (dextran 150 solution) having a similar sublimation resistance as the original solution of the molecule for use in the lyophilized test and engineering batches.

[0130] (1) Preparation of the stock solution: a) raw material freezing, the T-DM1 raw material composition is T-DM1 30 mg / mL, succinic acid 0.5 g / L, sodium succinate 1.559 g / L, and the solvent is ultrapure water; b) dialysis: fill the T-DM1 raw material into the dialysis cartridge, and add to the beaker containing succinic acid 0.5 g / L, sodium succinate 1.559 g / L, sucrose 60 g / L (solvent is ultrapure water), and stir; c) polysorbate 20 addition: add 10 g / kg of polysorbate 20 (solvent is ultrapure water), and the solution containing succinic acid 0.5 g / L, sodium succinate 1.559 g / L, sucrose 60 g / L (solvent is ultrapure water) to the T-DM1 concentration of 20 mg / mL, and the polysorbate 20 concentration of 0.02%; d) filtration: filter using a needle filter with a pore size of 0.22 μm, to obtain the stock solution.

[0131] (2) Preparation of the simulated material solution: add 30 g, 50 g, 70 g, 90 g of dextran 150 to each beaker, respectively, then add 0.5 g of succinic acid, 1.559 g of sodium succinate hexahydrate, 60 g of sucrose and 0.2 g of polysorbate 20 to each beaker, dissolve with ultrapure water, and then use ultrapure water to make up to 1 L, and filter using a needle filter with a pore size of 0.22 μm.

[0132] (3) Freeze dryer: use the Azbil Telstar Technology Telstar LyoBetaMini (0.18 m 2 ) freeze dryer for freeze drying.

[0133] (4) The freeze drying parameters are as follows:

[0134]

[0135] (5) Water loss determination: take 1 mL of the stock solution and different concentrations of the simulated material solution prepared above, respectively, and add to 2R vials, respectively, to determine the mass of the stock solution and different concentrations of the simulated material solution before freeze drying and the mass after freeze drying, and the difference between the two is the water loss of the stock solution and different concentrations of the simulated material solution.

[0136] (6) The formulations of the stock solution and different concentrations of the simulated material solution and their water loss are as follows:

[0137]

[0138]

[0139] (7) Analysis of variance: one-way analysis of variance

[0140] The F value of the 30, 50, 70, 90 mg / mL dextran 150 mimetic material solution group is calculated by using the above data through the following method:

[0141] (a) Calculate the within-group sample mean of the sample of the test mimetic material solution at different concentrations on the dehydration amount of the sample at each concentration

[0142]

[0143] Where Δm i,j is the dehydration amount of the sample of the jth test mimetic material solution at the ith concentration, n is the number of samples of the test mimetic material solution at the ith concentration, and 1≤j≤n, n≥2, k is the number of concentrations, and 1≤i≤k, k≥2;

[0144] (b) Calculate the total mean of the dehydration amount of the sample of the test mimetic material solution at all concentrations

[0145]

[0146] (c) Calculate the within-group sum of squares and the between-group sum of squares of the sample of the test mimetic material solution, wherein:

[0147] The within-group sum of squares SSE is calculated as

[0148]

[0149] The between-group sum of squares SSA is calculated as

[0150]

[0151] (d) Calculate the statistic F,

[0152]

[0153] The F value is 28.124, which is greater than the critical value F α (3.490) (where α=0.05) corresponding to the molecular degrees of freedom 3 and the denominator degrees of freedom 3×4 in the F distribution table, and it is considered that the concentration of the mimetic material solution has a significant effect on the dehydration amount, and the dehydration amount can be used for mimetic material screening.

[0154] Conclusion: When the concentration of the dextran 150 solution is 30-90 mg / mL, the concentration of the dextran 150 solution that is closest to the original solution in terms of the loss of water can be selected within the concentration range. When the concentration of the dextran 150 solution is 50 mg / mL, the loss of water thereof is closest to that of the original solution. Therefore, 50 mg / mL dextran 150, 10 mM succinic acid-sodium succinate, 6% (w / v) sucrose, and 0.02% (w / v) polysorbate 20 are used as the simulation material solution to fill the freeze dryer plate layer, and the simulation material solution is consistent with the GMP actual production batch in terms of the one-time drying endpoint.

[0155] Example 2

[0156] On the basis of Example 1, the least significant difference method is further used to perform multiple comparisons between each group of concentrations.

[0157]

[0158] wherein α = 0.05, t α / 2 is the critical value corresponding to the degree of freedom (4-1) x 4 in the t distribution table, i.e., 2.179. When If the value of t is greater than LSD, it is considered that there is a significant difference between the two groups.

[0159] Therefore, when the concentration of the dextran 150 solution is 30, 50, or 70 mg / mL, the data of each group has a significant difference.

[0160] Conclusion: On the basis of Example 1, it is proved that the concentration of the dextran 150 solution that is closest to the original solution in terms of the loss of water can be further selected within the concentration range of 30-70 mg / mL. When the concentration of the dextran 150 solution is 50 mg / mL, the loss of water thereof is closest to that of the original solution. Therefore, 50 mg / mL dextran 150, 10 mM succinic acid-sodium succinate, 6% (w / v) sucrose, and 0.02% (w / v) polysorbate 20 are used as the simulation material solution to fill the freeze dryer plate layer, and the simulation material solution is consistent with the GMP actual production batch in terms of the one-time drying endpoint.

[0161] Example 3

[0162] To prove the conclusions of Example 1 and Example 2, a full-load freeze drying test is performed, and an Azbil Telstar Technology Telstar LyoBeta Mini (0.18 m 2 ) freeze dryer is used for freeze drying, and the freeze drying parameters are as follows:

[0163]

[0164] The end of primary drying was determined by the double vacuum gauge end point, i.e. when the Pirani gauge and the capacitance vacuum gauge values were equal, the primary drying end point was considered to have been reached, and the results were as follows:

[0165]

[0166] When the ratio of the primary drying end point time of the simulated material solution to the stock solution was 0.8-1.2, preferably 0.9-1.1, the primary drying end point times of the two were considered to be close.

