CN111346225A

CN111346225A - Pharmaceutical formulations containing proteins

Info

Publication number: CN111346225A
Application number: CN201811574466.7A
Authority: CN
Inventors: 郭怀祖; 徐进; 曹嘉伟
Original assignee: Shanghai Biomabs Pharmaceuticals Co Ltd
Current assignee: Shanghai Biomabs Pharmaceuticals Co Ltd
Priority date: 2018-12-21
Filing date: 2018-12-21
Publication date: 2020-06-30

Abstract

The invention provides a protein-containing pharmaceutical preparation, which comprises an effective dose of an anti-PD-1 antibody or an antigen-binding fragment thereof, a buffer solution, a stabilizer and a surfactant.

Description

Pharmaceutical formulations containing proteins

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a pharmaceutical preparation containing protein, more particularly to a stable pharmaceutical preparation containing an anti-PD-1 antibody and an antigen binding fragment thereof.

Background

Programmed Cell Death Protein 1 (PD-1: Programmed Cell Death Protein 1, also known as CD279), a Cell surface receptor expressed on activated T cells and pre-B cells (pro-B cells), is an immune checkpoint Protein that has a negative regulatory effect on the immune system, promotes self-tolerance by inhibiting T Cell inflammatory activity, and more specifically, PD-1 prevents autoimmunity by promoting the apoptosis of antigen-specific T cells in lymph nodes and reducing the apoptosis of regulatory T cells, but can also prevent the immune system from killing cancer cells.

The "immune escape" of cancer cells is thought to be the major mechanism of tumorigenesis, progression and drug resistance. Tumors can protect themselves from the clearance of the immune system by directly or indirectly inhibiting the signaling of T cells. Immune checkpoint therapy (immuno checkpoint therapy) for tumors is a therapeutic approach to enhance anti-tumor immune responses by modulating T cell activity through a series of pathways, such as co-inhibitory or co-stimulatory signals.

PD-1 binds to 2 ligands (family B7 members PD-L1 and PD-L2). In mice, PD-L1 is widely expressed on various organs such as heart, lung, thymus, spleen, kidney and the like, and almost all mouse tumor cell lines, and is highly expressed on various human tumor cells; while the expression of PD-L2 is more restricted, mainly dendritic cells and a few tumor cell lines. Therefore, PD-1 and its ligand PD-L1 become the target of anti-tumor immunotherapy.

Inhibitors of programmed death protein-1 (PD-1) and its ligand (PD-L1) are the main drugs of immune checkpoint therapy, and the breadth, depth and persistence of the response are rare, and the inhibitors are the hot spots of tumor immunotherapy research in recent years. Currently marketed nivolumab (nivolumab) and parbolizumab (pembrolizumab) belong to PD-1 inhibitors, and are mainly used for treating malignant tumors such as melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, Hodgkin lymphoma, urothelial cancer, microsatellite high-instability cancer and the like.

PD-1 inhibitors (anti-PD-1 antibodies) are a class of drugs that block PD-1, activate the immune system to attack tumors, and are used to treat certain types of cancer.

Different antibody drugs need different solvent environments to maintain the stability in the processes of freezing, thawing, storage, transportation and the like, and even the antibody drugs with the same target point need different preparation formulas due to different amino acid sequences and post-translational modifications of the antibody.

Disclosure of Invention

The present invention provides a pharmaceutical formulation comprising a protein, and more particularly, a stable pharmaceutical formulation comprising an anti-PD-1 antibody and antigen-binding fragments thereof.

The invention provides a protein-containing pharmaceutical preparation, which is characterized by comprising an effective dose of an anti-PD-1 antibody or an antigen-binding fragment thereof, a buffer solution, a stabilizer and a surfactant, wherein the anti-PD-1 antibody or the antigen-binding fragment thereof has the amino acid sequence shown in SEQ ID NO:1 (heavy chain CDR1), SEQ ID NO: 2 (heavy chain CDR2), SEQ ID NO: 3 (heavy chain CDR3) and a light chain variable region comprising SEQ ID NO: 4 (light chain CDR1), SEQ ID NO: 5 (light chain CDR2) and SEQ ID NO: 6 (light chain CDR 3).

