CN113827717A

CN113827717A - anti-HER 2 monoclonal antibody freeze-dried preparation and preparation method thereof

Info

Publication number: CN113827717A
Application number: CN202010577294.XA
Authority: CN
Inventors: 杨泗兴; 张士荣; 苏研; 黄浩旻; 朱祯平
Original assignee: Sunshine Guojian Pharmaceutical Shanghai Co Ltd
Current assignee: Sunshine Guojian Pharmaceutical Shanghai Co Ltd
Priority date: 2020-06-23
Filing date: 2020-06-23
Publication date: 2021-12-24
Also published as: WO2021259101A1; CN115768799A

Abstract

The invention provides a stable freeze-dried preparation of an anti-HER 2 monoclonal antibody and a preparation method thereof. The invention greatly improves the defect of unstable protein solution by optimizing the formulation of the preparation and the freeze-drying process, and the medicament produced according to the formulation of the freeze-drying preparation and the preparation method has the stability of at least 6 months at 25 ℃ and the stability of at least 60 months at 5 ℃.

Description

anti-HER 2 monoclonal antibody freeze-dried preparation and preparation method thereof

Technical Field

The invention belongs to the field of biological pharmacy, and particularly relates to a freeze-dried preparation of an anti-HER 2 monoclonal antibody and a preparation method thereof.

Background

HER2/neu (human epidermal growth factor receptor 2), also known as erbB2, has tyrosine protein kinase activity and is a member of the human epidermal growth factor receptor family and is expressed at low levels in only a few normal tissues of adults. However, studies show that HER2 is overexpressed in various tumors, such as the overexpression is existed in about 30% of breast cancer patients and 16% of gastric cancer patients, and the overexpression of HER2 in tumors can remarkably promote the neogenesis of tumor vessels, the growth of tumors and enhance the invasion and metastasis capacity of tumors, and is an important indicator for poor prognosis of patients of the kind.

PCT patent application WO2020/025013 discloses a self-developed recombinant anti-HER 2 humanized monoclonal antibody 19H6-Hu of anti-HER 2 dimerization domain, which is capable of specifically binding to the functional domain DIII of the extracellular domain of human HER2 (HER2-ECD), is a recombinant humanized monoclonal antibody expressed in CHO cells by using DNA recombination technology, and is humanized from the heavy chain variable region and the light chain variable region of an anti-HER 2 murine antibody. In the intensive study of 19H6-Hu, the protein solution has more charge isomers and has a growing trend in the storage process, so that the stability needs to be improved through preparation optimization.

Disclosure of Invention

In order to solve the problems, the invention aims to provide a freeze-dried preparation of an anti-HER 2 monoclonal antibody and a preparation method thereof. The freeze-dried preparation consists of an anti-HER 2 monoclonal antibody, a buffer solution, a protein protective agent and a surfactant. The preparation method comprises the steps of subpackaging the liquid medicine, freezing-drying and the like.

In order to realize the purpose of the invention, the invention adopts the following technical scheme:

the invention provides a freeze-dried preparation of an anti-HER 2 monoclonal antibody, which comprises the anti-HER 2 monoclonal antibody, a buffer solution, a protein protective agent and a surfactant.

Wherein the concentration of the anti-HER 2 monoclonal antibody is 30-80mg/ml, and the anti-HER 2 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 1 and the heavy chain as set forth in SEQ ID NO: 2, or a light chain as shown in figure 2. Preferably, the concentration of the anti-HER 2 monoclonal antibody is 40 mg/ml.

Wherein the buffer solution is histidine-histidine hydrochloride buffer solution or phosphate buffer solution, and the concentration of the buffer solution is 10-20 mM. Preferably, the buffer is histidine-histidine hydrochloride buffer, and the concentration of the buffer is 10 mM.

Wherein the protein protective agent is trehalose or sucrose, and the concentration of the protein protective agent is 30-40 mg/ml. Preferably, the protein protective agent is trehalose, and the concentration of the protein protective agent is 30 mg/ml.

Wherein the surfactant is polysorbate 80, and the concentration of the surfactant is 0.01-1.0 mg/ml. Preferably, the concentration of the surfactant is 0.3 mg/ml.

Wherein the pH of the lyophilized preparation is 5.2-7.0. Preferably, the pH of the lyophilized formulation is 5.2-6.3. More preferably, the pH of the lyophilized formulation is 5.7-6.3.

The second aspect of the invention provides a preparation method of the freeze-dried preparation, which comprises the steps of subpackaging liquid medicine and freezing-drying.

