CN116474090A

CN116474090A - Pharmaceutical composition

Info

Publication number: CN116474090A
Application number: CN202310058933.5A
Authority: CN
Inventors: 宫皖晴; 任彤; 李纲
Original assignee: Maiwei Shanghai Biotechnology Co ltd
Current assignee: Maiwei Shanghai Biotechnology Co ltd
Priority date: 2022-01-24
Filing date: 2023-01-18
Publication date: 2023-07-25

Abstract

The invention discloses a pharmaceutical composition, which comprises the following components: 30-70mg/ml antibody; 15-25mM acidic buffer; 5-10% (w/v) stabilizer; and 0.02-0.1% (w/v) surfactant. The pharmaceutical composition has simple formula, stable protein system and convenient mass production, storage and transportation. The pharmaceutical composition of the embodiment of the invention is beneficial to enhancing the safety and effectiveness of antibody molecules.

Description

Pharmaceutical composition

Technical Field

The invention relates to the field of biological medicine preparation research, in particular to a novel composition of an artificially designed antibody, and particularly relates to a preparation method of a water needle composition of a humanized anti-PD-L1 and TGF-beta double-functional single-domain antibody-ligand capture Fc fusion protein medicine. The invention also relates to therapeutic and diagnostic uses of the diabody composition, in particular in the treatment, prevention and/or diagnosis of PD-L1/TGF-beta related diseases, such as cancer.

Background overview

TGF-beta and PD-L1 are important inhibitory molecules in tumor microenvironment, double inhibition of the TGF-beta and the PD-L1 possibly leads to enhancement of anti-tumor activity, and a plurality of bifunctional antibody clinical researches taking TGF-beta/PD-L1 as targets have been carried out at home and abroad, so that the combined use of the TGF-beta and the PD-L1 is a very promising treatment strategy. .

The anti-PD-L1 and TGF-beta bifunctional single domain antibody-ligand capture Fc fusion protein is a biological macromolecule, has a complex structure, and can generate physical changes such as aggregation, denaturation, precipitation and the like and chemical changes such as isomerization, deamidation, oxidation and the like in the production and storage processes. These changes affect the safety and efficacy of the product, and no suitable stable formulation exists on the market, which can maintain the stability of the bifunctional single domain antibodies of PD-L1 and TGF-beta for a longer period of time. Thus, there is a need for a stable formulation that ensures that the antibodies have the desired biological activity for treatment prior to entry into the patient.

Disclosure of Invention

The invention aims to solve the problems of aggregation, denaturation, precipitation and the like in the storage and transportation process of bispecific antibody molecules in the prior art, and particularly solves the technical problems of safety and effectiveness of a bifunctional single domain antibody-ligand capture Fc fusion protein composition of PD-L1 and TGF-beta. To this end, one object of the present invention is to prepare stable compositions of PD-L1/TGF-beta diabodies.

The present invention has been completed based on the following work of the inventors:

bispecific antibodies represent an emerging therapeutic strategy that can recognize and bind two different antigens simultaneously to perform specific functions, such as specifically linking immune cells to tumor cells, enhancing their killing; simultaneously combines two antigens on tumor cells, blocks double signal paths and reduces tumor drug resistance. Moreover, the dual function strategy potentially circumvents the limitations of combination immunotherapy. Compared with monoclonal antibodies, the bispecific antibodies have higher sensitivity and specificity, and have wide development and application prospects in the development of antitumor drugs.

The buffer system and pH of the composition are one of the relevant factors affecting the stability of antibody protein drugs. For the preservation of the antibodies, the present invention selects a pharmaceutically acceptable buffer as the solvent for the antibodies, including but not limited to a citric acid/sodium citrate mixture, an acetic acid/acetate mixture, and a histidine/histidine hydrochloride mixture. The inventor prefers the buffer system to be a mixture of citric acid and sodium citrate, and the citric acid buffer system has strong buffer capacity and protection effect on protein, and the concentration of the buffer solution is 20mM, and the pH value is 6.0. In order to prevent the decrease in the pharmaceutical activity caused by the degradation of protein, the inventors set the protein concentration to 50mg/ml. To increase the stability of the antibody composition, in some embodiments saccharides are added as stabilizers, including but not limited to mannitol, trehalose, and sucrose, with sucrose being preferred at a concentration of 7% (w/v).