[0167] As can be seen from the above table, compared with the placebo as the simulated material solution, the primary drying end point time of the 50mg / mL dextran 150 simulated material solution and the stock solution was closer (the ratio of the primary drying end point times of the two was about 1.15). Therefore, the simulated material solution screening can be completed within a few hours by the amount of water loss, and the simulated material solution screened is more suitable for filling the freeze dryer plate layer of the test batch and the engineering batch.

[0168] Example 4

[0169] This example took emicymuzumab (T-DM1) as the test object, and screened a simulated material solution (trehalose dihydrate solution) having similar sublimation resistance to the stock solution of the molecule for use in the test batch and the engineering batch of freeze drying.

[0170] (1) Preparation of the stock solution:

[0171] The stock solution was prepared as in Example 1.

[0172] (2) Preparation of the simulated material solution: 140g, 160g, 180g and 200g trehalose dihydrate were added to each beaker, respectively, and then 0.5g succinic acid, 1.559g sodium succinate hexahydrate, 60g sucrose and 0.2g polysorbate 20 were added to each beaker, and the super-pure water was dissolved and then made up to 1L with super-pure water, and filtered using a needle filter with a pore size of 0.22μm.

[0173] (3) Freeze dryer: an Azbil Telstar Technology Telstar LyoBetaMini (0.18m 2 ) freeze dryer was used for freeze drying.

[0174] (4) The freeze drying parameters were as follows:

[0175]

[0176]

[0177] (5) Water loss: 1 mL of the stock solution prepared as above and simulant solutions of different concentrations were taken and added to 2R vials. The mass of the stock solution and simulant solutions of different concentrations before and after freeze-drying was measured. The difference between the two was the water loss of the stock solution and simulant solutions of different concentrations.

[0178] (6) The formulas of the stock solution and the simulated material solutions of different concentrations and their water loss are as follows:

[0179]

[0180] (7) Analysis of variance: One-way analysis of variance

[0181] The single-factor variance analysis was performed as in Example 1, and the F value was 13.006, which is greater than the critical value F corresponding to the numerator degree of freedom 3 and the denominator degree of freedom 3×4 in the F distribution table. α (3.490), it is believed that the concentration of the simulated material solution has a significant effect on the water loss, and the water loss can be used to screen the simulated materials.

[0182] According to the trehalose dihydrate concentration and water loss, the Figure 1 shown.

[0183] Conclusion: When the concentration of the simulated material trehalose dihydrate solution is 140-200 mg / mL, the concentration of the simulated material trehalose dihydrate solution that is closest to the water loss of the original solution can be selected within this concentration range. Figure 1 It was found that when the concentration of the simulated material trehalose dihydrate solution was 190 mg / mL, its water loss was closest to that of the original solution. Therefore, 190 mg / mL trehalose dihydrate, 10 mM succinic acid-sodium succinate, 6% (w / v) sucrose, and 0.02% (w / v) polysorbate 20 were used as the simulated material solution to fill the freeze dryer plate layer. This simulated material solution maintained the same primary drying endpoint in the pilot batch, engineering batch, and actual GMP production batch.

[0184] Example 5

[0185] To prove the conclusion of Example 4, a freeze-drying test was carried out using a method similar to that of Example 3. Azbil Telstar Technology Telstar LyoBeta Mini (0.18 m 2 ) freeze dryer for freeze drying, with the double vacuum gauge coincidence as the end point, and the results are as follows:

[0186]

[0187] The primary drying end point times of the mock material solutions and the bulk solution are considered to be close when the ratio of the primary drying end point time of the mock material solution to the bulk solution is 0.8-1.2, preferably 0.9-1.1.

[0188] As can be seen from the above table, the primary drying end point time of the 190 mg / mL trehalose dihydrate mock material solution is closer to that of the bulk solution (the ratio of the primary drying end point time of the mock material solution to the bulk solution is about 0.85) than that of the placebo as the mock material solution. Therefore, the mock material solution screening can be completed within several hours by the loss of water, and the mock material solution screened is more suitable for use in the freeze-drying of the test and engineering batches to fill the freeze-dryer plate layers.

[0189] Example 6

[0190] In this example, bevacizumab was used as the test object, a pilot-scale freeze-dryer was used for freeze-drying, and a mock material solution (dextran 150 solution) having a similar sublimation resistance as the molecule was screened for use in the test and engineering batches for freeze-drying.

[0191] (1) Preparation of the bulk solution:

[0192] a) Freezing of the raw material: the bevacizumab raw material consisted of bevacizumab 35 mg / mL, histidine 0.44 g / L, hydrochloric acid histidine 3.59 g / L, and the solvent was ultrapure water; b) Dialsis: the bevacizumab raw material was filled into a dialysis cassette, and was added to a beaker containing histidine 0.44 g / L, hydrochloric acid histidine 3.59 g / L, and 80 g / L trehalose dihydrate (the solvent was ultrapure water), and was stirred; c) Addition of polysorbate 80: 10 g / kg of polysorbate 80 (the solvent was ultrapure water) and a solution containing histidine 0.44 g / L, hydrochloric acid histidine 3.59 g / L, and 80 g / L trehalose dihydrate (the solvent was ultrapure water) were added to a T-DM1 concentration of 20 mg / mL and a polysorbate 20 concentration of 0.02%; d) Filtration: filtration was performed using a needle filter with a pore size of 0.22 μm, and the bulk solution was obtained.

[0193] (2) Preparation of the mock material solutions: 10 g, 30 g, and 50 g of dextran 150 were added to each beaker, respectively, and then 0.44 g of L-histidine, 3.59 g of L-hydrochloric acid histidine monohydrate, 80 g of trehalose dihydrate, and 0.1 g of polysorbate 80 were added to each beaker, and ultrapure water was used to dissolve and dilute to 1 L, and filtration was performed using a needle filter with a pore size of 0.22 μm.