Wherein the split charging dosage of the liquid medicine is 30-420 mg/bottle. Preferably, the split dose of the liquid medicine is 210-420 mg/bottle. More preferably, the split dose of the liquid medicine is 210 mg/bottle.

Wherein, the freezing-drying comprises the steps of pre-freezing, primary drying, secondary drying and final drying.

Wherein the pre-freezing temperature in the box is 5 ℃, the pre-freezing temperature is-40 ℃, and the pre-freezing time is 180-185 min; the main drying temperature is-10 to-20 ℃, the time is 17 to 30 hours, and the vacuum degree is 200 and 300 mu bar; the secondary drying temperature is 0-15 ℃, the time is 5-15h, and the vacuum degree is 100-; the final drying temperature is 30 ℃, the time is 2.5-4h, and the vacuum degree is 20-60 mu bar.

Preferably, the pre-freezing temperature in the box is 5 ℃, the pre-freezing temperature is-40 ℃, and the pre-freezing time is 185 min; the main drying temperature is-15 ℃, the time is 26h, and the vacuum degree is 300 mu bar; the secondary drying temperature is 0-15 ℃, the time is 740min, and the vacuum degree is 200-300 mu bar; the final drying temperature is 30 ℃, the time is 2.5h, and the vacuum degree is 20-60 mu bar.

In a third aspect, the invention provides the use of the lyophilized formulation for the manufacture of a medicament for the treatment of cancer.

Wherein the cancer comprises breast cancer, gastric cancer or ovarian cancer.

Has the advantages that: the invention provides a stable freeze-dried preparation of an anti-HER 2 monoclonal antibody and a preparation method thereof. The invention greatly improves the defect of unstable protein solution by optimizing the formulation of the preparation and the freeze-drying process, and the medicament produced according to the formulation of the freeze-drying preparation and the preparation method has the stability of at least 6 months at 25 ℃ and the stability of at least 60 months at 5 ℃.

Drawings

FIG. 1 shows the analysis of the effect of pH, excipients and dosage forms on protein purity.

Figure 2 is DOE result analysis.

FIG. 3 shows the results of examination of buffers and protein protectants.

FIG. 4 is a 4-1 lyophilization curve.

FIG. 5 is a 4-2 lyophilization curve.

FIG. 6 is a 4-3 lyophilization curve.

FIG. 7 shows the IEC purity change at 5 ℃.

Detailed Description

The protein samples used in the following examples were derived from the anti-HER 2 monoclonal antibody 19H6-Hu disclosed in WO2020/025013, the heavy and light chain amino acid sequences of which are shown below.

19H6-Hu heavy chain amino acid sequence (SEQ ID NO: 1)

QVQLVQSGAEVKKPGASVKVSCKGSGYTFTDYAIHWVRQAPGQSLEWIGVFSIYYENINYNQKFKGRATMTVDKSTSTAYMELRSLRSDDTAVYYCARRDGGTINYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

19H6-Hu light chain amino acid sequence (SEQ ID NO: 2)

DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHIPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The detection method used in the following examples is illustrated below:

SEC purity, Polymer detection method:

mobile phase: 200mM phosphate buffer, pH 6.8. + -. 0.1. Filtering with 0.22 μm filter membrane, and ultrasonic degassing. A chromatographic column: TSK G3000SWxl, 7.8X 300mm 5 μm, TOSOH 08541. High performance liquid chromatograph: waters Alliance e 26952489 UV/visible light Detector, Dionex Ultimate 3000 VWD-3400(RS) Detector or other suitable HPLC system equipped with a UV Detector.

System applicability sample: the reference substance is diluted to the concentration of 5.0mg/ml by a mobile phase, centrifuged at 13000rpm for 10min, and the supernatant is taken and transferred to a sample bottle and placed in an HPLC sample tray. And (3) testing the sample: diluting the sample concentration to 5.0mg/ml with mobile phase, centrifuging at 13000rpm for 10min, taking supernatant, transferring to a sample bottle, and placing into HPLC sample plate. Chromatographic conditions are as follows: the column temperature is 25 +/-2 ℃; the sample temperature is 10 +/-2 ℃; detecting the wavelength UV 280 nm; the injection volume is 20 mu L; the flow rate was 0.5 ml/min.

