CN112516090B - Pharmaceutical composition of antibody coupled drug, freeze drying agent, preparation method and application - Google Patents
Pharmaceutical composition of antibody coupled drug, freeze drying agent, preparation method and application Download PDFInfo
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- CN112516090B CN112516090B CN201910879701.XA CN201910879701A CN112516090B CN 112516090 B CN112516090 B CN 112516090B CN 201910879701 A CN201910879701 A CN 201910879701A CN 112516090 B CN112516090 B CN 112516090B
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- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
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- 239000001509 sodium citrate Substances 0.000 description 1
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- 239000008227 sterile water for injection Substances 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
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- SPRBJFWIUVWXBT-UHFFFAOYSA-K tripotassium 2-hydroxypropane-1,2,3-tricarboxylate 2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [K+].[K+].[K+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O SPRBJFWIUVWXBT-UHFFFAOYSA-K 0.000 description 1
- SPOMEWBVWWDQBC-UHFFFAOYSA-K tripotassium;dihydrogen phosphate;hydrogen phosphate Chemical compound [K+].[K+].[K+].OP(O)([O-])=O.OP([O-])([O-])=O SPOMEWBVWWDQBC-UHFFFAOYSA-K 0.000 description 1
- LEAHFJQFYSDGGP-UHFFFAOYSA-K trisodium;dihydrogen phosphate;hydrogen phosphate Chemical compound [Na+].[Na+].[Na+].OP(O)([O-])=O.OP([O-])([O-])=O LEAHFJQFYSDGGP-UHFFFAOYSA-K 0.000 description 1
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Images
Classifications
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- A61K9/00—Medicinal preparations characterised by special physical form
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- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
- A61K31/537—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines spiro-condensed or forming part of bridged ring systems
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
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- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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Abstract
The invention discloses a pharmaceutical composition of an antibody coupled drug, a freeze drying agent, a preparation method and application. The composite material comprises the following components in percentage by mass: F0002-ADC is 3-20 mg/mL, pH buffer is 5-50 mM, protein protectant is 10-100 mg/mL, surfactant is 0.05-2.0 mg/mL, pH is 5-7, arginine hydrochloride is 0-50 mg/mL. The medicine composition (preparation) containing the F0002-ADC antibody coupled medicine greatly improves the problems of low medicine solubility (medicine solution is in a clear state) and poor patent medicine property; reducing the water content of the freeze-dried preparation; the long-term stability experiment proves that the formula can meet the clinical requirement of F0002-ADC. The pharmaceutical composition or the prepared freeze-drying agent has better application prospect in preparing medicines for treating CD30 positive tumors.
Description
Technical Field
The invention belongs to the field of pharmaceutical preparations, and in particular relates to a pharmaceutical composition containing an F0002-ADC antibody coupled drug, a freeze-dried preparation, a preparation method and application thereof.
Background
Lymphomas are the ten-top malignant tumors with incidence and mortality rates in China (Cancer statistics in China,2015.CA Cancer J Clin.2016;66 (2): 115-32.) and were newly developed 8.82 thousands of people and 5.21 thousands of people die in 2015 as measured by the national cancer center. Lymphomas are classified into Hodgkin Lymphomas (HL) and non-hodgkin lymphomas (NHL), 95% of which are classical hodgkin lymphomas (cHL), the incidence rate of HL in europe and america is 2-3 per 10 tens of thousands of people, accounting for about 20-30% of all lymphomas, belonging to rare diseases (Epidemiology and etiology of Hodgkin's lymphoma Ann Oncol.2002;13suppl 4:147-52), while HL in China is less, accounting for 8-9% of lymphomas only, and the incidence rate of HL in the year is about 0.6 per hundred thousands of people. CD30, one of the members of the tumor necrosis factor receptor superfamily, is a marker antigen for classical Hodgkin's lymphoma (cHL) and Anaplastic Large Cell Lymphoma (ALCL). Based on the high expression of CD30 on HL and ALCL cells, the United states Seattle Gene company developed a CD30 antibody-coupled drug Brentuximab vedotin (brentuximab-VC-MMAE, trade name ADCETRIS, code number SGN-35), which was clinically used for two-line treatment of HL and ALCL, with clinical effective rates (ORR) of 73% and 86% for HL and ALCL, respectively, and treatment remission reached an average of 6.7 and 12.6 months. In the single drug treatment relapse or refractory classical hodgkin lymphoma test, 34 patients (33%) among 102 treated patients obtained complete remission, a total 5-year survival estimate of 41% and a progression-free 5-year survival estimate of 22%; patients with complete remission were obtained with a 5 year survival estimate of 64% and a 5 year progression free survival estimate of 52% (the previous significant recurrence was still effective), effectively inhibiting the HL and ALCL that were either relapsing or refractory CD30 positive, and were on the market with FDA approval at month 8 2011, the first FDA approved treatment for hodgkin's lymphoma and the first targeted new drug specifically adapted for the treatment of ALCL since 1977 (N Engl J Med2010; 363:1812-21).
Although ADCETRIS has achieved great clinical success, it has been found that it has strong toxic and side effects and poor patient tolerance in clinical use. The reason for the stronger toxic and side effects of ADCETRIS is attributed to the "bystander effect" caused by MMAE. The ADCETRIS structure includes three parts: antibody cAC targeting CD30, an enzymatically degradable valine citrulline dipeptide (VC) linker, and the highly active tubulin inhibitor MMAE. ADCETRIS binds to CD30 endocytosis on cell membrane surface to enter cells, and active molecule MMAE is released by enzymolysis of VC linker specific to cathepsin B in lysosome, polymerization of tubulin is prevented, cell mitosis is inhibited, and tumor cells are killed. But MMAE has good cell permeability, and can enter other cells in the region after being released from apoptotic cells, thereby forming a bystander effect, namely, cells around the tumor cells can be nonspecifically killed after CD30 specific tumor cells are killed, and the clinical toxic and side effects are obvious.
Based on the therapeutic effect of ADCETRIS on CD30 positive lymphoma and the aim of improving the toxic and side effects thereof), shanghai Fudan Zhang biological medicine Co-Ltd and Shanghai cross-linking research and development Co-Ltd jointly develop a recombinant anti-human CD30 chimeric monoclonal antibody MCC-DM1 coupling agent F0002-ADC for injection, and the coupling agent adopts an enzyme non-degradable connector SMCC to couple DM 1. The F0002-ADC structure is as follows: f0002 mab, enzyme non-degradable linker SMCC, and highly active tubulin inhibitor DM1 (specific structure and preparation method can be seen from the antibody conjugate disclosed in chinese patent document CN 201810078006.9). After administration, F0002-ADC binds to cell membrane CD30, endocytozes into tumor cells, and is subjected to enzymolysis in lysosomes to release main active substances Lys-MCC-DM1, which can inhibit the formation of tubulin, inhibit mitosis and induce apoptosis, while Lys-MCC-DM1 released by cell lysis cannot pass through the cell membrane well, does not have bystander effect, only specifically kills CD30 positive tumor cells, and avoids damage to normal cells. Can avoid bystander effect of ADCETRIS, and embody better safety in non-clinical animal pharmacological and toxicological researches.
The antibody drug conjugate (antibody drug conjugate, ADC) connects monoclonal antibody or antibody fragment with cytotoxin with biological activity through chemical joint, makes full use of specificity of antibody to tumor cell or high expression antigen binding and high efficiency of cytotoxin, and avoids toxic and side effects to normal cells. This means that the antibody drug conjugate can bind tumor cells precisely and reduce the effect of killing normal cells compared to conventional chemotherapeutics. The therapeutic concept of ADCs is to use as a vehicle the delivery of cytotoxic drugs to tumor cells by binding and internalizing antibodies to target surface antigens.
The ADC drug consists of antibody (targeting moiety), linker and toxin three parts. Among these, the good targeting moiety determines the specificity of the ADC drug, which includes not only specific targeting binding, but also efficient endocytosis.
However, antibody drugs, particularly ADCs, have a larger molecular weight and a more complex structure than other chemical drugs, and are susceptible to degradation, polymerization, unwanted chemical modification, and the like, and become unstable. The ADC has a heterogeneous structure more complicated than that of unconjugated antibody, the hydrophobic physicochemical property of the antibody drug is greatly changed by introducing chemical drugs and linkers, and the ADC has extremely high requirements on the dissolution property and freeze-dried preparation of the ADC drug. In order to make antibody drug conjugates suitable for administration and maintain stability during storage and subsequent use, the study of stable formulations of antibody drugs is particularly important. It is a matter of urgency to provide a pharmaceutical (formulation) composition comprising F0002-ADC that is sufficiently stable and more suitable for administration.