[0194] (3) Freeze-dryer: freeze-drying was performed using an Azbil Telstar Technology Telstar LyoBetaMini (0.18 m 2 ) freeze-dryer.

[0195] (4) Freeze-drying parameters: The freeze-drying parameters were as follows:

[0196]

[0197] (5) Determination of water loss: 1 mL of the stock solution and different concentrations of the simulated material solution prepared above were taken into 2R carboy bottles, respectively, and the mass of the stock solution and different concentrations of the simulated material solution before freeze-drying and after freeze-drying was determined, respectively, and the difference between the two was the water loss of the stock solution and different concentrations of the simulated material solution.

[0198] (6) Formulation of the stock solution and different concentrations of the simulated material solution and their water loss were as follows:

[0199]

[0200]

[0201] (7) Analysis of variance: One-way analysis of variance

[0202] The one-way analysis of variance was performed as in Example 1, and the F value was 16.276, which was greater than the critical value F α (5.143) (wherein a = 0.05) corresponding to the numerator degree of freedom 2 and the denominator degree of freedom 2 x 3 in the F distribution table, and it was considered that the concentration of the simulated material solution had a significant effect on the water loss, and the water loss could be used for the screening of the simulated material.

[0203] Conclusion: When the concentration of the simulated material dextran 150 solution was 10-50 mg / mL, the concentration of the simulated material dextran 150 solution closest to the water loss of the stock solution could be selected within the concentration range. When the concentration of the simulated material dextran 150 solution was 50 mg / mL, its water loss was closest to that of the stock solution. Therefore, 50 mg / mL dextran 150, 20 mM histidine-histidine hydrochloride, 8% (w / v) trehalose dihydrate, and 0.01% (w / v) polysorbate 80 were used as the simulated material solution to fill the freeze-dryer plate layer, and the simulated material solution was consistent with the GMP actual production batch in the test batch and the engineering batch and the one-time drying end point.

[0204] Example 7

[0205] To prove the conclusion of Example 6, a full-load freeze-drying test was performed using a method similar to that of Example 3, and a Dongfenglong Lyo-0.5 freeze-dryer of pilot production scale was used for freeze-drying, and the double vacuum metering was combined as the end point, and the results were as follows:

[0206]

[0207] The primary drying endpoint times of the mock material solutions and the original solution are considered to be close when the ratio of the primary drying endpoint time of the mock material solution to the primary drying endpoint time of the original solution is 0.8-1.2, preferably 0.9-1.1.

[0208] As can be seen from the above table, the primary drying endpoint time of the 50 mg / mL dextran 150 mock material solution is closer to that of the original solution (the ratio of the primary drying endpoint time of the mock material solution to that of the original solution is about 0.95) than that of the placebo as a mock material solution. Therefore, the mock material solution screening can be completed within several hours by the loss of water amount, and the mock material solution screened is more suitable for filling the freeze dryer plate layer of the freeze-drying pilot batch and engineering batch.

[0209] Example 8

[0210] This example compares the method of the present application with the conventional method (determining the mock material concentration according to the similarity of the glass transition temperature (Tg’) to that of the original solution).

[0211] The theoretically calculated Tg’ temperatures of the original solution, the placebo and the dextran 150 mock material solutions with different concentrations are as follows:

[0212]

[0213] The calculation of Tg’ can be found in Jameel et al., Principles and Practices of Lyophilization in Product Development and Manufacturing, Chapter 2 Section 3.3 “Estimation of Tg’”. As can be seen from the above table, according to the conventional method, the 5 mg / mL dextran 150 mock material solution (its Tg’ is almost equal to that of the original solution) should be selected instead of the 50 mg / mL dextran 150 mock material solution (its Tg’ is -17.1, which is quite different from that of the original solution (-26.5)). However, according to Example 7 above, the primary drying endpoint time of the 50 mg / mL dextran 150 mock material solution is the closest to that of the original solution, which is the most suitable mock material solution for the original solution. This shows that the selection of the mock material solution by the theoretical calculation of Tg’ is not accurate. The method of the present application can better select the suitable mock material solution for filling the freeze dryer plate layer of the freeze-drying pilot batch and engineering batch.

[0214] The mock material solution selected according to Tg’ is subjected to full load freeze-drying test using the Dongfenglong Lyo-0.5 freeze dryer of pilot scale in production, and the end point is double vacuum metering, and the results are as follows:

[0215]

[0216]

[0217] Example 9

[0218] This example takes trastuzumab emtansine (T-DM1) as the test object, and screens a simulated material solution (dextran 20 solution) with similar sublimation resistance to the original solution of the molecule for the test batch and engineering batch of freeze-drying.

[0219] (1) Preparation of the original solution:

[0220] The original solution was prepared as in Example 1.

[0221] (2) Preparation of the simulated material solution: 30 g, 50 g and 70 g of dextran 20 were added to each beaker, respectively, followed by the addition of 0.5 g of succinic acid, 1.559 g of sodium succinate hexahydrate, 60 g of sucrose and 0.2 g of polysorbate 20 in each beaker. After dissolving in ultrapure water, the volume was made up to 1 L with ultrapure water, and filtration was performed using a needle filter with a pore size of 0.22 μm.

[0222] (3) Freeze dryer: freeze-drying was performed using an Azbil Telstar Technology Telstar LyoBetaMini (0.18 m 2 ) freeze dryer

[0223] (4) The freeze-drying parameters were as follows:

[0224]

[0225] (5) Determination of the loss on drying: 1 mL of the original solution and the simulated material solutions of different concentrations prepared above were taken into 2R vials, respectively, and the mass of the original solution and the simulated material solutions of different concentrations before freeze-drying and after freeze-drying was determined, respectively. The difference between the two was the loss on drying of the original solution and the simulated material solutions of different concentrations.