Integration was performed using chromatography software and peak area normalization was used to calculate the peak area percentage of each peak. Acceptance criteria for system suitability: the separation degree of polymers and monomers of 6-needle system applicability samples is more than or equal to 1.5, the retention time RSD of a main peak is less than or equal to 1.0%, the peak area RSD of the main peak is less than or equal to 2.0%, the asymmetry of the main peak is less than or equal to 2.0, and the number of theoretical plates is more than or equal to 4000. The test article reports the results: the SEC purity of the sample is reported as the peak area percentage of the monomer main peak and the polymer content as the peak area percentage of the polymer peak.

The IEC purity detection method comprises the following steps:

mobile phase A: 20mM phosphate buffer, pH 6.5. + -. 0.05. Filtering with 0.22 μm filter membrane, and ultrasonic degassing. Mobile phase B: 20mM phosphate buffer +200mM sodium chloride, pH 6.5. + -. 0.05. Filtering with 0.22 μm filter membrane, and ultrasonic degassing. A chromatographic column: propac WCX-10, 4X 250mm, Thermo Dionex 054993. High performance liquid chromatograph: waters Alliance e2695, Dionex Ultimate series 3000 or other suitable HPLC system equipped with an ultraviolet detector.

System applicability sample: the reference substance is diluted to 1.0mg/ml by mobile phase, centrifuged for 10min at 13000rpm, and the supernatant is transferred to a sample bottle and placed in an HPLC sample tray. And (3) testing the sample: diluting the sample concentration to 1.0mg/ml with mobile phase, centrifuging at 13000rpm for 10min, taking supernatant, transferring to a sample bottle, and placing into an HPLC sample tray. Chromatographic conditions are as follows: the column temperature is 30 +/-2 ℃; the sample temperature is 10 +/-2 ℃; the detection wavelength is UV 214 nm; the injection volume is 20 mu L; the flow rate was 1.0 ml/min. The mobile phase gradient was as follows:

purity analysis: and calculating the peak area percentages of a main peak, an acid peak area and an alkali peak area on the sample map by using a peak area normalization method. The IEC purity results are reported as peak area percentages of the main peak.

The starting components used in the following examples are commercially available, unless otherwise noted.

The following examples and experimental examples are intended to further illustrate the present invention, but are not intended to limit the present invention in any way.

Example 1 Effect of pH, excipients, and dosage forms on protein purity

This example examines the effect of different ranges of pH (4.0-7.0), different buffer types (20mM acetate buffer, 20mM phosphate buffer, 20mM histidine-histidine hydrochloride buffer), different protein concentrations (30-80mg/ml), different polysorbate 80 concentrations (0.2-2.0mg/ml), different protein protectants (sucrose, trehalose), different formulations (injections, lyophilisates) on protein SEC purity, IEC purity. The formulations of 10 groups were prepared according to Table 1, with a fill volume of 1ml for injection, a dose of 30mg, a fill volume of 5.3ml for lyophilization, and a dose of 420mg, with the lyophilization parameters shown in Table 2. The samples were taken at 40 ℃ for examination and the SEC purity and the IEC purity were determined on

days

0, 3, 5, 7 and 10.

TABLE 1 Effect of pH, excipients, and dosage forms on protein purity

TABLE 2, 1-9, 1-10 lyophilization parameters used in the lyophilization groups

Step (ii) of	Temperature of sheet layer (. degree. C.)	Vacuum degree (ubar)	Time (min)
				Put into box	5	\	\
Prefreezing	-40	\	5
				Prefreezing	-40	\	180
Drying	-20	250	40
				Drying	-20	250	540
Drying	-10	250	25
				Drying	-10	250	120
Drying	10	250	40
				Drying	10	250	300
Drying	30	60	20
				Drying	30	60	220

TABLE 3 test results of the influence of pH, excipients, and dosage forms on protein purity

As can be seen from the results of Table 3 and FIG. 1, the SEC purity and IEC purity of 1-9 and 1-10 are significantly superior to those of the other groups, indicating that the lyophilized preparation has better stability than the injection; as can be seen from the results of IEC purity, 1-4, 1-5, 1-6, 1-7 and 1-8 are superior to 1-1, 1-2 and 1-3 except for the freeze-dried group, indicating that the lower pH value is not favorable for the IEC purity; the difference between the two groups 1-1 and 1-2 is small, which shows that the influence of different polysorbate 80 contents is not obvious, and 0.2-2.0mg/ml meets the requirement. 1-8 and 1-7 are not obvious in difference, which indicates that the concentration of the protein 80mg/ml is not obvious in difference with the concentration of the protein 30mg/ml, and the protein is subpackaged into 1.0-5.3ml according to 2-20ml penicillin bottles, and the dosage can be 30-420 mg/bottle; the differences of the groups 1-4, 1-5, 1-6 and 1-7 are not obvious, which shows that the histidine-histidine hydrochloride buffer solution and the phosphate buffer solution have no obvious difference and are superior to the acetate buffer solution; 1-9 and 1-10 did not differ significantly, indicating that there was no significant difference between sucrose and trehalose.