The protein-containing pharmaceutical preparation as described above, wherein the effective dose of the anti-PD-1 antibody has the amino acid sequence of SEQ ID NO:8, having the amino acid sequence of the heavy chain shown in SEQ ID NO:10, or a light chain amino acid sequence set forth in seq id no.

The protein-containing pharmaceutical preparation is characterized by comprising 1-20 g/L of an effective dose of the anti-PD-1 antibody or the antigen-binding fragment thereof.

The pharmaceutical preparation containing the protein, which is characterized by containing an effective dose of 10g/L of the anti-PD-1 antibody.

The pharmaceutical preparation containing the protein is characterized in that the buffer solution is a histidine buffer solution with the concentration of 0-20 mM/L.

The pharmaceutical preparation containing a protein as described above, wherein the buffer is a 20mM/L histidine-HCl buffer.

The pharmaceutical preparation containing protein is characterized in that the stabilizer is 0-80 g/L of trehalose.

The pharmaceutical preparation containing protein as described above, wherein the stabilizer is trehalose at 40 g/L.

The pharmaceutical preparation containing the protein is characterized in that the surfactant is 0-2 g/L of polysorbate 20.

The pharmaceutical preparation containing a protein as described above, wherein the surfactant is polysorbate 20 at 0.5 g/L.

The pharmaceutical preparation containing the protein is characterized in that the pH value of the pharmaceutical preparation is 5.8-6.5.

The pharmaceutical preparation containing a protein as described above, wherein the pH of the pharmaceutical preparation is 6.0.

Use of a pharmaceutical formulation comprising a protein as described above in the manufacture of a medicament for modifying the immune response of a subject.

The use of a pharmaceutical formulation comprising a protein as described above in the manufacture of a medicament for inhibiting the growth of tumor cells.

The stability of a protein pharmaceutical formulation may vary depending on the nature of the protein it comprises, and the stable protein-containing pharmaceutical formulation of the present invention is also suitable for use in protein pharmaceutical formulations of anti-PD-1 antibodies and antigen-binding fragments thereof.

The anti-PD-1 antibody of the preferred embodiment has the amino acid sequence of SEQ ID NO:8, having the amino acid sequence of seq id NO:10, or a light chain amino acid sequence as described herein. After the anti-PD-1 antibody described in the preferred embodiment is expressed, the protein structure thereof is different from that of the existing anti-PD-1 antibody, and the anti-PD-1 antibody obtained by the preparation method described in the preferred embodiment has no glycosylation at the Fc terminal, and is more hydrophobic than the existing anti-PD-1 antibody. Thus, the stable protein formulation requirements of the anti-PD-1 antibodies described in the preferred embodiments of the invention are different from those of existing formulations.

The glycosylation in the antibody structure can cause adverse reaction and cause side effects such as hypersensitivity in the clinical medication process. In order to reduce unnecessary side effects in the clinical medication process, glycosylation modification of the antibody is inevitable, and a stable pharmaceutical preparation for the antibody with the therapeutic effect after glycosylation modification is necessary. In a preferred embodiment of the present invention, a stable protein pharmaceutical formulation for an aglycosylated anti-PD-1 antibody is provided.

The anti-PD-1 antibody of the preferred embodiment has the amino acid sequence of SEQ ID NO:8, having the amino acid sequence of seq id NO:10, or a light chain amino acid sequence as described herein. The structure of the protein of the anti-PD-1 antibody in the preferred embodiment is different from that of the existing anti-PD-1 antibody after the anti-PD-1 antibody is expressed, and therefore, the requirements for a stable protein formulation of the anti-PD-1 antibody in the preferred embodiment of the present invention are different from those of the existing formulation. In a preferred embodiment, the Fc terminal of the anti-PD-1 antibody has no glycosylation and has stronger hydrophobicity than the existing anti-PD-1 antibody.

Glycosylation in the antibody can cause adverse reaction and cause hypersensitivity in the clinical medication process. Therefore, it is necessary to provide a stable pharmaceutical formulation for glycosylation engineered antibodies. In a preferred embodiment of the present invention, a stable protein formulation for an anti-PD-1 antibody free of glycosylation is provided.