[0226] (6) The formulations of the original solution and the simulated material solutions of different concentrations and their loss on drying were as follows:

[0227]

[0228]

[0229] (7) Analysis of variance: single-factor analysis of variance

[0230] Single-factor analysis of variance was performed as in Example 1, and the F value was 69.537, which was greater than the critical value F α(4.256) (where a = 0.05), the concentration of the simulated material solution was considered to have a significant effect on the loss on drying, and the loss on drying can be used for the screening of the simulated material.

[0231] Conclusion: When the concentration of the simulated material dextran 20 solution is 30-70 mg / mL, the concentration of the simulated material dextran 20 solution that is closest to the loss on drying of the stock solution can be selected within the concentration range. When the concentration of the simulated material dextran 20 solution is 30 mg / mL, it is closest to the loss on drying of the stock solution. Therefore, 30 mg / mL dextran 20, 10 mM succinic acid-sodium succinate, 6% (w / v) sucrose, and 0.02% (w / v) polysorbate 20 are used as the simulated material solution to fill the freeze dryer plate layer, and the simulated material solution is consistent with the GMP actual production batch in the test batch and the engineering batch and the one-time drying end point.

[0232] Example 10

[0233] In this example, trastuzumab emtansine (T-DM1) is used as the test object, and a simulated material solution (dextran 70 solution) with similar sublimation resistance to the stock solution of the molecule is screened for use in the test batch and the engineering batch of the freeze-dried product.

[0234] (1) Preparation of the stock solution:

[0235] The stock solution is prepared as in Example 1.

[0236] (2) Preparation of the simulated material solution: 30 g, 50 g, and 70 g of dextran 70 are added to each beaker, respectively, and then 0.5 g of succinic acid, 1.559 g of sodium succinate hexahydrate, 60 g of sucrose, and 0.2 g of polysorbate 20 are added to each beaker. After dissolving in ultrapure water, the volume is adjusted to 1 L with ultrapure water, and filtered using a needle filter with a pore size of 0.22 μm.

[0237] (3) Freeze dryer: Azbil Telstar Technology Telstar LyoBetaMini (0.18 m 2 ) freeze dryer is used for freeze drying

[0238] (4) The freeze drying parameters are as follows:

[0239]

[0240]

[0241] (5) Water loss determination: 1 mL of the stock solution and different concentrations of the simulated material solution prepared above were taken into 2R Schlenk flasks, respectively, and the mass of the stock solution and different concentrations of the simulated material solution before freeze-drying and after freeze-drying was determined, respectively, and the difference between the two was the water loss of the stock solution and different concentrations of the simulated material solution.

[0242] (6) The formulations of the stock solution and different concentrations of the simulated material solution and their water loss are as follows:

[0243]

[0244] (7) Analysis of variance: single factor analysis of variance

[0245] The single factor analysis of variance was carried out as in Example 1, and the F value was 36.469, which was greater than the critical value F α (4.256) (wherein a = 0.05), and it was considered that the concentration of the simulated material solution had a significant effect on the water loss, and the water loss could be used for the screening of the simulated material.

[0246] Conclusion: When the concentration of the simulated material dextran 70 solution was 30-70 mg / mL, the concentration of the simulated material dextran 70 solution closest to the water loss of the stock solution could be selected within this concentration range. When the concentration of the simulated material dextran 70 solution was 30 mg / mL, its water loss was closest to that of the stock solution. Therefore, 30 mg / mL dextran 70, 10 mM succinic acid-sodium succinate, 6% (w / v) sucrose, and 0.02% (w / v) polysorbate 20 were used as the simulated material solution to fill the freeze-dryer plate layer, and the simulated material solution was consistent with the GMP actual production batch in the test batch and the engineering batch and the one-time drying end point.

[0247] Example 11

[0248] To prove the conclusion of Example 10, a full-load freeze-drying test was carried out using a method similar to that of Example 3, using an Azbil Telstar Technology Telstar LyoBeta Mini (0.18 m 2 ) freeze-dryer to freeze-dry, with double vacuum weighing as the end point, and the results were as follows:

[0249]

[0250] When the ratio of the one-time drying end point time of the simulated material solution to the stock solution was 0.8-1.2, preferably 0.9-1.1, it was considered that the one-time drying end point times of the two were close.

[0251] As can be seen from the above table, compared with placebo as a simulation material solution, the one-time drying end point time of 30 mg / mL dextran 70 simulation material solution is closer to that of the stock solution (the ratio of the one-time drying end point time of the two is about 0.88). Therefore, the simulation material solution screening can be completed within a few hours by the water loss amount, and the simulation material solution screened is more suitable for filling the freeze dryer plate layer of the test batch and engineering batch for freeze drying.

[0252] Example 12

[0253] This example takes trastuzumab emtansine (T-DM1) as a test object, and screens a simulation material solution (only containing bovine serum albumin as a simulation material) with similar sublimation resistance to the stock solution of the molecule for test batches and engineering batches for freeze drying.

[0254] (1) Preparation of the stock solution:

[0255] The stock solution was prepared as in Example 1.

[0256] (2) Preparation of the simulation material solution: 20 g, 40 g and 60 g of bovine serum albumin were added to each beaker, respectively, and dissolved in ultrapure water, and then the volume was adjusted to 1 L with ultrapure water, and filtered using a needle filter with a pore size of 0.22 μm.

[0257] (3) Freeze dryer: freeze drying was performed using an Azbil Telstar Technology Telstar LyoBetaMini (0.18 m 2 ) freeze dryer.

[0258] (4) The freeze drying parameters were as follows:

[0259]

[0260]

[0261] (5) Water loss amount determination: 1 mL of the stock solution and simulation material solutions with different concentrations prepared as above were taken into 2R cellophaned bottles, respectively, and the mass of the stock solution and simulation material solutions with different concentrations before freeze drying and after freeze drying was determined, respectively, and the difference between the two was the water loss amount of the stock solution and simulation material solutions with different concentrations.