Disclosure of Invention
The invention aims to overcome the defects of lack of a drug (preparation) composition with good solubility and good stability for F0002-ADC antibody coupled drugs in the prior art, and provides a drug composition containing the F0002-ADC antibody coupled drugs, a freeze-dried preparation, a preparation method and application thereof.
The invention solves the technical problems through the following technical proposal.
The development of ADC pharmaceutical formulation formulations is similar to traditional antibody drugs, but the antibody-small molecule conjugated form introduces new quality attributes, thus compromising both monoclonal antibody and small molecule properties during formulation selection. Firstly, the main framework of the ADC is a monoclonal antibody, so that the stability of the monoclonal antibody needs to be considered in the preparation research process; secondly, the influence of the small molecule drugs on the physicochemical properties of the whole ADC is fully considered. For example, since conjugated Payload is typically a strongly hydrophobic small molecule, it tends to reduce the overall solubility of the ADC drug, directly affecting the drug properties. The F0002-ADC preparation is found in the process development process, and the F0002-ADC sample has milky flocculent insoluble matter with poor solubility at a slightly high concentration (> 5 mg/mL). The invention selects proper cosolvent types, cosolvent, pH value buffer, protective agent, surfactant dosage and specific pH value range, and the prepared pharmaceutical composition containing F0002-ADC antibody coupled medicine has better solubility and better stability, and the prepared freeze-dried preparation has lower water content.
The invention provides a pharmaceutical composition, which comprises the following components:
when the concentration of the F0002-ADC is 2-4 mg/mL, the dosage of arginine hydrochloride is 0-50 mg/mL; when the concentration of the F0002-ADC is 4-20 mg/mL, the dosage of arginine hydrochloride is 20-50 mg/mL.
In the invention, the F0002-ADC is an antibody conjugate, and the structural general formula of the antibody conjugate is Ab-L m -Y n ；
Wherein Ab is anti-human CD30 antibody cAC, an active fragment thereof or variant thereof;
the Ab is only linked to the L;
y is a cytotoxic agent maytansinoid DM1;
n is 2-5, and m is more than or equal to n; said Y being connected only to said L; part of L is connected with Ab and Y at two ends respectively, and the rest of L is connected with Ab only;
when both ends of the L are respectively connected with the Ab and the Y, the L is
The left end of the thiol compound forms an amide bond with the amino group of the lysine residue of the Ab, and the right end of the thiol compound forms a thioether bond with S in the DM1 thiol;
when the L is linked only to the Ab, the L is
The left end forms an amide bond with the amino group of the lysine side chain of the Ab.
The antibody conjugate typically has m of 2 to 10. Preferably, m is equal to n, and the structural general formula of m is Ab- (L-Y) n, and the structure is as follows:
More preferably, the variant of the anti-human CD30 antibody cAC has a similarity to the amino acid sequence of cAC of no less than 90% and a lysine-related mutation of no more than 80%.
Even more preferably, n=3 to 4; most preferably, n=3.6.
In the present invention, the F0002-ADC can be prepared by methods conventional in the art. For example, the F0002-ADC may be prepared according to method one or method two disclosed in Chinese patent document CN 201810078006.9. In some embodiments, the preparation is performed by example 3 disclosed in chinese patent document CN 201810078006.9.
In the invention, the dosage of arginine hydrochloride is required to be in the range of 50mg/mL, because arginine hydrochloride is solubilized and simultaneously has adverse effects on freeze drying, and a loose and complete freeze-dried powder cake is difficult to obtain by using a formulation of an arginine hydrochloride preparation with higher concentration.
In the present invention, when the concentration of the F0002-ADC is 2 to 4mg/mL, the arginine hydrochloride is preferably used in an amount of 5 to 45mg/mL, for example, 10mg/mL, 20mg/mL, 30mg/mL or 40mg/mL.
When the concentration of the F0002-ADC is 4 to 20mg/mL, the arginine hydrochloride is preferably used in an amount of 25 to 45mg/mL, for example, 20mg/mL, 25mg/mL, 30mg/mL, 35mg/mL or 40mg/mL.
In the present invention, the amount of the F0002-ADC is preferably 5 to 18mg/mL, for example, 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, 9mg/mL, 10mg/mL, 12mg/mL or 15mg/mL.
In the invention, the arginine hydrochloride plays a role of a cosolvent in the pharmaceutical composition.
In the present invention, the pH buffer may be a pH buffer conventionally used in the art, and may be, for example, a citrate buffer, an acetate buffer, a succinate buffer, a histidine buffer, a phosphate buffer, or Bis-Tris (Bis (2-hydroxyethyl) amino (trimethylol) methane) buffer. The preferred buffer of the present invention is citrate buffer.
In the present invention, the pH buffer is preferably used in an amount of 10 to 45mM, more preferably 15 to 40mM, for example 20mM, 25mM, 30mM or 35mM. As is known in the art, "mM" refers to mmol/L. In the present invention, the concentration refers to the concentration of the acid ions in the pH buffer, for example in the acetate buffer, which refers to the concentration of the acetate ions; in the acetate buffer, the concentration refers to the concentration of phosphate ions.
In the present invention, the protein protecting agent may be a protecting agent conventionally used in the art for protecting proteins, preferably sucrose and/or trehalose.
In the present invention, the amount of the protein protecting agent is preferably 20 to 90mg/mL, more preferably 30 to 80mg/mL, for example 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL or 80mg/mL.
In the present invention, the surfactant may be a surfactant conventionally used in the art, preferably polysorbate 20 and/or polysorbate 80.
In the present invention, the surfactant is preferably used in an amount of 0.1 to 1.8mg/mL, more preferably 0.2 to 1.5mg/mL, for example, 0.2mg/mL, 0.5mg/mL, 1.0mg/mL or 1.2mg/mL.
In the present invention, the solvent in the pharmaceutical composition may be conventional in the art, preferably one or more of purified water, water for injection and physiological saline, more preferably water for injection.
In the present invention, the pH is preferably 5.0 to 6.6, for example 5.0, 5.5 or 6.0. The pH of the pharmaceutical composition may be adjusted by methods conventional in the art, for example, by adding sodium hydroxide such that the pH is within the aforementioned range. For example, in certain embodiments, the pharmaceutical composition is brought to a pH of 5.0 by the addition of 0.8mg/mL sodium hydroxide.
In some embodiments, the pharmaceutical compositions may be formulated under the following numbers 1-24, respectively:
In other embodiments, the pharmaceutical compositions may be formulated under the following numbers 1-12, respectively:
in other embodiments, the pharmaceutical compositions may be formulated with the following numbers 1-10, respectively:
the pharmaceutical compositions of the present invention may be prepared by methods conventional in the art.
The invention also provides a preparation method of the pharmaceutical composition, which comprises the following steps: after carrying out centrifugal liquid exchange on the protein-containing liquid, concentrating until the concentration of the F0002-ADC meets the requirements;
wherein the protein solution is a solution containing the F0002-ADC; in the centrifugal liquid exchange process, the adopted replacement buffer solution comprises the pH value buffer, the protein protecting agent, the surfactant and the arginine hydrochloride, wherein the pH value of the replacement buffer solution is as described above.
In the present invention, the initial buffer solution in the protein solution may be conventional in the art, preferably 25mM citric acid-sodium citrate buffer, 150mM NaCl,2mM EDTANa 2 ，pH 6.0±0.1。
In the present invention, the concentration of F0002-ADC in the protein solution may be conventional in the art, for example, 5-15 mg/mL. In a specific embodiment, for example, 10mg/mL.
In the present invention, the operation and conditions of the centrifugal liquid exchange can be conventional in the art, preferably performed in ultrafiltration-concentration tubes, more preferably in 30kD ultrafiltration-concentration tubes. As known in the art, the centrifugation is performed by replacing the initial buffer solution in the protein solution with the replacement buffer. In the centrifugal liquid exchange process, the rotating speed can be RCF:3200 Xg. According to the general knowledge in the art, the centrifugal liquid exchange operation is only aimed at a laboratory research and development stage, and an ultrafiltration membrane bag system is adopted for liquid exchange in the subsequent pilot plant test and production stage.
In the present invention, the ratio of the volume of the substitution buffer to the volume of the protein solution used in the centrifugal liquid exchange process may be conventional in the art, and is preferably 5 to 15 times, for example 10 times.
In the present invention, the substitution buffer may be prepared by a method conventional in the art, for example, by uniformly mixing the pH buffer, the protein protectant, the surfactant and the arginine hydrochloride, and then adjusting the pH to meet the aforementioned requirements.