The invention confirms possible preparation prescription conditions (auxiliary material addition, pH and the like) through multiple times of multi-parameter condition screening, and finally obtains the optimal preparation formula of the anti-PD-1 antibody. In a preferred embodiment, the pharmaceutical formulation, preferably the stable formulation, containing the protein anti-PD-1 antibody having a therapeutic effect consists of 10g/L of the protein anti-PD-1 antibody having a therapeutic effect, 20mM/L of histidine-HCl buffer, 40g/L of trehalose, 0.5g/L of polysorbate 20, and pH 6.0.

The formulation screening of the preparation depends on various evaluation means for comprehensive evaluation, and the used evaluation means comprises the following steps: freeze-thaw test, influence factors (illumination, high temperature) and the like, and the detection method used for evaluation comprises the following steps: SEC/IEX/CE/LC-MSSubunit/particle size distribution/DSC analysis, and the like, through comprehensive evaluation and balancing of each index parameter, the components and conditions of the formula and the amount of each component are finally confirmed, and meanwhile, a basis is provided for the range of each condition.

Abbreviations involved in the present invention have the following meanings:

R-CE-SDS: reduce Capillary Electrophoresis, reducing the Capillary ElectroPhoresis purity; NR-CE-SDS: Non-Reduce Capillary ElectroPhoresis purity; NR/R-CE-chip-SDS: capillary Electrophoresis, non-reducing/reducing Capillary Electrophoresis; SEC-UPLC: Size-ExclusionChromatograPhy, Size exclusion liquid chromatography aggregation; IEX-HPLC/UPLC: ion exchange chromatography, charge heteroplasmon distribution; IEX: ion Exchange Chromatography, Ion Exchange liquid Chromatography; HIC-HPLC/UPLC: hydrophic Interaction-high Performance Liquid ChromatograPhy/Ultra Performance Liquid ChromatograPhy; LC-MS Subunit: liquid Chromatography-Mass Spectrum Subunit, Liquid phase-Mass Spectrometry; DSC: differential Scanning calirometer, thermal analysis; UFT-NC: ultrafiltration, Ultrafiltration concentrate containing the target protein; UFT-EB: ultrafiltration exchange buffer, Ultrafiltration displacement buffer; TM value: melt Temperature.

C: cycle, Cycle; h: hour, hours; d: day, Day; m: month, Month; UV: UV Stability Test/UVExposed Sample, ultraviolet Test, total illumination not less than 200W/hr cm 2; l1: light Stability Test/Photostability Exposed Sample, wherein the total illumination is not less than 1.2-106 h in the illumination Test; l2: DarkControl, protected from light; p1: upper Position; p2: horizontal Position; s1: ShakingStress, shock; s3: saccharoification Test, Saccharification Test; o2: oxidation Test, Oxidation Test; t2: 2-8 ℃; t3: 25 +/-2 ℃ and 75% +/-5% humidity; t4: 40 +/-2 ℃ and 75% +/-5% humidity; t5: freezing and thawing at a temperature of below-18 ℃ to 25 +/-2 ℃; t7: freezing and thawing at a temperature of below-70 ℃ to 25 +/-2 ℃; t8: room temperature; t10: 50 ℃ plus or minus 2 ℃ and humidity of 75 percent plus or minus 5 percent.

Drawings

FIG. 1 shows a mass spectrum of a light chain of an anti-PD-1 antibody prepared in example 1 in FIG. 1;

FIG. 2 shows a mass spectrum of the heavy chain of the anti-PD-1 antibody prepared in example 1 in FIG. 2;

FIG. 3 shows a mass spectrum of a heavy chain of a conventional anti-PD-1 antibody Opdivo in FIG. 3;

FIG. 4 shows a mass spectrum of the heavy chain of Keytruda, a conventional anti-PD-1 antibody, as shown in FIG. 4.

Detailed Description

Example 1: preparation of anti-PD-1 antibody

Artificially synthesizing a nucleotide sequence of a recombinant anti-PD-1 antibody, carrying out enzyme digestion on the nucleotide sequence, connecting the nucleotide sequence with a plasmid pcDNA3.1(+) (Invitrogen company), constructing an eukaryotic expression vector containing a light chain and a heavy chain of the anti-PD-1 antibody, wherein SEQ ID NO 7 and SEQ ID NO 8 respectively show a heavy chain nucleotide and an amino acid sequence of the anti-PD-1 antibody, SEQ ID NO 9 and SEQ ID NO 10 respectively show a light chain nucleotide and an amino acid sequence of the anti-PD-1 antibody, and inoculating 3 × 10 to a 3.5cm tissue culture dish⁵CHO-K1 cells (ATCC CRL-9618) were transfected when cultured to 90% -95% confluence.