[0262] (6) The formulations of the stock solution and simulation material solutions with different concentrations and their water loss amounts were as follows:

[0263]

[0264] (7) Analysis of variance: one-way analysis of variance

[0265] The F value is 61.355, which is greater than the critical value F of the F distribution table corresponding to the molecular degrees of freedom 2 and the denominator degrees of freedom 3x3 α (4.256) (wherein a = 0.05), it is considered that the concentration of the simulated material solution has a significant effect on the loss of water, and the loss of water can be used for the screening of the simulated material.

[0266] Conclusion: When the concentration of the simulated material bovine serum albumin solution is 20-60 mg / mL, the concentration of the simulated material bovine serum albumin solution closest to the loss of water of the original solution can be selected in the concentration range. When the concentration of the simulated material bovine serum albumin solution is 40 mg / mL, the loss of water thereof is closest to that of the original solution. Therefore, 40 mg / mL bovine serum albumin is used as a simulated material solution to fill the plate layer of the freeze dryer, and the simulated material solution is consistent with the GMP actual production batch in the test batch and the engineering batch and the one-time drying endpoint.

[0267] Example 13

[0268] To prove the conclusion of Example 12, a full-load freeze-drying test was performed using a method similar to that of Example 3, using an Azbil Telstar Technology Telstar LyoBeta Mini (0.18 m 2 ) freeze dryer to freeze-dry, with a double vacuum metering end point, and the results are as follows:

[0269]

[0270] When the ratio of the one-time drying endpoint time of the simulated material solution to the original solution is 0.8-1.2, preferably 0.9-1.1, it is considered that the one-time drying endpoint times of the two are close.

[0271] As can be seen from the above table, compared with the placebo as the simulated material solution, the one-time drying endpoint time of the 40 mg / mL bovine serum albumin simulated material solution and the original solution is closer (the ratio of the one-time drying endpoint times of the two is about 0.97). Therefore, the screening of the simulated material solution can be completed within a few hours by the loss of water, and the simulated material solution screened is more suitable for filling the plate layer of the freeze dryer for the test batch and the engineering batch.

[0272] Example 14

[0273] This example takes trastuzumab emtansine (T-DM1) as the test object, and screens a simulated material solution (containing only polydextran 70 as the simulated material) with similar sublimation resistance to the original solution of the molecule for the test batch and the engineering batch for freeze-drying.

[0274] (1) Preparation of the original solution:

[0275] The stock solution was prepared as in Example 1.

[0276] (2) Preparation of simulated material solutions: 30 g, 50 g and 70 g of polysucrose 70 were added to each beaker, respectively, and dissolved in ultrapure water. The volume was made up to 1 L with ultrapure water, and filtered using a needle filter with a pore size of 0.22 μm.

[0277] (3) Freeze dryer: a Telstar LyoBeta Mini (0.18 m 2 ) freeze dryer from Azbil Telstar Technology was used for freeze drying.

[0278] (4) The freeze drying parameters were as follows:

[0279]

[0280] (5) Determination of water loss: 1 mL of the stock solution and simulated material solutions of different concentrations prepared above were taken into 2R vials, respectively, and the mass of the stock solution and simulated material solutions of different concentrations before freeze drying and after freeze drying was determined, respectively. The difference between the two was the water loss of the stock solution and simulated material solutions of different concentrations.

[0281] (6) The formulations of the stock solution and simulated material solutions of different concentrations and their water loss were as follows:

[0282]

[0283]

[0284] (7) Analysis of variance: one-way analysis of variance

[0285] One-way analysis of variance was performed as in Example 1, and the F value was 29.235, which was greater than the critical value F α (4.256) in the F distribution table corresponding to the numerator degree of freedom 2 and the denominator degree of freedom 3 x 3 (where α = 0.05), and it was considered that the concentration of the simulated material solution had a significant effect on the water loss, and the water loss could be used for the screening of simulated materials.

[0286] Conclusion: When the concentration of the simulated material polysucrose 70 solution was 30-70 mg / mL, the simulated material polysucrose 70 solution with the water loss closest to that of the stock solution could be selected within this concentration range. When the concentration of the simulated material polysucrose 70 solution was 70 mg / mL, its water loss was closest to that of the stock solution. Therefore, 70 mg / mL polysucrose 70 was used as the simulated material solution to fill the freeze dryer plate, and the simulated material solution was consistent with the GMP actual production batch in terms of the one-time drying end point of the test batch and the engineering batch.

[0287] Example 15

[0288] This example used trastuzumab emtansine (T-DM1) as the test subject, and screened simulated material solutions (containing only sucrose as the simulated material) having similar sublimation resistance to the original solution of the molecule for use in the test batch and engineering batch of lyophilization.

[0289] (1) Preparation of the original solution:

[0290] The original solution was prepared as in Example 1.

[0291] (2) Preparation of the simulated material solution: 240 g, 260 g, 280 g, and 300 g of sucrose were added to each beaker, respectively, and dissolved in ultrapure water, and then ultrapure water was added to make up to 1 L, and filtered using a needle filter with a pore size of 0.22 μm.

[0292] (3) Lyophilizer: Lyophilization was performed using an Azbil Telstar Technology Telstar LyoBetaMini (0.18 m 2 ) lyophilizer.

[0293] (4) The lyophilization parameters were as follows:

[0294]

[0295] (5) Determination of the loss on drying: 1 mL of the original solution and the simulated material solutions of different concentrations prepared above were taken into 2R

[0296] (6) The formulations of the original solution and the simulated material solutions of different concentrations and their loss on drying were as follows:

[0297]

[0298] (7) Analysis of variance: One-way analysis of variance

[0299] One-way analysis of variance was performed as in Example 1, and the F value was 21.691, which was greater than the critical value F α (3.490) in the F distribution table corresponding to the numerator degrees of freedom 3 and the denominator degrees of freedom 3 x 4 (where α = 0.05), and it was considered that the concentration of the simulated material solution had a significant effect on the loss on drying, and the loss on drying could be used for simulated material screening.