Thus, according to one aspect of an embodiment of the present invention, there is provided a pharmaceutical composition comprising:

30-70mg/ml antibody;

15-25mM acidic buffer;

5-10% (w/v) stabilizer; and

0.02-0.1% (w/v) surfactant.

The pharmaceutical composition provided by the embodiment of the invention has the advantages of simple formula, stable protein system, convenience for large-scale production, storage and transportation, effectively solves the problems that the antibody molecules are easy to generate physical changes such as aggregation, denaturation, precipitation and the like and chemical changes such as isomerization, deamidation, oxidation and the like in the production, storage and transportation processes, and obviously improves the safety and the effectiveness of the antibody molecules in the pharmaceutical composition.

In addition, the antibody or antigen-binding fragment according to the above embodiment of the present invention may have the following additional technical features:

the dosage form of the pharmaceutical composition according to the embodiments of the present invention is an injection, preferably a liquid injection. That is, the pharmaceutical composition is a water injection preparation of the antibody.

According to an embodiment of the invention, the pharmaceutical composition, the antibody is a single domain antibody. The single domain antibody has small molecular weight and good tissue penetrability, and for some solid tumors, the composition of the embodiment of the invention can better reach the deep layer of tumor tissues and achieve the treatment effect.

According to an embodiment of the invention, the antibody is directed against PD-L1 and/or TGF- β. The antibody composition of the embodiment of the invention can be a PD-L1 antibody preparation, a TGF-beta antibody preparation, an antibody preparation containing two antibodies of PD-L1 and TGF-beta, and a PD-L1/TGF-beta double antibody preparation, so that the problems that the anti-PD-L1 and TGF-beta double-function antibody molecules are easy to generate physical changes such as aggregation, denaturation and precipitation and chemical changes such as isomerization, deamidation and the like in the production, storage and transportation processes due to complex structures are effectively solved, and the safety and the effectiveness of the antibody molecules in the composition of the embodiment of the invention are remarkably improved.

According to an embodiment of the invention, the antibody is a fusion protein. The present example is directed to the development of pharmaceutical compositions, diagnosis and treatment of booster tumors, directed to bifunctional single domain antibody-ligand trap Fc fusion proteins against PD-L1 and TGF-beta.

According to an embodiment of the invention, the antibody against TGF- β is a TGF- βrii mutant having a mutation at one or more amino acid residue positions selected from the group consisting of: bit 6, bit 12, bit 20. Compared with wild type TGF-beta RII, TGF-beta RII mutant can bind TGF-beta, has less fragmentation and/or degradation when expressed recombinantly, is convenient for large-scale production, and is a more stable quality antibody/TGF-beta RII bifunctional protein. See patent 202010721371.4 for details of such antibodies against TGF- β.

According to an embodiment of the invention, the TGF- βrii mutant has one or several mutations relative to wild-type TGF- βrii selected from the group consisting of: Q6N, D12T, G20T. The mutant includes more than two functional fragments, and the functional fragments also include antibodies or antigen binding portions thereof, receptors or ligand binding portions thereof, cytokines or fragments thereof, cytotoxins or variants thereof, markers or tracers, and the like, and are capable of binding TGF-beta 1, TGF-beta 2 and TGF-beta 3. According to some embodiments of the invention, the sequence of the TGF-beta RII mutant is shown in SEQ ID NO. 9 and the corresponding nucleotide sequence is shown in SEQ ID NO. 8.

According to an embodiment of the invention, the antibody CDR1-CDR3 directed against PD-L1 comprises an amino acid sequence having more than 75% identity to the amino acid sequence shown in SEQ ID NO 1-3, respectively.