And (3) carrying out amplification culture on the high-expression clone obtained by screening by using a serum-free culture medium: and (2) aiming at serum-free culture, repeated comparison and optimization are carried out, a culture medium and a supplement culture medium suitable for the anti-PD-1 antibody are finally determined, the expression quantity of the engineering cells in the optimized culture medium is more than 30pg/cell. And further separating and purifying to obtain the anti-PD-1 antibody, wherein compared with the existing anti-PD-1 antibody, the Fc end of the anti-PD-1 antibody obtained by the preparation method has no glycosylation site and does not contain glycosylation modification.

The anti-PD-1 antibody obtained by the preparation method is subjected to mass spectrum detection, wherein a light chain mass spectrum of the anti-PD-1 antibody is shown in figure 1, and a heavy chain mass spectrum of the anti-PD-1 antibody is shown in figure 2. The Opdivo heavy chain mass spectrum of the existing anti-PD-1 antibody is shown in FIG. 3, and the Keytruda heavy chain mass spectrum is shown in FIG. 4.

As can be seen from mass spectrum analysis, the anti-PD-1 antibody obtained by the preparation method has no glycosylation modification detected in the heavy chain mass spectrum of the antibody obtained by the invention compared with the prior anti-PD-1 antibody Opdivo and Keytruda. The anti-PD-1 antibody heavy chain mass spectrogram provided by the invention has no glycosyl group detected in a picture 2, an Opdivo heavy chain mass spectrogram has obvious G0F glycosylation modification in a picture 3, and Keytruda heavy chain mass spectrogram has the same G0F glycosylation modification detected in a picture 4. (see fig. 2, 3, 4).

Example 2: preparation of samples

The anti-PD-1 antibody obtained in example 1 is prepared into different formulations according to different components and different proportions, and a sample of the formulation is subjected to detection of a preparation stability related experiment.

Preparing 360g/L of trehalose dihydrate mother liquor, 180g/L of mannitol mother liquor and 50g/L of polysorbate 20 mother liquor. Sample preparations for different formulation formulations were performed for different pH, protein concentration with therapeutic effect, trehalose concentration, mannitol concentration, polysorbate 20(PS20) concentration according to the formulation method in table 1.

UFT-NC anti-PD-1 antibody samples were taken 50mg, 100mg to 50ml in a centrifuge tube, dissolved using 20mM/L histidine-hcl buffer, polysorbate 20: 0.025g and 2g of mannitol, and the volume is fixed to 50ml, so that the final concentration of each component is as follows: therapeutic protein anti-PD-1 antibody concentrations were 10mg/ml, 20mg/ml, histidine buffer 20mM, polysorbate 20: 0.5g/L, 1g/L, 2g/L and 40g/L of mannitol.

The sample preparation is divided into 160 parts according to different preparation formula compositions, and detailed sample preparation formula components and the proportion thereof are shown in table 1. Wherein UFT-NC is: an ultrafiltration concentrate containing an anti-PD-1 antibody; UFT-EB is: 20mM histidine-hydrochloric acid buffer.

TABLE 1 sample prescription screening condition configuration Table

Example 3: formulation stability test-repeated freeze-thaw test

After the sample grouping of the embodiment 2, the repeated freeze-thaw tests of 1 cycle, 2 cycles, 3 cycles and 8 cycles are respectively carried out on the No. 1 to No. 160 samples, and the conditions of the repeated freeze-thaw tests are as follows: t5: repeated freeze thawing at-18 deg.c to 25 deg.c +/-2 deg.c.

After repeated freeze thawing, the sample solution is subjected to sample stability detection, and after repeated freeze thawing tests, SEC-UPLC detection is performed, and the detection results are shown in Table 2.

TABLE 2 Freeze-thaw test SEC-UPLC detection results

According to the test results shown in Table 2, the sample containing 40g/L mannitol has a significant increase in the area of the polymer peak with the increase of the number of freeze thawing, and therefore, in a stable pharmaceutical preparation of an anti-PD-1 antibody, the presence of mannitol in the adjuvant needs to be avoided.