[0300] Conclusion: When the concentration of the simulated material sucrose solution is 240-300 mg / mL, the concentration of the simulated material sucrose solution that is closest to the original solution in terms of the amount of water loss can be selected within this concentration range. When the concentration of the simulated material sucrose solution is 240 mg / mL, the amount of water loss thereof is closest to that of the original solution. Therefore, 240 mg / mL sucrose is used as the simulated material solution to fill the freeze dryer plate, and the simulated material solution is consistent with the GMP actual production batch in terms of the primary drying endpoint of the test batch and the engineering batch.

[0301] Example 16

[0302] To prove the conclusion of Example 15, a full-load freeze-drying test was performed using a method similar to that of Example 3, using an Azbil Telstar Technology Telstar LyoBeta Mini (0.18 m2) freeze dryer to perform freeze-drying, with a double vacuum meter as the endpoint, and the results are as follows: 2 ) freeze dryer to perform freeze-drying, with a double vacuum meter as the endpoint, and the results are as follows:

[0303]

[0304] When the ratio of the primary drying endpoint time of the simulated material solution to that of the original solution is 0.8-1.2, preferably 0.9-1.1, the primary drying endpoint times of the two are considered to be close.

[0305] As can be seen from the above table, the primary drying endpoint time of the 240 mg / mL sucrose simulated material solution is very close to that of the original solution (the ratio of the primary drying endpoint times of the two is about 0.95). Therefore, the selection of the simulated material solution can be completed within a few hours by the amount of water loss, and the simulated material solution selected is more suitable for use in filling the freeze dryer plate of the test batch and the engineering batch for freeze-drying.

[0306] Example 17

[0307] This example uses enfortumab vedotin as the test subject to select a simulated material solution (dextran 20 solution) having a similar sublimation resistance to the original solution of the molecule for use in the test batch and the engineering batch for freeze-drying.

[0308] (1) Preparation of the stock solution: a) Raw material freezing. b) Ultrafiltration: Fill 15 mL of Enanumab stock solution into the ultrafiltration centrifuge tube, centrifuge at 3500 rpm for 30 minutes, after centrifugation, add a solution containing 0.5 g / L succinic acid, 1.559 g / L sodium succinate, 60 g / L sucrose (solvent is ultrapure water) to 15 mL, repeat 7 times, then concentrate the sample to 30 mg / mL; c) Polysorbate 20 addition: add a solution containing 10 g / kg polysorbate 20 (solvent is ultrapure water), and a solution containing 0.5 g / L succinic acid, 1.559 g / L sodium succinate, 60 g / L sucrose (solvent is ultrapure water) to the concentration of Enanumab to 20 mg / mL, the concentration of polysorbate 20 to 0.02% liquid; d) Filtration: filter using a needle filter with a pore size of 0.22 μm.

[0309] (2) Preparation of the simulated material solution:

[0310] Prepare the simulated material solution as in Example 9

[0311] (3) Freeze dryer: use Azbil Telstar Technology Telstar LyoBetaMini (0.18 m 2 ) freeze dryer for freeze-drying.

[0312] (4) The freeze-drying parameters are as follows:

[0313]

[0314]

[0315] (5) Water loss determination: take 1 mL of the stock solution and different concentrations of the simulated material solution prepared above, respectively, add to 2R cellophanes, respectively, determine the mass of the stock solution and different concentrations of the simulated material solution before freeze-drying and the mass after freeze-drying, the difference between the two is the water loss of the stock solution and different concentrations of the simulated material solution.

[0316] (6) The formulations of the stock solution and different concentrations of the simulated material solution and their water loss are as follows:

[0317]

[0318] (7) Analysis of variance: one-way analysis of variance

[0319] Perform one-way analysis of variance as in Example 1, get F value 69.537, the value is greater than the critical value F α (4.256) in the F distribution table corresponding to the numerator degrees of freedom 2, denominator degrees of freedom 3x3 (where α = 0.05), it is considered that the concentration of the simulated material solution has a significant effect on the water loss, and the water loss can be used for simulated material screening.

[0320] Conclusion: When the concentration of the dextran 20 solution is between 30-70 mg / mL, the concentration of the dextran 20 solution that is closest to the original solution in terms of the amount of water loss can be selected within this range. When the concentration of the dextran 20 solution is 50 mg / mL, it is closest to the original solution in terms of the amount of water loss. Therefore, 50 mg / mL dextran 20, 10 mM succinic acid-sodium succinate, 6% (w / v) sucrose, 0.02% (w / v) polysorbate 20 can be used as the simulation material solution to fill the freeze dryer plate layer, which is consistent with the GMP actual production batch in terms of the primary drying endpoint for the test batch and the engineering batch.

[0321] Example 18

[0322] To prove the conclusion of Example 17, a full-scale freeze-drying test was performed using a production pilot-scale Dongfenglong Lyo-0.5 freeze dryer to freeze-dry, with the double vacuum metering endpoint, using a method similar to that of Example 3, and the results were as follows:

[0323]

[0324] When the ratio of the primary drying endpoint time of the simulation material solution to the original solution is 0.8-1.2, preferably 0.9-1.1, the primary drying endpoint times of the two are considered to be close.

[0325] As can be seen from the above table, the primary drying endpoint time of the 50 mg / mL dextran 20 simulation material solution is very close to that of the original solution (the ratio of the primary drying endpoint times of the two is about 1.09). Therefore, the simulation material solution screening can be completed within a few hours by the amount of water loss, and the simulation material solution screened is more suitable for filling the freeze dryer plate layer for the freeze-drying test batch and the engineering batch.

[0326] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Any or all of the features discussed above and throughout this application can be combined in various embodiments of the present application. Furthermore, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting.