In the present invention, the operation and conditions of the concentration may be conventional in the art. In the concentration process, as known in the art, only the F0002-ADC is concentrated, and the concentration of the solute in the substitution buffer solution is unchanged and the pH value is unchanged. Concentrating to the concentration of the F0002-ADC to meet the requirement, and taking out the pharmaceutical composition by a pipette. As is known in the art, during the laboratory development phase, an ultrafiltration tube is typically used to perform the experiment, a pipette is used to remove the drug, and during the subsequent pilot and production phases, an ultrafiltration membrane pack system is typically used to remove the drug using a pump system.
The invention also provides a method for preparing a freeze-dried preparation containing the F0002-ADC antibody coupled drug, which comprises the step of freeze-drying the pharmaceutical composition.
The pharmaceutical composition is generally packaged in penicillin bottles and lyophilized by a lyophilizer to obtain a lyophilized preparation. In some embodiments, a method of preparing a lyophilized formulation comprising an F0002-ADC antibody conjugated drug, wherein the lyophilization comprises the steps of pre-freezing, primary sublimation, and analytical drying in sequence.
The pre-freezing stage aims at enabling the products to be at the same low temperature level, synchronously cooling in the cooling process, freezing in a short time period as much as possible, and improving uniformity. The parameters of the prefreezing stage may be conventional in the art, preferably set in sequence as: precooling below 5 ℃ for 1 hour, precooling below-5 ℃ for 1.5 hours, regulating the temperature of the plate layer to below-45 ℃, controlling the overall down-regulating speed to below 1 ℃/min, keeping for more than 2 hours, slowly raising the plate temperature to not more than-25 ℃ for annealing, keeping for 2 hours, quickly cooling to below-45 ℃ and keeping for more than 5 hours. And an annealing operation of-45 ℃ to-25 ℃ to-45 ℃ is added in the pre-freezing stage, so that the appearance of the product is optimized, and the primary sublimation efficiency is improved.
The parameters of the primary sublimation can be conventional in the art, and are preferably set in sequence as: the temperature of the plate layer is set to be minus 33 ℃ or below, and the overall temperature rising rate is not more than 0.5 ℃/min. The temperature of the cold trap is ensured not to exceed 50 ℃ in the process, and the temperature rise is delayed when the temperature of the cold trap exceeds 50 ℃. The temperature of the product is controlled to be not higher than-25 ℃ before the waterline disappears. After the temperature of the plate layer reaches a set value, the waterline of the observation window is lowered, and the time is prolonged by more than one time after the waterline disappears, and the heat is preserved for at least 40 hours. Then slowly heating to 3-6 ℃. After the temperature of the plate layer reaches the set temperature, the plate layer is kept for more than 5 hours, and the maximum time can be 10 hours. If the temperature of the plate layer is below minus 30 ℃, the temperature is continuously and slowly increased by 3-5 ℃. Hold for 5 hours. In the whole sublimation process, the vacuum degree is controlled to be 0.08-0.18 mbar.
The parameters of the analytical drying may be conventional in the art, and are preferably set as follows: after the sublimation was completed, the plate was first warmed to-5℃and held for 5 hours. The temperature of the plate is slowly raised to 5 ℃ and kept for 2 to 5 hours. Then the temperature of the plate is raised to 15 ℃ and kept for 2 to 5 hours. Finally, the plate temperature was raised to 25 ℃. The vacuum degree is controlled between 0.08 and 0.18mbar. After the plate temperature had risen to 25℃a final vacuum of 0.01mbar was applied. When the temperature of the plate layer reaches 25 ℃, the heat preservation is carried out for more than 6 hours.
And (3) generally, carrying out analysis and drying, and then taking out from the box. The endpoint is determined by a pressure rise test as known in the art. For example, endpoint determination criteria: the rise in the front box pressure was less than 0.05mbar within 5 minutes. If the test is not passed, the drying time is extended until the pressure rise test is passed. Vacuum tamponade, and capping after discharging.
The invention also provides a freeze-dried preparation containing the F0002-ADC antibody coupling drug prepared by the method.
In some embodiments, there is also provided a lyophilized formulation wherein the lyophilized formulation upon reconstitution may form any of the aforementioned pharmaceutical compositions, preferably reconstituted using one or more of purified water, water for injection and physiological saline, more preferably reconstituted using water for injection.
The invention also provides an application of the pharmaceutical composition or the freeze-dried preparation in preparing medicines for treating CD30 positive tumors.
Preferably, the CD30 positive tumor is a lymphoma. More preferably, the lymphoma is hodgkin's lymphoma (cHL), anaplastic large cell lymphoma, diffuse tissue cell lymphoma, or cutaneous T-cell lymphoma. Most preferably, the lymphoma is hodgkin's lymphoma.
The invention also provides a method of treating and/or preventing CD30 positive tumors comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition or lyophilized formulation according to the foregoing.
Preferably, the CD30 positive tumor is a lymphoma. More preferably, the lymphoma is hodgkin's lymphoma (cHL), anaplastic large cell lymphoma, diffuse tissue cell lymphoma, or cutaneous T-cell lymphoma. Most preferably, the lymphoma is hodgkin's lymphoma.
The present invention also provides an article of manufacture comprising a container containing the pharmaceutical composition or lyophilized formulation described above. The container is preferably a glass bottle or a liquid storage bag.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.
The reagents and materials used in the present invention are commercially available.
The invention has the positive progress effects that:
the medicine composition (preparation) containing the F0002-ADC antibody coupled medicine greatly improves the problems of low medicine solubility (medicine solution is in a clear state) and poor patent medicine property (compared with ADCETRIS); reducing the water content of the freeze-dried preparation; the long-term stability experiment proves that the formula can meet the clinical requirement of F0002-ADC.
Drawings
Fig. 1 shows the SEC monomer trend during long-term storage of the lyophilized formulation prepared in example 5 at a temperature of 4 ℃.
Fig. 2 shows the SEC polymer trend during long-term storage of the lyophilized formulation prepared in example 5 at a temperature of 4 ℃.
FIG. 3 shows the tendency of the lyophilized preparation prepared in example 5 to reduce CE-SDS immunoglobulin during long-term storage at 4 ℃.
FIG. 4 shows the trend of non-reduced CE-SDS immunoglobulins of the lyophilized preparation prepared in example 5 during long-term storage at 4 ℃.
FIG. 5 shows the relative activity profile of the lyophilized formulation prepared in example 5 during long-term storage at a temperature of 4 ℃.
FIG. 6 shows the relative binding activity profile of the lyophilized formulation prepared in example 5 during long-term storage at a temperature of 4 ℃.
FIG. 7 shows the moisture content change during long-term storage at a temperature of 4℃of the lyophilized preparation prepared in example 5.
FIG. 8 shows the protein content change during long-term storage at a temperature of 4℃of the lyophilized preparation prepared in example 5.
Fig. 9 is a graph showing the variation of SEC monomer during long-term storage of the lyophilized formulation prepared in example 5 at 25 ℃.
Fig. 10 is a graph showing the tendency of SEC polymers during long-term storage at 25 ℃ for the lyophilized formulation prepared in example 5.
FIG. 11 shows the tendency of the lyophilized preparation prepared in example 5 to reduce CE-SDS immunoglobulin during long-term storage at 25 ℃.
FIG. 12 shows the low molecular weight distribution of non-reduced CE-SDS during long-term storage at 25℃of the lyophilized preparation prepared in example 5.
FIG. 13 shows the relative activity profile of the lyophilized formulation prepared in example 5 during long-term storage at 25 ℃.
FIG. 14 shows the relative binding activity profile of the lyophilized formulation prepared in example 5 during long-term storage at 25 ℃.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
1. Interpretation of the terms
In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
"pharmaceutical composition" means a mixture comprising one or more of the compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity. As used herein, the terms "pharmaceutical composition" and "formulation" are not intended to be mutually exclusive.
The pharmaceutical composition provided by the invention can achieve a stable effect: a pharmaceutical composition wherein the antibody substantially retains its physical and/or chemical stability and/or biological activity after storage, preferably the pharmaceutical composition substantially retains its physical and chemical stability and its biological activity after storage. The shelf life is generally selected based on the predetermined shelf life of the pharmaceutical composition. There are a number of analytical techniques for measuring protein stability that measure stability after storage at a selected temperature for a selected period of time.
By "freeze-dried formulation" is meant a pharmaceutical composition in liquid or solution form or a formulation obtained after a liquid or solution formulation has been subjected to a freeze-drying step in vacuo.