After the mannitol-containing test sample is removed, the other 128 samples are subjected to 8-cycle repeated freeze-thaw tests, and the results of SEC-UPLC detection show that the protein aggregates are not significantly increased in the mannitol-removed other samples (the results are shown in Table 2).

Thus, mannitol cannot be present in a stable formulation containing an effective dose of anti-PD-1 antibody.

Example 4: formulation stability test-high temperature, light test

After screening in example 3, 32 samples containing mannitol adjuvant were removed, and 128 samples remained. The remaining 128 groups of samples were subjected to high temperature, light tests under T4P 1: the temperature is 40 ℃, the humidity is 60 percent, and the device is placed upright; the lighting conditions were T2L1P 2: the temperature is 2-8 ℃, the humidity is 60-75%, and the lamp is horizontally placed under illumination.

The sample is placed for 0-10 days after high temperature and illumination, and is sampled after high temperature illumination on the 5 th day and the 10 th day respectively, the parameters of the polymer, the acid variant, the alkaline variant and the like in each sample are detected, and the detection results are shown in the table 3(5 days) and the table 4(10) after SEC/CE-chip-SDS/IEX detection.

TABLE 3 SEC/CE-chip-SDS/IEX test results after the samples were left for 5 days at high temperature and under illumination

The samples were allowed to stand at high temperature and under illumination for 5 days, and the samples were sampled for SEC/CE-chip-SDS/IEX detection, and the detection results are shown in Table 3.

According to the analysis of the detection results in Table 3, the samples were allowed to stand for 5 days under the conditions of high temperature and light influence, and the results of SEC/CE-chip-SDS/IEX purity detection and R-CE-chip-SDS detection of small molecule fragments showed that the purity, small molecule fragments and acidic variants of the anti-PD-1 antibody with therapeutic effect in the formulation of 8 sample preparations of 48, 49, 53, 54, 88, 89, 93 and 94 were less changed than those of other samples.

After 5 days of high temperature and light test, 8 samples of 48, 49, 53, 54, 88, 89, 93 and 94 are screened out to continue the high temperature and light test, the samples are placed to the 10 th day, the samples are sampled to detect the parameters of the polymer, the acid variant, the alkaline variant and the like in each sample, and the detection result of SEC/CE-chip-SDS/IEX is shown in Table 4.

TABLE 4 SEC/CE-chip-SDS/IEX test results after the samples were left for 10 days at high temperature and under illumination

Through detection of high-temperature and illumination tests for 0-10 days and analysis of test results, the optimal combined formula for screening the pharmaceutical preparation containing the anti-PD-1 antibody is as follows: the concentration of the protein anti-PD-1 antibody with the therapeutic effect is 10g/L, the pH value is 6.0-6.2, the trehalose is 40-80 g/L, and the polysorbate 20 is 0.5-1 g/L.

Example 5: optimization of pharmaceutical formulation containing anti-PD-1 antibody

According to the test results of the above examples, conditions such as pH, trehalose dihydrate concentration, and polysorbate 20 concentration in the formulation were further refined and adjusted.

Preparing 360g/L of trehalose dihydrate mother liquor, 180g/L of mannitol mother liquor and 50g/L of polysorbate 20 mother liquor. Sample preparations for different formulation formulations were performed for different pH, protein concentration with therapeutic effect, trehalose concentration, mannitol concentration, polysorbate 20(PS20) concentration according to the configuration in table 5.

The sample preparation method is shown in table 5.

TABLE 5 optimal configuration table of sample preparation formula

Samples containing an effective dose of a therapeutically effective protein anti-PD-1 antibody concentration of 10g/L were grouped: the pH value is 6.0, 6.1 and 6.2, the trehalose is 40g/L, 60g/L and 80g/L, the polysorbate 20 is 0.5g/L and 1g/L, the total number is 18 groups, and the serial number is 161-178.

The samples 161-178 are tested by high temperature and illumination for 0-10 days, and are sampled on the 5 th day and the 10 th day respectively, and SEC/CE-chip-SDS/IEX detection is carried out on the content of protein anti-PD-1 antibody with treatment effect, the content of small molecular protein, the content of polymer, acid variant, alkaline variant and the like, and the detection results are shown in tables 6 and 7.

Table 6 shows the results of high-temperature SEC/CE-chip-SDS/IEX detection for 5 days and 10 days, and Table 7 shows the results of illumination SEC/CE-chip-SDS/IEX detection for 5 days and 10 days.