Furthermore, according to an embodiment of the present invention, the amino acid sequence of the heavy chain variable region of the antibody against PD-L1 has an amino acid sequence having 75% or more identity with the amino acid sequence shown in SEQ ID NO. 4.

Specifically, modifications, deletions, insertions or substitutions of one or more amino acids may be made to the CDRs to obtain related mutants of the CDRs which are amino acid sequences having 75, 80, 85, 90, 95, 98, or more than 99% identity to the CDRs shown in SEQ ID NOS.1-3. CDR1 is mutated and according to some embodiments of the invention, the antibody CDR1 directed against PD-L1 comprises an amino acid sequence as shown in SEQ ID NO. 5. The amino acid sequence of the heavy chain variable region of the antibody aiming at PD-L1 has an amino acid sequence with more than 75 percent of identity with the amino acid sequence shown in SEQ ID NO. 6, and the corresponding nucleotide sequence is shown in SEQ ID NO. 7.

According to an embodiment of the invention, the amino acid sequence of the heavy chain variable region of the antibody against PD-L1 is shown in SEQ ID NO. 4. The PD-L1 antibody sequence is prepared by extracting PBMC from a human PD-L1 immune camel to construct a phage surface display VHH antibody library, screening and identifying to obtain an anti-human PD-L1 specific single variable domain antibody 2-2F2, and preparing a chimeric antibody chF2 and a humanized modified antibody hzF2 mutant on the basis. The affinity of the hzF mutant can reach or even exceed that of the starting single variable domain antibody 2-2F2, the binding of PD-1 and PD-L1 can be blocked in vitro, and the growth of tumors can be inhibited in vivo tests of tumor-bearing mice. See patent CN202010324761.8 for details of the antibodies against PD-L1.

According to an embodiment of the present invention, the acidic buffer is at least one of a citric acid/sodium citrate buffer, an acetic acid/acetate buffer and a histidine/histidine salt buffer, preferably a citric acid/sodium citrate buffer. In one embodiment of the invention, a citric acid/sodium citrate buffer is used; in one embodiment of the invention, an acetic acid/acetate buffer is used; in one embodiment of the invention, histidine/histidine salt buffer is employed. The buffer system of the composition is one of the key factors affecting the stability of antibody protein drugs, and the three buffers are helpful for capturing the stability of Fc fusion protein drugs by using bifunctional single domain antibody-ligand of anti-PD-L1 and TGF-beta, so that the tumor inhibition effect is improved, wherein the effect of the buffer solution of citric acid/sodium citrate is better.

According to an embodiment of the invention, the stabilizer is a saccharide. The main component of the antibody is protein, and protein stabilizing agent is often required to be added in order to keep the activity of the antibody.

According to an embodiment of the invention, the saccharide is at least one of mannitol, trehalose and sucrose, preferably sucrose. In one embodiment of the invention, mannitol is used as a stabilizer; in one embodiment of the invention, trehalose is employed as a stabilizer; in one embodiment of the present invention, sucrose is used as a stabilizer. Sucrose not only can be used as a protective agent and an excipient of the antibody, but also has the function of regulating the osmotic pressure molar concentration, thereby being beneficial to capturing the stability of Fc fusion protein medicines by the bifunctional single domain antibody-ligand of PD-L1 and TGF-beta and improving the tumor inhibiting effect.

According to an embodiment of the invention, the surfactant is polysorbate 80 (PS 80) or polysorbate 20.

According to an embodiment of the invention, the pH of the pharmaceutical composition is 5.0-6.5, preferably 5.7-6.3. The pH screening helps to better preserve the antibody composition and to better produce efficacy after entry into the human body. The pH of the pharmaceutical composition according to the embodiment of the present invention may be any value between 5.0, 5.1, 5.2, 5.3, etc., 5.0 and 6.5, preferably any value between 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, etc., 5.7 and 6.3.

According to an embodiment of the invention, the concentration of the antibody is 40-60mg/ml. The concentration of the pharmaceutical composition of the embodiment of the invention can be any concentration between 40 and 60mg/ml, and the proper concentration is favorable for the protein to play a role, and the PD-L1/TGF-beta double antibody has better stability under the concentration.