Claims

1. A method for screening a simulated material solution containing a stock solution of an active ingredient, the method comprising: (1) preparing a stock solution containing the active ingredient; (2) Selecting a simulated material to be tested and preparing solutions of the simulated material to be tested with different concentrations; (3) Under the same freeze-drying conditions, the stock solution prepared in step (1) and the simulated material solution to be tested prepared in step (2) are freeze-dried respectively; (4) Determine the water loss of each sample after freeze-drying; (5) determining whether the simulated material solutions of different concentrations to be tested can be used as alternative simulated material solutions for the stock solution by statistically analyzing the water loss; (6) If the judgment result of step (5) is that the candidate simulating material solution can be used as the original solution, the simulating material solution to be tested with a concentration closest to the water loss of the original solution is selected as the simulating material solution of the original solution.

2. The method according to claim 1, wherein the active ingredient is a biopharmaceutical molecule, preferably one or more substances selected from the following: proteins, polypeptides, nucleic acids or any derivatives thereof (such as fusions or conjugates), For example, the biopharmaceutical molecule is a protein, optionally, the protein has the following post-translational modifications: glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting groups / blocking groups, modification with aliphatic chains / fatty acid chains, polyethylene glycol modification, proteolytic cleavage or modification with non-naturally occurring amino acids, and / or the protein has a quaternary structure, preferably the protein is an immunoglobulin or a fragment or derivative thereof, preferably the protein is an antibody or an antigen-binding fragment thereof, such as rituximab, trastuzumab, bevacizumab, cetuximab, pembrolizumab, nivolumab, pertuzumab, atezolizumab, Adalimumab, ustekinumab, dupilumab, secukinumab, edixetumab, ocrelizumab, ofatumumab, alizumab, edoxetine, denosumab, aducanumab, alemtuzumab, atumumab, atezolizumab, anetuzumab, avelumab, bapilumab, basiliximab, betumumab, bermedizumab, besolumab, belotuzumab, vebrutuximab, vebrutuximab, bodalumab, cemiprilizumab, simpanumab, clituzumab, clenezumab, daclizumab, daratumumab, dinutuximab, dotalizumab, durvalumab, edrecolomab , elotuzumab, epavacumab, enroku, epratuzumab, edamasumab, gemtuzumab, gemtuximab, gonelizumab, ibritumomab tiuxetan, inelizumab, infliximab, inotuzumab, ogamicin inotuzumab, ipilimumab, isatuzumab, rosopetuzumab, labetuzumab, lencaneluzumab, lantuzumab, moglizumab, nexitozumab, nimotuzumab, natalizumab, nasituximab, olaramumab, ogavuzumab, panitumumab, pollotuzumab, vepotuzumab, punituzumab, rituximab, ramucirumab, certolizumab pegol, goxetine monoclonal antibody, ceritumomab, siltuximab, solanezumab, tancitumomab, tetuzumab, tetuzumab, tiplanezumab, tocilizumab, tositumomab, vedolizumab, futumomab, zegetelumab, zalumab or zalumab or an antigen-binding fragment of the above antibodies, such as a bispecific antibody such as belintoumab, emicizumab, terituzumab, BVX001, motuzumab, zetuzumab, ervantuzumab, grofituzumab, zenidatuzumab, catumaxomab, cardunilizumab, icoretumumab, taquitozumab, enatuzumab, faricizumab, bimegilide, ivocizumab; Alternatively, preferably the protein is an immunoconjugate, such as an antibody-drug conjugate such as gemtuzumab ozogamicin, vebutuximab, emtansine trastuzumab, emtansine trastuzumab biosimilar, ogaiituzumab, detrastuzumab, vepotuzumab, veentuzumab, goxatuzumab, belantamab mafudotin, Akalux, vedicizumab, tarantuzumab, tisotumomab, sometuximab, epratuzumab SN38, ZW-49, such as an antibody-radionuclide conjugate, an antibody-immunostimulatory conjugate, an antibody-degrader conjugate, an antibody-fragment conjugate, an antibody-oligonucleotide conjugate, an antibody-cell conjugate or an antibody-biopolymer conjugate, Alternatively, preferably, the protein is a fusion protein, such as aflibercept, tetacept, belatacept, rilocept, romiplostim, abatacept, alefacept, etanercept, dulaglutide, Alternatively, preferably, the protein is selected from one or more of the following substances: cyclic peptides such as syfovre, GLP-1RA such as liraglutide, semaglutide, exenatide, etc., hormones such as growth hormone, insulin, estrogen, etc., cytokines such as interferon, interleukin, etc., blood products such as immunoglobulin, albumin, etc., Alternatively, for example, the biopharmaceutical molecule is a nucleic acid molecule, such as an unmodified nucleic acid molecule or a chemically modified nucleic acid molecule, such as a naturally occurring modified nucleic acid molecule, such as a glycosylated or methylated nucleic acid molecule, or an artificially modified derivative, such as a biotinylated p nucleic acid molecule, a locked nucleic acid, a peptide nucleic acid, etc.

3. The method according to claim 1 or 2, wherein the mimetic material is selected from one or more of the following substances: sugars, such as monosaccharides, disaccharides or polysaccharides; proteins; polymers; organic acids; alcohols; and amino acids; Preferably, the compound is selected from one or more of the following substances: sugars, such as monosaccharides, disaccharides (such as lactose, sucrose or trehalose) or polysaccharides (such as dextran, polysucrose, sorbitol, mannitol, hydroxyethyl starch); proteins (such as human serum albumin, bovine serum albumin or other protein-like substances); polymers (such as polyethylene glycol or polyvinylpyrrolidone (PVP)); organic acids (such as citric acid); and amino acids (such as histidine or glycine); More preferably, one or more substances are selected from the following: dextran, such as dextran 10, dextran 20, dextran 40, dextran 70, dextran 150 or dextran 400; bovine serum albumin; human serum albumin; polysucrose, such as polysucrose 70, polysucrose 400; hydroxyethyl starch, such as hydroxyethyl starch 130 or hydroxyethyl starch 200; polyethylene glycol, such as polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 1000, polyethylene glycol 1500, polyethylene glycol 3350, polyethylene glycol 4000 or polyethylene glycol 6000; polyvinyl pyrrolidone (PVP), such as PVP K30 or PVP K90; sucrose; trehalose / trehalose dihydrate; citric acid; mannitol; sorbitol; histidine; glycine; and lactose; Further preferably, it is selected from one or more of the following substances: dextran, bovine serum albumin, polysucrose, sucrose, trehalose / trehalose dihydrate; More preferably, the compound is selected from one or more of the following substances: dextran 20, dextran 70, dextran 150, bovine serum albumin, polysucrose 70, sucrose, and trehalose dihydrate.