Example 2 DOE investigation of pH, surfactants

In this example, the pH and the polysorbate 80 amount were examined by DOE design, the pH was set to 5.2-6.2, and the polysorbate 80 content was set to 0.01-1.0 mg/ml. The protein content is 40mg/ml, histidine-histidine hydrochloride buffer solution is 20mM, and the dosage of trehalose is 40 mg/ml. The filling volume is 1ml, and the dosage is 40 mg/bottle (solution). 10 groups of formulations were placed at 40 ℃ for 4 weeks and sampled for SEC purity, IEC purity, insoluble particles at 0, 1, 2, 3, 4 weeks, respectively.

TABLE 4 DOE design formula table

TABLE 5 DOE results

The results of the DOE groups in Table 5 were subjected to linear regression analysis, and the slope of each group was subjected to DOE model analysis using JMP, and the results are shown in FIG. 2. As can be seen from FIG. 2, the shaded area in the figure is the optimal range for screening. The preferred range of pH may be 5.2-6.2 and the preferred amount of polysorbate 80 may be 0.01-1.0mg/ml, wherein a lower range of polysorbate 80 is used when a higher range of pH is used, and a higher range of polysorbate 80 is used when a lower range of pH is used.

Example 3 buffer concentration, protein protectant concentration examination

This example examines the buffer concentration and the concentration of the protein protectant. As shown in Table 6, 5 samples were prepared with a histidine-histidine hydrochloride buffer in the range of 5-20mM, trehalose at 20-40mg/ml, pH 6.0. The samples were prepared and lyophilized at a volume of 5.3ml for a dose of 210 mg/vial, with the lyophilization parameters shown in Table 7. After freeze-drying, the mixture is placed at 40 ℃, and samples are taken at 0, 1, 2, 3 and 4 weeks to detect the SEC purity and the IEC purity.

TABLE 6 buffer concentration, protein protectant concentration examination

TABLE 7 examination of buffer, protein protectant lyophilization parameters

TABLE 8 buffer, protein protectant results

As can be seen from the results of Table 8 and FIG. 3, the SEC purity and IEC purity were poor in the 3-1 group and poor in the 3-4 group. The IEC purity of 3-2 groups is the best, and the SEC purity of 3-5 groups is the best. From the above results, it is found that the preferable buffer concentration is 10 to 20mM, and the preferable trehalose concentration is 30 to 40 mg/ml.

Example 4 lyophilization Process investigation

The lyophilization process was examined according to the lyophilization parameters of table 9, respectively. The freeze-dried formulation comprises 40mg/ml of protein, 10mM of histidine-histidine hydrochloride buffer solution, 30mg/ml of trehalose, 800.3 mg/ml of polysorbate and pH 6.0. And 5.3ml of penicillin bottles with the dosage of 210 mg/bottle are adopted for filling. And (5) inspecting the freeze-drying curve and the appearance of the sample after freeze-drying.

TABLE 9 examination of lyophilization Process

As can be seen from the results of FIGS. 4-6, the drying time was insufficient for the 4-1 lyophilization curve, the product curve did not intersect the temperature line of the sheet layer, which may result in incomplete sublimation of water, while the 4-2 and 4-3 curves meet the requirement, with the 4-2 intersection occurring at about 1020min (17h) at the earliest, indicating a main drying time of at least 17 h. The appearance of the three freeze-dried samples meets the requirements. Thus, preferred lyophilization parameter conditions are as follows: putting the mixture into a box at 5 ℃, and pre-freezing the mixture for 3 hours at the temperature of minus 40 ℃; the main drying temperature is-15 to-20 ℃, the vacuum degree is 200 to 300 mu bar, and the time is more than 17 hours; drying for 5-10 h at 0-15 ℃ and 100-300 μ bar vacuum degree; final drying at 30 deg.C under 60 μ bar for 4 h.

Example 5 stability study

As shown in Table 10, a batch of the drug is prepared according to the preferred formula protein of the invention, 40mg/ml, histidine-histidine hydrochloride 10mM, trehalose 30mg/ml, polysorbate 800.3 mg/ml, pH6.0 +/-0.3, and the filling volume is 5.3ml, and the dosage is 210 mg/per unit. And (4) after freeze-drying, respectively standing at 25 ℃ and 5 ℃ for stability investigation, wherein detection items comprise SEC purity and IEC purity.