According to an embodiment of the invention, the concentration of the acidic buffer is 15-25mM. The buffer concentration of the pharmaceutical composition of the embodiment of the invention can be any concentration between 15 and 25mM, for example, 18, 20, 22, 24mM, etc., and the proper buffer concentration is helpful for maintaining the pH under which the PD-L1/TGF-beta diabody has better stability.

According to an embodiment of the invention, the concentration of the stabilizer is 6-8% (w/v). The stabilizer concentration of the pharmaceutical composition of the embodiment of the invention can be any concentration between 6 and 8% (w/v), such as 6.5%, 7% or 7.5%, and the like, and the proper stabilizer concentration is favorable for better maintenance stability of protein, and PD-L1/TGF-beta double antibody has better stability under the stabilizer concentration.

According to an embodiment of the invention, the concentration of the surfactant is 0.02-0.1% (w/v). The concentration of the surfactant of the pharmaceutical composition provided by the embodiment of the invention can be any concentration between 0.02 and 0.1% (w/v), and the proper concentration of the surfactant is favorable for maintaining the protein to exert stability, so that the PD-L1/TGF-beta diabody has better stability under the concentration of the surfactant.

According to an embodiment of the invention, the pharmaceutical composition is selected from one of the following combinations:

(1) 50mg/ml of the antibody, 20mM of the citric acid/sodium citrate buffer, 5% (w/v) of the mannitol, 0.02% (w/v) of the polysorbate 80, and pH 6.0;

(2) 50mg/ml of the antibody, 20mM of the citric acid/sodium citrate buffer, 3% (w/v) of the mannitol, 0.02% (w/v) of the polysorbate 80, and pH 6.0;

(3) 50mg/ml of the antibody, 20mM of the citric acid/sodium citrate buffer, 5% (w/v) of the trehalose, 0.02% (w/v) of the polysorbate 80, and pH 6.0;

(4) 50mg/ml of the antibody, 20mM of the citric acid/sodium citrate buffer, 5% (w/v) of the sucrose, 0.02% (w/v) of the polysorbate 80, and pH 6.0;

(5) 50mg/ml of the antibody, 20mM of the citric acid/sodium citrate buffer, 7% (w/v) of the sucrose, 0.05% (w/v) of the polysorbate 80, and pH 6.0;

(6) 50mg/ml of the antibody, 20mM of the histidine/histidine salt buffer, 5% (w/v) of the mannitol, 0.02% (w/v) of the polysorbate 80, and pH 6.0;

(7) 50mg/ml of the antibody, 20mM of the histidine/histidine salt buffer, 5% (w/v) of the mannitol, 0.02% (w/v) of the polysorbate 80, and pH 6.5;

(8) 50mg/ml of the antibody, 20mM of the citric acid/sodium citrate buffer, 5% (w/v) of the mannitol, 0.02% (w/v) of the polysorbate 80, and pH 6.5;

(9) 50mg/ml of the antibody, the 20mM acetic acid/acetate buffer, 5% (w/v) mannitol, 0.02% (w/v) polysorbate 80, and pH5.5.

According to the embodiment of the invention, the inventor finds that the bi-functional single domain antibody-ligand capturing Fc fusion protein medicine stable composition for resisting PD-L1 and TGF-beta, which is obtained by the embodiment of the invention, consists of bi-specific protein, buffer solution, protective agent and surfactant, has simple and reasonable process, low cost and strong stability, particularly has remarkable effect on preventing and treating tumors, and has low side effect.

For a better understanding of the invention, some terms are first defined. Other definitions are set forth throughout the detailed description.

The term "specific" refers to determining the presence or absence of a protein in a heterogeneous population of the protein and/or other organism. Thus, under the specified conditions, a particular ligand/antigen binds to a particular receptor/antibody and does not bind in significant amounts to other proteins present in the sample.