The antibody drug conjugate (antibody drug conjugate, ADC) is characterized in that monoclonal antibodies or antibody fragments are connected with cytotoxins with biological activity or small molecule drugs with cell killing activity through stable chemical joint compounds, so that the specificity of the antibodies on tumor cell specificity or high expression antigen binding and the high efficiency of cytotoxins are fully utilized, and the toxic and side effects on normal cells are avoided. This means that the antibody drug conjugate can bind tumor cells precisely and reduce the effect on normal cells compared to conventional chemotherapeutics.
"antibody conjugated drug F0002-ADC (anti-CD 30-MCC-DM 1)" refers to an antibody conjugated drug targeting anti-CD 30 antibody conjugated DM 1.
"pH buffer" refers to a buffer that is resistant to pH changes by the action of its acid-base conjugate components. The buffer of the present invention has a pH of about 5.0 to 7.0, preferably about 5.0 to 6.6, such as 5.0, 5.5 or 6.0. Examples of buffers that control the pH in this range include citrate buffer, acetate buffer, succinate buffer, histidine buffer, phosphate buffer, or Bis-Tris (Bis (2-hydroxyethyl) amino (trimethylol) methane) buffer. The preferred buffer of the present invention is citrate buffer.
A "phosphate buffer" is a buffer that includes phosphate ions. Examples of phosphate buffers include potassium dihydrogen phosphate-dipotassium hydrogen phosphate, potassium dihydrogen phosphate-disodium hydrogen phosphate, sodium dihydrogen phosphate-dipotassium hydrogen phosphate, and the like. The preferred phosphate buffer of the present invention is sodium dihydrogen phosphate-disodium hydrogen phosphate.
A "succinate buffer" is a buffer that includes succinate ions. Examples of succinate buffers include sodium succinate-succinate, histidine succinate, potassium succinate-succinate, calcium succinate-succinate salt, and the like. The preferred succinate buffer of the present invention is sodium succinate-succinate.
A "citrate buffer" is a buffer that includes citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-sodium hydroxide, and the like. A preferred citrate buffer of the present invention is citric acid-sodium citrate.
An "acetate buffer" is a buffer that includes acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, acetic acid-potassium acetate, acetic acid-sodium hydroxide, sodium acetate-hydrochloric acid, and the like. The preferred acetate buffer of the present invention is acetic acid-sodium acetate.
Examples of "histidine buffer" include histidine-hydrochloric acid, histidine-histidine hydrochloride, and the like. A preferred histidine buffer of the present invention is histidine-hydrochloric acid.
"Bis-Tris buffer". A preferred combination of the present invention is Bis-Tris-HCl.
WFI: water for injection, water for injection.
The term "treatment" or its equivalent, when used in reference to, for example, cancer, refers to a procedure or process used to reduce or eliminate the number of cancer cells in a patient or to alleviate symptoms of cancer. "treating" of cancer or another proliferative disorder does not necessarily mean that the cancer cells or other disorder will actually be eliminated, that the number of cells or disorders will actually be reduced or that the symptoms of the cancer or other disorder will actually be reduced. In general, methods of treating cancer are performed even with low likelihood of success, but are still considered to induce an overall beneficial course of action, given the patient's medical history and estimated survival expectancy.
An "effective amount" comprises an amount sufficient to ameliorate or prevent a symptom or condition of a medical disorder. An effective amount is also meant to be an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on the following factors: for example, the condition to be treated, the general health of the patient, the route and dosage of administration, and the severity of the side effects. An effective amount may be the maximum dose or regimen that avoids significant side effects or toxic effects.
"Tm value" refers to the temperature at which a protein is heat denatured, i.e., the temperature at which half of the protein is unfolded, at which time the spatial structure of the protein is destroyed, so that the higher the Tm value, the higher the thermal stability of the protein.
2. Materials and methods
Preparation of 2.1F0002-ADC
Can be prepared by methods conventional in the art. For example, F0002-ADC can be prepared according to method one or method two disclosed in Chinese patent document CN 201810078006.9. In an embodiment, the preparation was carried out by example 3 disclosed in chinese patent document CN 201810078006.9.
2.2 major instrumentation and reagents
Freeze dryer TELSTAR LyoBeta 6PL; a high performance liquid analysis system Waters E2695; column TOSOH TSKgel G3000SWXL (5 μm,7.8 mm. Times.300 mm); capillary electrophoresis analysis system Beckman Coulter PA800plus; coulomb method kar moisture meter Metrohm 831KF Coulometer; multifunctional microplate reader Molecular Devices SpectraMax m e. Citric acid was purchased from Hunan Huari pharmaceutical Co., ltd; sucrose was purchased from merck company; arginine hydrochloride was purchased from Shanghai, inc. of amino acids, inc.; polysorbate 20 was purchased from J.T. Baker.
2.3. Size Exclusion Chromatography (SEC) purity analysis)
Chromatographic column: TSKgel G3000SWXL (5 μm,7.8 mm. Times.300 mm); mobile phase: 0.2mol/L K 2 HPO 4 0.2mol/L KCl,15% isopropanol, pH 5.5; flow rate: 0.6ml/min; sample loading amount: 200 μg; column temperature: 25 ℃; sample cell temperature: 5 ℃; detection wavelength: 280nm. And (3) carrying out data processing on experimental results by using a Waters E2695 high performance liquid analysis system, and calculating the purity by using an area normalization method.
2.4 capillary electrophoresis (CE-SDS) purity analysis
Diluting the test sample to 1mg/ml with SDS sample buffer (0.1 mol/L Tris-HCl buffer, containing 1% SDS, pH 9.0), respectively sucking 95 μl of the diluted protein solution, 2 μl of 10kDa internal standard, 5 μl of beta-mercaptoethanol, adding all into a 0.5ml centrifuge tube, mixing by vortex oscillation, placing in a constant temperature mixer for incubation at 70deg.C for 10 min, cooling to room temperature, taking 100 μl, and processing the sample with Beckman Coulter PA plus capillary electrophoresis analysis system with bandwidth: 10nm, sample cell temperature: capillary temperature 10 ℃): 25 ℃, detection wavelength: 220nm. The original map is imported into Empower software and calculated according to an area normalization method.
2.5 analysis of moisture content
The method is referred to as coulometric titration in the first method of general rule 0832 of the 2015 edition of the pharmacopoeia of the people's republic of China, based on the Karl-Fei Xiushi reaction, a permanent titration method (general rule 0701 of the 2015 edition of the pharmacopoeia of the people's republic of China) is applied to determine a test article, and the moisture is determined according to the principle of quantitative reaction of iodine and sulfur dioxide with water in pyridine and methanol solution. The sample to be tested is smashed rapidly, about 20mg of the sample is weighed precisely and added into a moisture tester for reaction. 3 bottles of test substance were measured for each batch, 2 times per bottle.
2.6 relative binding Activity assay
hCD30 antigen was coated at 108ng/ml in 96-well elisa plate and allowed to stand at 2-8 ℃ overnight. The 3% BSA was blocked at room temperature for 2 hours. Uniformly mixing F0002-ADC sample (20000 ng/ml-1.4 ng/ml) with biotinylated F0002 antibody in equal volume, adding the mixture into an ELISA plate, competing for binding hCD30 antigen, and performing 200 revolutions at room temperatureIncubate for 1 hour. HRP-labeled streptavidin was used as an enzyme-linked, and incubated for 1 hour under the above conditions. TMB color development, 1mol/L H 2 SO 4 And (5) terminating. As for the microplate reader m2e, readings were made at a wavelength of 450nm (detection)/650 nm (reference). And selecting a four-parameter fitting mode to analyze the data.
2.7 analysis of biological Activity
Karpas299 cells were diluted to 5X 10 with RPMI160 medium containing 10% fetal bovine serum 4 Per ml,100 μl/well was seeded in 96-well cell culture plates. A gradient diluted F0002-ADC sample (50 ng/ml to 0.3 ng/ml) was then added. At 37 ℃,5% CO 2 After 77 hours of incubation in the incubator of (2), 30 μl/well of alamar blue dye was added and incubation was continued for 19 hours. After mixing at room temperature for half an hour, a reading was made at a wavelength of 530nm (excitation)/590 nm (emission) in the microplate reader m2 e. And selecting a four-parameter fitting curve to perform data analysis on the result.
Example 1
The formulation and dissolution of each pharmaceutical composition are shown in Table 1. In Table 1, the concentration of citric acid-sodium citrate in each formulation of the pharmaceutical composition was 10mM. Part of the pharmaceutical composition can obviously improve the solubility of the F0002-ADC, and the pharmaceutical solution is in a clear state, so that the development of a stable preparation is facilitated. Arginine hydrochloride of 20-50 g/L is added into the formula as the first choice for improving the solubility.