TABLE 10 stability batch lyophilization parameters

Step (ii) of	Temperature of sheet layer (. degree. C.)	Vacuum degree (ubar)	Time (min)
				Put into box	5	\	\
Prefreezing	-40	\	5
				Prefreezing	-40	\	180
Evacuation	\		300					\
				Drying	-15	300	60
Drying	-15	300	1500
				Drying	0	300	60
Drying	0	300	480
				Drying	15	200	20
Drying	15	200	180
				Drying	30	60	20
Drying	30	60	60
				Drying	30	20	10
Drying	30	20	60

TABLE 11 stability results

As can be seen from the results in Table 11, the SEC purity and IEC purity varied little after standing at 25 ℃ for 6 months and met the quality standard requirements. As can be seen from the results in Table 11 and FIG. 7, there was no significant change in SEC purity after 6 months at 5 ℃ and the drug expiration date calculated for IEC purity according to the ICH guidelines was 91 months. Therefore, the stability of the medicine produced according to the formula and the preparation method of the invention can support the effective period of 60 months.

Sequence listing

<110> Sansheng Guojian pharmaceutical industry (Shanghai) GmbH

<120> anti-HER 2 monoclonal antibody freeze-dried preparation and preparation method thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 447

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile

35 40 45

Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 2

<211> 219

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Ile Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

Claims

1. A lyophilized formulation of an anti-HER 2 monoclonal antibody, said lyophilized formulation comprising: anti-HER 2 monoclonal antibody, buffer solution, protein protectant, and surfactant; wherein the concentration of the anti-HER 2 monoclonal antibody is 30-80mg/ml, and the anti-HER 2 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 1 and the heavy chain as set forth in SEQ ID NO: 2; the buffer solution is histidine-histidine hydrochloride buffer solution or phosphate buffer solution, and the concentration of the buffer solution is 10-20 mM; the protein protective agent is trehalose or sucrose, and the concentration of the protein protective agent is 30-40 mg/ml; the surfactant is polysorbate 80, and the concentration of the surfactant is 0.01-1 mg/ml; the pH of the freeze-dried preparation is 5.2-7.0.

2. The lyophilized formulation of claim 1, wherein said anti-HER 2 monoclonal antibody is present at a concentration of 40 mg/ml; the buffer solution is histidine-histidine hydrochloride buffer solution, and the concentration of the buffer solution is 10 mM; the protein protective agent is trehalose, and the concentration of the protein protective agent is 30 mg/ml; the concentration of the surfactant is 0.3 mg/ml; the pH of the lyophilized preparation is 5.2-6.3, and preferably, the pH of the lyophilized preparation is 5.7-6.3.

3. The method for preparing a lyophilized formulation according to claim 1 or 2, comprising the steps of liquid medicine dispensing and freeze-drying.

4. The method of claim 3, wherein the amount of the liquid medicine dispensed is 30-420 mg/bottle.

5. The method for preparing the compound of claim 4, wherein the split dose of the drug solution is 210 mg/bottle and 420 mg/bottle, preferably 210 mg/bottle.

6. The method of claim 3, wherein the freeze-drying step comprises the steps of pre-freezing, primary drying, secondary drying, and final drying.

7. The method as claimed in claim 6, wherein the pre-freezing temperature is 5 ℃, the pre-freezing temperature is-40 ℃, and the pre-freezing time is 180-185 min; the main drying temperature is-10 to-20 ℃, the time is 17 to 30 hours, and the vacuum degree is 200 and 300 mu bar; the secondary drying temperature is 0-15 ℃, the time is 5-15h, and the vacuum degree is 100-; the final drying temperature is 30 ℃, the time is 2.5-4h, and the vacuum degree is 20-60 mu bar.

8. The method according to claim 7, wherein the prefreezing temperature is 5 ℃, the prefreezing temperature is-40 ℃, and the prefreezing time is 185 min; the main drying temperature is-15 ℃, the time is 26h, and the vacuum degree is 300 mu bar; the secondary drying temperature is 0-15 ℃, the time is 740min, and the vacuum degree is 200-300 mu bar; the final drying temperature is 30 ℃, the time is 2.5h, and the vacuum degree is 20-60 mu bar.

9. Use of the lyophilized formulation of claim 1 or 2 for the preparation of a medicament for the treatment of cancer.

10. The use of claim 9, wherein the cancer comprises breast cancer, gastric cancer or ovarian cancer.