TABLE 6 high temperature SEC/CE-chip-SDS/IEX test results

TABLE 7 illumination SEC/CE-chip-SDS/IEX test results

After the above tests, the monomer peak area, acidic variant, basic variant, purity and other parameters were analyzed comprehensively, and 5 conditions 161, 162, 164, 165 and 167 were screened better, and the 5 samples 161, 162, 164, 165 and 167 were subjected to DSC thermal analysis, and the thermal stability of the protein anti-PD-1 antibody having therapeutic effect under the preparation conditions was tested, and the results are shown in table 8.

TABLE 8 DSC thermal stability analysis

After the thermal stability detection analysis, samples under the 5 conditions of 161, 162, 164, 165 and 167 are obtained by screening and have better thermal stability.

After a series of screening, the optimal preparation formula is determined as follows: 10g/L of protein anti-PD-1 antibody with therapeutic effect, 20mM/L histidine-hydrochloric acid buffer solution, 40g/L of trehalose, 0.5g/L of polysorbate 20 and 6.0 of pH value.

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50 55 60

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

65 70 75 80

Pro Val Glu Ala Asp Asp Thr Ala Asn Tyr Phe Cys Gln Gln Ser Gln

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

Claims

1. A protein-containing pharmaceutical formulation comprising an effective dose of an anti-PD-1 antibody or antigen-binding fragment thereof, a buffer, a stabilizer, and a surfactant, wherein the anti-PD-1 antibody or antigen-binding fragment thereof has an amino acid sequence of SEQ ID NO:1 (heavy chain CDR1), SEQ ID NO: 2 (heavy chain CDR2), SEQ ID NO: 3 (heavy chain CDR3) and a light chain variable region comprising SEQ ID NO: 4 (light chain CDR1), SEQ ID NO: 5 (light chain CDR2) and SEQ ID NO: 6 (light chain CDR 3).

2. The protein-containing pharmaceutical formulation according to claim 1, wherein the effective dose of the anti-PD-1 antibody has the amino acid sequence of SEQ ID NO:8, having the amino acid sequence of the heavy chain shown in SEQ ID NO:10, or a light chain amino acid sequence set forth in seq id no.

3. The protein-containing pharmaceutical preparation according to claim 1, wherein the anti-PD-1 antibody or an antigen-binding fragment thereof is contained in the pharmaceutical preparation at an effective dose of 1 to 20 g/L.

4. The protein-containing pharmaceutical formulation according to claims 2 and 3, wherein the anti-PD-1 antibody is contained in the pharmaceutical formulation at an effective dose of 10 g/L.

5. The protein-containing pharmaceutical formulation according to claim 1, wherein the buffer is a histidine buffer of 0 to 20 mM/L.

6. The protein-containing pharmaceutical formulation according to claim 5, wherein the buffer is 20mM/L histidine-HCl buffer.

7. The protein-containing pharmaceutical preparation according to claim 1, wherein the stabilizer is trehalose at a concentration of 0 to 80 g/L.

8. The protein-containing pharmaceutical formulation according to claim 7, wherein the stabilizer is trehalose at 40 g/L.

9. The protein-containing pharmaceutical preparation according to claim 1, wherein the surfactant is polysorbate 20 at 0-2 g/L.

10. The protein-containing pharmaceutical formulation according to claim 9, wherein the surfactant is polysorbate 20 at 0.5 g/L.

11. The protein-containing pharmaceutical formulation according to claim 1, wherein the pharmaceutical formulation has a pH of 5.8 to 6.5.

12. The protein-containing pharmaceutical formulation according to claim 11, wherein the pharmaceutical formulation has a pH of 6.0.

13. Use of a protein-containing pharmaceutical formulation according to claims 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12 in the manufacture of a medicament for modifying an immune response in a subject.

14. Use of a protein-containing pharmaceutical preparation according to claims 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12 for the preparation of a medicament for inhibiting tumor cell growth.

15. Use of a protein-containing pharmaceutical formulation according to claim 4 in the manufacture of a medicament for modifying the immune response of a subject.

16. Use of a protein-containing pharmaceutical formulation according to claim 4 in the manufacture of a medicament for inhibiting the growth of tumor cells.