The preparation method of the pharmaceutical compositions comprises the following steps (except for final lyophilization, F0002-ADC is in aqueous solution):
(1) Preparing a replacement buffer solution: uniformly mixing adjuvants except for antibody, including citric acid-sodium citrate, sucrose, arginine hydrochloride, polysorbate 20 or polysorbate 80, and adjusting pH to meet the requirements of table 1;
(2) The protein solution (25 mM citrate buffer, 150mM NaCl,2mM EDTANa2,pH 6.0 + -0.1, antibody F0002-ADC concentration 10 mg/mL) was subjected to centrifugation (i.e., the initial buffer solution in the protein solution was replaced with the replacement buffer solution prepared in step (1)) with a 30kD ultrafiltration concentration tube, and RCF:3200 Xg, the volume of the exchanged liquid is 10 times of the volume of the protein liquid. After completion of the exchange, the F0002-ADC was further concentrated to the concentrations specified by the numbers in Table 1 (for example, the concentration of the antibody was 3mg/mL as exemplified by the number 1) (in the course of concentrating the protein, as known in the art, only the antibody was concentrated, the solute concentration in the exchange buffer was unchanged, and the pH value was unchanged), and the sample was withdrawn from the pipette to obtain a pharmaceutical composition.
The pharmaceutical compositions numbered 1 to 6 in table 1 are clear, but because the concentration of the antibody is too low, the amount of the coating material used during lyophilization is too large, which is not beneficial to cost control and clinical application convenience. The pharmaceutical compositions of the groups 7 to 8, 13 to 16, 20 to 21, 26 to 27 and 32 to 33 have no arginine hydrochloride or too little arginine hydrochloride, and all have turbidity in solubility observation. If the turbidity is the phenomenon, the medicine has poor solubility, is precipitated or precipitated, and can not be clinically used. The pharmaceutical compositions with serial numbers 34-35 are clear, but have relatively high pH value, so that the PayLoad small molecules are easy to fall off, the stability of the pharmaceutical composition is poor, the effective period is short, and the development of freeze-drying and drying preparations is not facilitated.
Table 1 pharmaceutical composition formulations and dissolution conditions thereof
Note that: "clear" generally means that the system is transparent and free of visible foreign matter by manual visual inspection.
Example 2
The water content of each pharmaceutical composition formulation and its lyophilized formulation is shown in table 2. In the formulations of the pharmaceutical compositions in Table 2, the concentration of polysorbate 20 was 0.2mg/mL and the pH was 5.0. Each pharmaceutical composition was prepared as described in example 1.
The preparation method of the freeze-dried preparation comprises the following steps:
the pharmaceutical composition containing F0002-ADC is packaged in injection bottles, and the injection bottles containing the samples are placed in a Lyobeta6PL freeze dryer to begin freeze drying. Pre-freezing: precooling below 5 ℃ for 1 hour. Precooling below-5 ℃ for 1.5 hours. The temperature of the plate layer is regulated to be lower than-45 ℃, the overall down-regulating speed is controlled to be lower than 1 ℃/min, and the plate layer is kept for more than 2 hours. Slowly raising the temperature of the plate to be no more than minus 25 ℃ for annealing, and keeping for 2 hours. Rapidly cooling to below-45 ℃ and keeping for more than 5 hours.
Primary sublimation: the temperature of the plate layer is set to be minus 33 ℃ or below, and the overall temperature rising rate is not more than 0.5 ℃/min. The temperature of the cold trap is ensured not to exceed 50 ℃ in the process, and the temperature rise is delayed when the temperature of the cold trap exceeds 50 ℃. The temperature of the product is controlled to be not higher than-25 ℃ before the waterline disappears. After the temperature of the plate layer reaches a set value, the waterline of the observation window is lowered, and the time is prolonged by more than one time after the waterline disappears, and the heat is preserved for at least 40 hours. Then slowly heating to 3-6 ℃. After the temperature of the plate layer reaches the set temperature, the plate layer is kept for more than 5 hours, and the maximum time can be 10 hours. If the temperature of the plate layer is below minus 30 ℃, the temperature is continuously and slowly increased by 3-5 ℃. Hold for 5 hours. In the whole sublimation process, the vacuum degree is controlled to be 0.08-0.18 mbar.
And (5) analysis and drying: after the sublimation was completed, the plate was first warmed to-5℃and held for 5 hours. The temperature of the plate is slowly raised to 5 ℃ and kept for 2 to 5 hours. Then the temperature of the plate is raised to 15 ℃ and kept for 2 to 5 hours. Finally, the plate temperature was raised to 25 ℃. The vacuum degree is controlled between 0.08 and 0.18mbar. After the plate temperature had risen to 25℃a final vacuum of 0.01mbar was applied. When the temperature of the plate layer reaches 25 ℃, the heat preservation is carried out for more than 6 hours.
And (5) discharging: and judging the end point by the pressure rise test. Endpoint determination criteria: the rise in the front box pressure was less than 0.05mbar within 5 minutes. If the test is not passed, the drying time is extended until the pressure rise test is passed. Vacuum tamponade, and capping after discharging.
Table 2 each pharmaceutical composition formulation and water content of lyophilized formulation thereof
As can be seen from table 2, the concentration of the citric acid-sodium citrate buffer greatly affects the water content of the lyophilized sample of the final formulation, and reducing the concentration of the citric acid-sodium citrate buffer may help to reduce the water content of the lyophilized product.
Example 3F0002-ADC lyophilized Dry formulation stability test
The following examples describe tests for analyzing the stability of F0002-ADC, the properties examined including differential scanning calorimetry, DSC. DSC is an important tool for researching protein stability, heat is required to be absorbed in the unfolding process of protein molecules, and the DSC method is used for analyzing the stability of antibody molecules under a certain preparation system by linearly increasing the temperature, measuring the melting temperature of the protein molecules and simultaneously measuring the difference value between the heat capacity rise of a protein solution and the heat capacity rise of a blank solution. The melting temperature (Tm value) of the protein in the formulations with different proportions is examined by a DSC method, and the results show that the Tm values and differences between the formulations before and after freeze-drying and between the formulations are not obviously different under the conditions of different concentrations and types of buffering agents, sucrose or trehalose with a certain concentration and arginine-containing pH value of 6.0.
The water content of each pharmaceutical composition formulation and the Tm values before and after lyophilization of the lyophilized formulation are shown in table 3. In Table 3, the pH of each formulation of the pharmaceutical compositions was 6.0, the F0002-ADC concentration was 5mg/mL, and the polysorbate 20 concentration was 0.2mg/mL. The water content refers to the water content of the lyophilized formulation of each pharmaceutical composition.
TABLE 3 stability analysis of lyophilized dry formulations of F0002-ADC
The Tm value measured in DSC is a prediction of long-term stability, and generally the higher the Tm, the better the stability. The Tm values of the various groups of conditions in table 3 were not significantly different, indicating consistent long term stability results for each formulation, and no significant differences. However, in the pharmaceutical compositions numbered 3, 6, 8, and 9 in table 3, arginine hydrochloride was absent or less used in the formulation, and was cloudy prior to lyophilization, and was also cloudy after lyophilization reconstitution, and was not clinically useful.
EXAMPLE 4 Freeze/thaw stability of pharmaceutical compositions
The formulation of the pharmaceutical composition is as follows: 5mg/mLF0002-ADC,30mg/mL arginine hydrochloride, 10mM citric acid-sodium citrate, 60mg/mL sucrose, 0.2mg/mL polysorbate 20, pH 5.0. The specific preparation method is the same as in example 1.
The biological macromolecule sample is subjected to freeze thawing, and the temperature and the local concentration of the sample are changed, so that the aggregation state of the antibodies can be influenced. The aforementioned pharmaceutical compositions were subjected to 0, 5, 10 freeze-thaw cycles, respectively, and characterized by SEC, IEC, CE-SDS, relative activity, and DM1 residues. The characterization results are shown in table 4 below, indicating that the high concentration stable formulations developed did not change in aggregation.
Table 4 analysis of repeated freeze-thaw stability of pharmaceutical compositions
EXAMPLE 5 Freeze-dried formulation
The formulation of the pharmaceutical composition is as follows: 5mg/mL F0002-ADC,30mg/mL arginine hydrochloride, 10mM citric acid-sodium citrate (2.1 mg/mL, 0.6mg/mL sodium citrate), 60mg/mL sucrose, 0.2mg/mL polysorbate 20, pH 5.0. The specific preparation method is the same as in example 1.
The pharmaceutical composition was prepared according to the preparation method of example 2 as a freeze-dried formulation.