4. The method according to any one of claims 1 to 3, wherein the active ingredient is an immunoglobulin or a fragment or derivative thereof, such as a monoclonal antibody or an antibody-drug conjugate (ADC); and the mimetic material is selected from one or more of the following substances: dextran, bovine serum albumin, polysucrose, sucrose, trehalose / trehalose dihydrate.

5. The method of claim 4, wherein the active ingredient is selected from one or more of the following: trastuzumab, bevacizumab, enatuzumab, zetuzumab, zenidatuzumab, evocituzumab, emtansine trastuzumab, emtansine trastuzumab biosimilar, detrastuzumab, vepotuzumab, or ZW-49.

6. The method according to claim 4 or 5, wherein the simulation material is selected from one or more of the following substances: dextran 20, dextran 70, dextran 150, bovine serum albumin, polysucrose 70, sucrose, and trehalose dihydrate.

7. The method according to any one of claims 1 to 6, wherein the stock solution and the simulated material solution further comprise one or more substances selected from the group consisting of a buffer, a lyoprotectant, an amino acid, a stabilizer, a preservative, an antioxidant, an osmotic pressure regulator, an adjuvant, a chelating agent, an acid-base regulator, a filler, and a surfactant.

8. The method according to any one of claims 1 to 7, wherein in step (2), a substance with a molecular weight close to the relative molecular weight of the active ingredient is selected as the simulating material, or a component in the stock solution other than the active ingredient is selected as the simulating material.

9. The method according to any one of claims 1 to 8, wherein in step (2), the concentration range of the simulated material solution to be tested is selected according to the molecular weight and concentration of the active ingredient in the stock solution prepared in step (1).

10. The method according to any one of claims 1 to 9, wherein in step (2), 2 to 10 concentrations of the simulated material solution to be tested are prepared, preferably 3 to 8 concentrations of the simulated material solution to be tested, more preferably 4 to 7 concentrations of the simulated material solution to be tested, for example 2, 3, 4, 5 or 6 concentrations of the simulated material solution to be tested.

11. The method according to any one of claims 1 to 10, wherein in step (2), at least 3 portions, preferably at least 5 portions, and more preferably at least 10 portions of the simulated material solution of each concentration are prepared.

12. The method according to any one of claims 1 to 11, wherein in step (2), the concentration gradient is 20 mg / mL, preferably 10 mg / mL, more preferably 5 mg / mL.

13. The method according to any one of claims 1 to 12, wherein in step (3), the stock solution prepared in step (1) and the simulated material solution prepared in step (2) are prepared in a container, such as a vial, and the specifications of the vial are preferably 2R, 6R, 10R, 20R, 30R, 50R, preferably 2R, 6R, 10R, and more preferably 2R.

14. The method according to claim 13, wherein the amount of the stock solution prepared in step (1) and the simulated material solution prepared in step (2) in the vial is about 1 / 3 to about 1 / 2 of the vial size.

15. The method according to any one of claims 1 to 14, wherein in step (3), freeze drying is stopped when the primary drying is about 30% to about 80%, preferably about 40% to about 60%, complete.

16. The method according to any one of claims 1 to 15, wherein the statistical analysis comprises: (a) Calculate the mean of the water loss of the samples of the simulated material solution of different concentrations prepared in step (2) at each concentration. where Δm i,j is the water loss of the jth sample of the simulated material solution to be tested at the i-th concentration, n is the number of samples of the simulated material solution to be tested at the i-th concentration, and 1≤j≤n, n≥2, k is the number of concentrations, and 1≤i≤k, k≥2; (b) Calculate the total mean water loss of the samples at all concentrations of the simulated material solution to be tested (c) Calculate the within-group sum of squares and between-group sum of squares of the samples of the simulated material solution to be tested, where: The within-group sum of squares SSE is calculated as The between-group sum of squares SSA is calculated as (d) Calculate the statistic F, (e) When F is greater than the critical value F corresponding to the numerator degrees of freedom k-1 and the denominator degrees of freedom (n-1)×k in the F distribution table α When determining that the simulated material solutions to be tested with different concentrations can be used as candidate simulated material solutions for the stock solution, the significance level α corresponding to the F distribution table is 0.05, preferably 0.

025.

17. The method of claim 16, wherein the statistical analysis further comprises: (f) calculating the absolute value of the difference between the mean of the water loss of the samples of the simulated material solution to be tested at the i-th concentration and the mean of the water loss of the samples of the simulated material solution to be tested at the j-th concentration (g) Calculate the least significant difference (LSD) of the samples of the simulated material solution: Among them, t α / 2 is the critical value corresponding to the degree of freedom (n-1)×k in the t-distribution table, wherein the significance level α corresponding to the t-distribution table is 0.05, preferably 0.025, (h) When When the concentration is greater than LSD, the simulated material solutions to be tested with the ith and jth concentrations are further selected from the candidate simulated material solutions determined in step (e) as the candidate simulated material solutions for the stock solution.

18. A simulated material solution obtained by screening according to the method according to any one of claims 1 to 17.

19. Use of the method according to any one of claims 1 to 17 or the simulant solution according to claim 18 in freeze-drying process research, development and pilot batches or engineering batches, in particular freeze-drying process research, development and pilot batches or engineering batches of biopharmaceutical molecules.

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