F0002-ADC is a drug conjugate comprising an anti-CD 30 antibody covalently linked to DM 1. The F0002-ADC was formulated as a freeze-dried formulation for injection after reconstitution and filling into glass vials. The lyophilized formulation was reconstituted with 5mL sterile water for injection (WFI) and provided a 20mg/mL F0002-ADC solution for injection. The composition of F0002-ADC per vial (lyophilized powder, i.e., lyophilized formulation) and per 1.0mL (reconstituted solution) is described in table 5 below, with the lyophilized formulation (formulation comprising buffer, sugar, surfactant, co-solvent, and anti-CD 30 antibody drug conjugate) for single use, and no preservative. The reconstituted drug was diluted with 0.9% physiological saline (sodium chloride injection, USP) for administration by infusion.
TABLE 5
Note that: (1) in Table 5, the pH of the solution after reconstitution was 5.0; (2) "amount reconstituted" refers to the concentration of each component per mL of solution after reconstitution with 5 mL.
Example 6 stability of lyophilized Dry formulations at 4℃
The stable lyophilized formulation prepared in example 5 above was subjected to long term stability experiments at 4 ℃ and tested after reconstitution with WFI at time points 0, 6, 12, 24 months, respectively. And characterized by appearance, SEC purity, CE-SDS, binding activity, conjugation ratio DAR, protein content, particle size, pH, free DM1, and water content.
(1) Long-term storage at 4 ℃ does not affect the appearance of the lyophilizate and reconstituted solution:
the appearance of the lyophilizate and reconstituted solution was visually assessed to confirm that the lyophilizate was virtually free of macroscopic foreign particulate matter and contained no moisture in the packaging material. The appearance of the lyophilisate meets the above criteria at the initial time point and after reconstitution after storage at 4 ℃ for up to 36 months. After reconstitution after storage at 4 ℃ for up to 36 months, the reconstituted solution is a colorless to yellowish solution and virtually free of macroscopic particulate matter.
(2) SEC purity of conjugate stored for long term at 4 ℃):
size exclusion chromatography of the reconstituted solution was performed using size exclusion-high performance liquid chromatography (SEC-HPLC). SEC-HPLC the purity of F0002-ADC was determined using size exclusion HPLC. Macromolecules are equally separated during gel filtration HPLC according to reduced molecular size. Purity was determined by comparing the area of the main peak of the F0002-ADC with the total area of the sample chromatogram (excluding buffer related peaks). The method is capable of resolving high molecular weight aggregates and truncated antibody species from the main peak of the F0002-ADC. In addition to the high molecular weight species, namely, the polymer (%) and the low molecular weight species (%), the main peak, namely, the monomer (%), was measured.
Fig. 1 shows the SEC monomer trend during long-term storage of the lyophilized formulation prepared in example 5 at a temperature of 4 ℃. Fig. 2 shows the SEC polymer trend during long-term storage of the lyophilized formulation prepared in example 5 at a temperature of 4 ℃. As can be seen from fig. 1-2, at the initial time point, SEC monomer was 98.8%, polymer was 1.2%, low molecular weight species was 0.0%; after reconstitution after 3 months of storage at 4 ℃, the SEC monomer was 98.8%, the polymer was 1.2%, and the low molecular weight species was 0.0%; after reconstitution after 6 months of storage at 4 ℃, the SEC monomer was 98.7%, the polymer was 1.3%, and the low molecular weight species was 0.0%; after reconstitution after 9 months of storage at 4 ℃, the SEC monomer was 98.7%, the polymer was 1.3%, and the low molecular weight species was 0.0%; after reconstitution after 12 months of storage at 4 ℃, the SEC monomer was 98.7%, the polymer was 1.3% and the low molecular weight species was 0.0%; after 18 months of reconstitution at 4 ℃, the SEC monomer was 98.8%, the polymer was 1.2%, and the low molecular weight species was 0.0%; after 24 months of reconstitution at 4 ℃, the SEC monomer was 98.7%, the polymer was 1.3% and the low molecular weight species was 0.0%; after reconstitution after 36 months of storage at 4 ℃, the SEC monomer was 98.8%, the polymer was 1.2% and the low molecular weight species was 0.0%.
(3) Long-term storage at 4 ℃ did not alter the CE-SDS purity of the conjugate. After reconstitution at the initial time point and after storage at 4 ℃ for up to 24 months, the reconstituted solution was subjected to capillary gel electrophoresis (CE-SDS) to determine the solution purity:
FIG. 3 shows the tendency of the lyophilized preparation prepared in example 5 to reduce CE-SDS immunoglobulin during long-term storage at 4 ℃. FIG. 4 shows the trend of non-reduced CE-SDS immunoglobulins of the lyophilized preparation prepared in example 5 during long-term storage at 4 ℃. As can be seen from FIGS. 3 to 4, at the initial time point, the CE-SDS immunoglobulin content was 96.5%, and the low molecular weight species was 2.9%. After reconstitution after 3 months of storage at 4 ℃, the CE-SDS immunoglobulin content was 96.1%, the low molecular weight species was 4.0%. After reconstitution after 6 months of storage at 4 ℃, the CE-SDS immunoglobulin content was 97.3%, the low molecular weight species was 3.0%. After reconstitution after 9 months of storage at 4 ℃, the CE-SDS immunoglobulin content was 97.1%, the low molecular weight species was 2.9%. After reconstitution after 12 months of storage at 4 ℃, the CE-SDS immunoglobulin content was 96.6%, the low molecular weight species was 3.0%. After reconstitution after 18 months of storage at 4 ℃, the CE-SDS immunoglobulin content was 96.4%, the low molecular weight species was 3.2%. After 24 months of storage at 4℃the CE-SDS immunoglobulin content was 96.5% and the low molecular weight species was 3.5%. After reconstitution after 36 months of storage at 4 ℃, the CE-SDS immunoglobulin content was 96.8%, the low molecular weight species was 3.2%.
(4) Long-term storage at 4 ℃ does not alter the activity of the solution:
determining the binding of the reconstituted solution and the reference to Karpas 299 cells at the initial time point and after reconstitution after storage at 4 ℃ for up to 36 months, resulting in biological activity (relative specific activity); the binding of the reconstituted solution and the reference to rhCD30 was measured to obtain relative binding activity.
FIG. 5 shows the relative activity profile of the lyophilized formulation prepared in example 5 during long-term storage at a temperature of 4 ℃. FIG. 6 shows the relative binding activity profile of the lyophilized formulation prepared in example 5 during long-term storage at a temperature of 4 ℃. As can be seen from fig. 5 to 6, at the initial time point, the relative specific activity was 95% and the relative binding activity was 104%. After reconstitution after 3 months of storage at 4 ℃, the relative specific activity was 90% and the relative binding activity was 109%. After reconstitution after 6 months of storage at 4 ℃, the relative specific activity was 104%, the relative binding activity was 94%. After reconstitution after 9 months of storage at 4 ℃, the relative specific activity was 88%, the relative binding activity was 98%. After reconstitution after 12 months of storage at 4 ℃, the relative specific activity was 92% and the relative binding activity was 96%. After reconstitution after 18 months of storage at 4 ℃, the relative specific activity is 88% and the relative binding activity is 100%. After reconstitution after 24 months of storage at 4 ℃, the relative specific activity was 92% and the relative binding activity was 94%. After reconstitution after 36 months of storage at 4 ℃, the relative specific activity was 92% and the relative binding activity was 102%.
(5) Long-term storage at 4 ℃ does not affect the water content of the solution:
water content was assessed by Karl-Fischer titration. Karl-Fischer titration uses electrical quantity or volume titration to determine trace amounts of water in a sample, expressed as a percentage.
FIG. 7 shows the moisture content change during long-term storage at a temperature of 4℃of the lyophilized preparation prepared in example 5. As can be seen from fig. 7, the water content was 2.2% at the initial time point. After reconstitution after 3 months of storage at 4 ℃, the water content was 2.0%. After reconstitution after 6 months of storage at 4 ℃, the water content was 1.6%. After reconstitution after 9 months of storage at 4 ℃, the water content was 1.9%. After reconstitution after 12 months of storage at 4 ℃, the water content was 1.7%. After reconstitution after 18 months of storage at 4 ℃, the water content was 1.6%. After reconstitution after 24 months of storage at 4 ℃, the water content was 2.1%. After reconstitution after 36 months of storage at 4 ℃, the water content was 1.7%.
(6) Long-term storage at 4 ℃ does not affect the coupling ratio DAR of the drug:
hydrophobic Interaction Chromatography (HIC) was performed on the reconstituted solution to determine the drug-to-antibody ratio (DAR) over a 36 month period. HIC was used to separate proteins based on their relative hydrophobicity, the HIC method being a measurement of the hydrophobicity of F0002-ADC. The binding component is eluted from the ligand by decreasing the salt concentration of the mobile phase with a gradient in order of increasing hydrophobicity. For F0002-ADC, the first peak eluted from the column was unconjugated antibody. The remaining peaks represent an increased number of drug-linker molecules per antibody. Thus, the number of drug-linkers per antibody was determined by peak retention time and relative peak area. The drug-to-antibody ratio (DAR) of all reconstituted solutions was measured to be 3.3-3.9 at the initial time point and after reconstitution after storage at 4 ℃ for up to 36 months.
(7) Long-term storage at 4 ℃ does not affect the protein content of the solution:
the protein content of the reconstituted solution was determined. The protein concentration of the ADC was determined spectrophotometrically using wavelengths of 252nm and 280 nm. FIG. 8 shows the protein content change during long-term storage at a temperature of 4℃of the lyophilized preparation prepared in example 5. As can be seen from fig. 8, at the initial time point, the protein content of the reconstituted solution was 26.3mg. After reconstitution after 3 months of storage at 4 ℃, the protein content of the reconstituted solution was 26.4mg; after reconstitution after 6 months of storage at 4 ℃, the protein content of the reconstituted solution was 26.5mg; after reconstitution after 9 months of storage at 4 ℃, the protein content of the reconstituted solution was 26.5mg; after reconstitution after 12 months of storage at 4 ℃, the protein content of the reconstituted solution was 26.3mg; after reconstitution after 18 months of storage at 4 ℃, the protein content of the reconstituted solution was 26.3mg. After reconstitution after 24 months of storage at 4 ℃, the protein content of the reconstituted solution was 26.4mg. After reconstitution after 24 months of storage at 4 ℃, the protein content of the reconstituted solution was 26.7mg.
(8) Long-term storage at 4 ℃ does not affect particle contamination in the solution:
particle contamination was assessed by measuring microscopic particles. Acceptable standards (USP (US pharmacopoeia) and european pharmacopoeia standards) were met by determining the number of particles exceeding 10 μm in each container (not exceeding 6000). At the initial time point, the number of particles exceeding 10 μm in each container was 920. After reconstitution after 3 months of storage at 4 ℃, the number of particles exceeding 10 μm in each container was 377. After reconstitution after 6 months of storage at 4 ℃, the number of particles exceeding 10 μm in each container was 345. After reconstitution after 9 months of storage at 4 ℃, the number of particles exceeding 10 μm in each container was 67. After reconstitution after 12 months of storage at 4 ℃, the number of particles exceeding 10 μm in each container was 72. After reconstitution after 24 months of storage at 4 ℃, the number of particles exceeding 10 μm in each container was 272. After reconstitution after 36 months of storage at 4 ℃, the number of particles exceeding 10 μm in each container was 822. Thus, the number of particles exceeding 10 μm in each container at all time points meets the acceptable criteria.
Acceptable standards (USP (US pharmacopoeia) and european pharmacopoeia standards) were also met by determining the number of particles exceeding 25 μm in each container (NM 600). At the initial time point, the number of particles exceeding 25 μm in each container was 10. After reconstitution after 3 months of storage at 4 ℃, the number of particles exceeding 25 μm in each container was 23. After reconstitution after 6 months of storage at 4 ℃, the number of particles exceeding 25 μm in each container was 3. After reconstitution after 9 months of storage at 4 ℃, the number of particles exceeding 25 μm in each container was 8. After reconstitution after 12 months of storage at 4 ℃, the number of particles exceeding 25 μm in each container was 8. After reconstitution after 24 months of storage at 4 ℃, the number of particles exceeding 25 μm in each container was 25. After reconstitution after 36 months of storage at 4 ℃, the number of particles exceeding 25 μm in each container was 5. Thus, the number of particles exceeding 25 μm in each container at all time points meets the acceptable criteria.
(9) Long-term storage at 4 ℃ does not affect the pH of the solution:
the pH of the reconstituted solution was determined at the initial time point and after reconstitution after 3 months, 6 months, 12 months, 24 months, 36 months of storage at 4 ℃. The results indicated that the pH of the reconstituted solution was 5.0 at each time point.
(10) Long-term storage at 4 ℃ does not affect the osmotic pressure of the solution:
the osmolarity of the reconstituted solution is determined at the initial time point and after reconstitution after 36 months of storage at 4 ℃ by measuring the average milliosmolarity value (mOsmol/kg) of solute per kilogram of solvent. At the initial time point, the osmolality was 465mOsmol/kg. At 36 months, the osmolality was 465mOsmol/kg.
(11) The content of free small molecule DM1 is unchanged after long-term storage at 4 ℃:
the DM1 content in the reconstituted solution was measured at the initial time point and after reconstitution after 36 months of storage at 4 ℃, at which point the free small molecule DM1 content was 1.2%. At 36 months, the free small molecule DM1 content was 1.2%.
The long-term stability data of the pharmaceutical composition for 36 months show that each test item data has no significant change, and the validity period of the pharmaceutical composition can reach 3 years under the condition.
Example 7 stability of lyophilized Dry formulations at 25℃
The stable lyophilized formulation prepared in example 5 above was subjected to long term stability experiments at 25 ℃ and tested after reconstitution with WFI at time points 0, 1, 3 and 6 months, respectively. And characterized by SEC, CE-SDS, binding activity, and moisture content.
(1) Long-term storage at 25 ℃ does not affect the appearance of the lyophilizate and reconstituted solution:
the appearance of the lyophilizate and reconstituted solution was visually assessed to confirm that the lyophilizate was virtually free of macroscopic foreign particulate matter and contained no moisture in the packaging material. The appearance of the lyophilisate meets the above criteria at the initial time point and after reconstitution after storage at 25 ℃ for up to 6 months. After reconstitution after storage at 25 ℃ for up to 6 months, the reconstituted solution is a colorless to yellowish solution and virtually free of macroscopic particulate matter.
(2) Long term storage at 25 ℃ does not affect SEC purity of conjugate
Size exclusion chromatography of the reconstituted solution was performed using size exclusion-high performance liquid chromatography (SE-HPLC). SE-HPLC the purity of F0002-ADC was determined using size exclusion HPLC. Macromolecules are equally separated during gel filtration HPLC according to reduced molecular size. Purity was determined by comparing the area of the main peak of the F0002-ADC with the total area of the sample chromatogram (excluding buffer related peaks). The method is capable of resolving high molecular weight aggregates and truncated antibody species from the main peak of the F0002-ADC. In addition to the high molecular weight species, namely, the polymer (%) and the low molecular weight species (%), the main peak, namely, the monomer (%), was measured.
Fig. 9 is a graph showing the variation of SEC monomer during long-term storage of the lyophilized formulation prepared in example 5 at 25 ℃. Fig. 10 is a graph showing the tendency of SEC polymers during long-term storage at 25 ℃ for the lyophilized formulation prepared in example 5. As can be seen from fig. 9 to 10, at the initial time point, the SEC monomer was 98.7%, the polymer was 1.3%, and the low molecular weight species was 0.0%. After reconstitution after 1 month of storage at 25 ℃, the SEC monomer was 98.7%, the polymer was 1.3% and the low molecular weight species was 0.0%. After reconstitution after 2 months of storage at 25 ℃, the SEC monomer was 98.7%, the polymer was 1.3% and the low molecular weight species was 0.0%. After reconstitution after 3 months of storage at 25 ℃, the SEC monomer was 98.7%, the polymer was 1.3% and the low molecular weight species was 0.0%. After reconstitution after 6 months of storage at 25 ℃, the SEC monomer was 98.7%, the polymer was 1.3% and the low molecular weight species was 0.0%.
(3) Long-term storage at 25 ℃ does not alter the CE-SDS purity of the conjugate
FIG. 11 shows the tendency of the lyophilized preparation prepared in example 5 to reduce CE-SDS immunoglobulin during long-term storage at 25 ℃. FIG. 12 shows the low molecular weight distribution of non-reduced CE-SDS during long-term storage at 25℃of the lyophilized preparation prepared in example 5. As can be seen from fig. 11 to 12, the purity of the reconstituted solution was measured by capillary gel electrophoresis (CE-SDS) at the initial time point and after reconstitution after storage at 25 ℃ for up to 6 months. At the initial time point, the CE-SDS immunoglobulin content was 97.1%, and the low molecular weight species was 3.1%. After reconstitution after 1 month of storage at 25 ℃, the CE-SDS immunoglobulin content was 97.1%, the low molecular weight species was 3.0%. After reconstitution after 2 months of storage at 25 ℃, the CE-SDS immunoglobulin content was 97.1%, the low molecular weight species was 3.1%. After reconstitution after 3 months of storage at 25 ℃, the CE-SDS immunoglobulin content was 96.9%, the low molecular weight species was 3.2%. After reconstitution after 6 months of storage at 25 ℃, the CE-SDS immunoglobulin content was 96.6%, the low molecular weight species was 3.2%.
(4) Does not change the activity of the solution after long-term storage at 25 DEG C
Determining the binding of the reconstituted solution and the reference to Karpas 299 cells at the initial time point and after reconstitution after storage at 25 ℃ for up to 6 months, resulting in biological activity (relative specific activity); the binding of the reconstituted solution and the reference to rhCD30 was measured to obtain relative binding activity.
FIG. 13 shows the relative activity profile of the lyophilized formulation prepared in example 5 during long-term storage at 25 ℃. FIG. 14 shows the relative binding activity profile of the lyophilized formulation prepared in example 5 during long-term storage at 25 ℃. As can be seen from fig. 13 to 14, at the initial time point, the relative specific activity was 106% and the relative binding activity was 102%. After reconstitution after 1 month of storage at 25 ℃, the relative specific activity was 98%, the relative binding activity was 94%. After reconstitution after 2 months of storage at 25 ℃, the relative specific activity was 92% and the relative binding activity was 104%. After reconstitution after 3 months of storage at 25 ℃, the relative specific activity was 97%, the relative binding activity was 104%. After reconstitution after 6 months of storage at 25 ℃, the relative specific activity was 102% and the relative binding activity was 100%.
Claims (20)
1. A freeze-dried formulation, characterized in that it is made of a pharmaceutical composition comprising the following components:
Arginine hydrochloride and the dosage thereof is 20-50 mg/mL;
the F0002-ADC is an antibody conjugate, and the structural general formula of the antibody conjugate is Ab-L m -Y n ；
Wherein Ab is anti-human CD30 antibody cAC, an active fragment of anti-human CD30 antibody cAC, or a variant of anti-human CD30 antibody cAC; the variant of the anti-human CD30 antibody cAC10 has a similarity to the amino acid sequence of cAC of no less than 90% and a lysine-related mutation of no more than 80%;
the Ab is only linked to the L;
y is a cytotoxic agent maytansinoid DM1;
n is 2-5, and m is more than or equal to n; said Y being connected only to said L; part of L is connected with Ab and Y at two ends respectively, and the rest of L is connected with Ab only;
when both ends of the L are respectively connected with the Ab and the Y, the L is
The left end of the thiol compound forms an amide bond with the amino group of the lysine residue of the Ab, and the right end of the thiol compound forms a thioether bond with S in the DM1 thiol;
when the L is linked only to the Ab, the L is
The left end of the amino acid form an amide bond with the amino group of the lysine side chain of the Ab;
the protein protecting agent is sucrose and/or trehalose; the surfactant is polysorbate 20 and/or polysorbate 80.
2. A lyophilized formulation according to claim 1,
M of the antibody conjugate is 2-10;
and/or the pH buffer is citrate buffer, acetate buffer, succinate buffer, histidine buffer, phosphate buffer or Bis-Tris buffer;
and/or the solvent in the pharmaceutical composition is one or more of purified water, water for injection and physiological saline.
3. The lyophilized formulation according to claim 2, wherein m is equal to n and has the general structural formula Ab- (L-Y) n, which has the structure shown below:
and/or, the pH buffer is a citrate buffer;
and/or the solvent in the pharmaceutical composition is water for injection.
4. The lyophilized formulation according to claim 1, wherein n = 3-4.
5. The lyophilized formulation according to claim 4, wherein n = 3.6.
6. The lyophilized formulation according to claim 1, wherein arginine hydrochloride is used in an amount of 25 to 45mg/mL;
the dosage of the F0002-ADC is 5-18 mg/mL;
and/or the dosage of the pH buffer is 10-45 mM;
and/or the dosage of the protein protective agent is 20-90 mg/mL;
and/or the dosage of the surfactant is 0.1-1.8 mg/mL;
And/or the pH is 5.0, 5.5 or 6.0.
7. The lyophilized formulation of claim 6, wherein arginine hydrochloride is present in an amount of 20mg/mL, 25mg/mL, 30mg/mL, 35mg/mL, or 40mg/mL;
the dosage of the F0002-ADC is 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, 9mg/mL, 10mg/mL, 12mg/mL or 15mg/mL;
and/or the dosage of the pH buffer is 15-40 mM;
and/or the dosage of the protein protective agent is 30-80 mg/mL;
and/or the dosage of the surfactant is 0.2-1.5 mg/mL.
8. The lyophilized formulation according to claim 6, wherein the pH buffer is used in an amount of 20mM, 25mM, 30mM or 35mM;
and/or the protein protectant is used in an amount of 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL or 80mg/mL;
and/or the surfactant is used in an amount of 0.2mg/mL, 0.5mg/mL, 1.0mg/mL, or 1.2mg/mL.
9. The lyophilized formulation according to claim 1, wherein the components of the pharmaceutical composition are any one of the following numbers 7 to 21:
alternatively, the components of the pharmaceutical composition are numbered 24:
alternatively, the components of the pharmaceutical composition are any one of the following numbers 1 to 12:
Alternatively, the components of the pharmaceutical composition are any one of the following numbers 1 to 10:
10. the freeze-dried formulation according to any one of claims 1 to 9, wherein the process for preparing the pharmaceutical composition comprises the steps of: after the protein liquid is subjected to centrifugal liquid exchange, concentrating to meet the concentration compounding requirement of the F0002-ADC;
wherein the protein solution is a solution containing the F0002-ADC; the replacement buffer solution adopted in the centrifugal liquid exchange process comprises the pH value buffer, the protein protecting agent, the surfactant and the arginine hydrochloride, wherein the pH value of the replacement buffer solution is as claimed in any one of claims 1 to 9.
11. The lyophilized formulation according to claim 10, wherein the initial buffer solution in the protein solution is: 25mM citric acid-sodium citrate buffer, 150mM NaCl,2mM EDTANa 2 ，pH 6.0±0.1；
And/or, the concentration of the F0002-ADC in the protein liquid is 5-15 mg/mL;
and/or the centrifugal liquid exchange operation is performed in an ultrafiltration concentration tube;
and/or, in the centrifugal liquid exchange process, the volume ratio of the adopted replacement buffer solution to the protein liquid is 5-15.
12. The lyophilized formulation according to claim 11, wherein the concentration of said F0002-ADC in said protein solution is 10mg/mL;
and/or, the operation of centrifugal liquid exchange is carried out in a 30kD ultrafiltration concentration tube;
and/or, in the centrifugal liquid exchange process, the volume ratio of the adopted replacement buffer solution to the protein solution is 10.
13. A process for the preparation of a lyophilized formulation as claimed in any one of claims 1 to 12 comprising the step of lyophilizing said pharmaceutical composition.
14. The method of claim 13, wherein the freeze-drying process comprises a prefreezing stage, a primary sublimation and a analytical drying step in sequence.
15. Use of a lyophilized formulation according to any one of claims 1 to 12 for the preparation of a medicament for the treatment of CD30 positive tumors.
16. The use of claim 15, wherein the CD30 positive tumor is a lymphoma.
17. The use of claim 16, wherein the lymphoma is hodgkin's lymphoma, anaplastic large cell lymphoma, diffuse tissue cell lymphoma, or cutaneous T-cell lymphoma.
18. The use of claim 17, wherein the lymphoma is hodgkin's lymphoma.
19. An article comprising a container containing a lyophilized formulation according to any one of claims 1 to 12.
20. The article of claim 19, wherein the container is a glass bottle or a liquid storage bag.
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| cAC10-vcMMAE, an anti-CD30–monomethyl auristatin E conjugate with potent and selective antitumor activity;Joseph A. Francisco等;《BLOOD》;第102卷(第4期);全文 * |
| Lysosomal trafficking and cysteine protease metabolism confer target-specific cytotoxicity by peptide-linked anti-CD30-auristatin conjugates;May S Kung Sutherland等;《J Biol Chem》;第281卷(第15期);全文 * |
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Address after: 201210 Shanghai Zhangjiang High Tech Park of Pudong New Area Cailun Road No. 308 Patentee after: SHANGHAI FUDAN-ZHANGJIANG BIO-PHARMACEUTICAL Co.,Ltd. Patentee after: Shanghai Shangyao Cross Linked Pharmaceutical Technology Co.,Ltd. Address before: 201210 Shanghai Zhangjiang High Tech Park of Pudong New Area Cailun Road No. 308 Patentee before: SHANGHAI FUDAN-ZHANGJIANG BIO-PHARMACEUTICAL Co.,Ltd. Patentee before: SHANGHAI JIAOLIAN PHARMACEUTICAL RESEARCH DEVELOPMENT Co.,Ltd. |