The term "antibody" herein is intended to include full length antibodies and any antigen-binding fragment (i.e., antigen-binding portion) or single chain thereof. Full length antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains, the heavy and light chains being linked by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (VL) and a light chain constant region. The light chain constant region is composed of one domain CL. VH and VL regions can also be divided into hypervariable regions called Complementarity Determining Regions (CDRs) which are separated by more conserved Framework Regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains comprise binding domains that interact with antigens. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various immune system cells (e.g., effector cells) and the first component of the traditional complement system (C1 q).

The term "bispecific antibody" (bispecific antibodies), an antibody structure that binds to different epitopes on the same or different antigens. Thus, bispecific antibodies are able to bridge two different molecules, serving to recruit effector molecules, effector cells, viruses, and drug carrier systems to target structures. The characteristic that bispecific antibodies can simultaneously recognize two different molecules (receptors and/or ligands) improves the selectivity and functional affinity of the antibodies.

The term "monoclonal antibody" or "mab" or "monoclonal antibody composition" refers to a preparation of antibody molecules of single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

The term "antigen-binding fragment" of an antibody (or simply an antibody portion) as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been demonstrated that the antigen binding function of an antibody can be performed by fragments of full length antibodies. Examples of binding fragments contained in the "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH 1; (ii) A F (ab') 2 fragment, a bivalent fragment comprising two Fab fragments disulfide-bridged at the hinge region; (iii) an Fd fragment consisting of VH and CH 1; (iv) Fv fragments consisting of single arm VL and VH of the antibody; (v) dAb fragments consisting of VH (Ward et al., (1989) Nature 341:544-546); (vi) an isolated Complementarity Determining Region (CDR); and (vii) nanobodies, a heavy chain variable region comprising a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined by recombinant methods via a synthetic linker that makes both single protein chains, in which the VL and VH regions pair to form a monovalent molecule (known as a single chain Fc (scFv); see, e.g., bird et al, (1988) Science 242:423-426;and Huston et al, (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883). These single chain antibodies are also intended to be included in the term meaning. These antibody fragments can be obtained by common techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as the whole antibody.

Antigen binding fragments of the invention include those capable of specifically binding to an antigen. Examples of antibody binding fragments include, for example, but are not limited to, fab ', F (ab') 2, fv fragments, single chain Fv (scFv) fragments, and single domain fragments.

The Fab fragment contains the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments in the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab 'fragments are generated by cleavage of disulfide bonds at the hinge cysteines of the F (ab') 2 pepsin digestion products. Additional chemical coupling of antibody fragments is known to those of ordinary skill in the art. Fab and F (ab') 2 fragments lack the fragment crystallizable (Fc) region of intact antibodies, clear more rapidly from the circulation of animals, and may have less non-specific tissue binding than intact antibodies (see, e.g., wahl et al, 1983, j. Nucleic. Med. 24:316).

As generally understood in the art, an "Fc" region is a fragment of an antibody that does not comprise an antigen-specific binding region, a crystallizable constant region. In IgG, igA and IgD antibody isotypes, the Fc region consists of two identical protein fragments, the second and third constant domains (CH 2 and CH3 domains, respectively) derived from the two heavy chains of the antibody. IgM and IgE Fc regions contain three heavy chain constant domains (CH 2, CH3 and CH4 domains) in each polypeptide chain.

"Single domain fragment" sdA (single domain antibody) consists of a single VH or VL domain for which the antigen shows sufficient affinity. In a specific embodiment, the single domain fragment is camelized (see, e.g., riechmann,1999,Journal of Immunological Methods 231:25-38).

The term "immunoglobulin sequence" is used as a generic term and includes full-size antibodies, individual chains thereof, and all parts, domains or fragments thereof (including but not limited to antigen-binding domains or fragments, such as VHH domains or VH/VL domains, respectively). Furthermore, the term "sequence" (e.g. in terms of "immunoglobulin sequence", "antibody sequence", "variable domain sequence", "VHH sequence" or "protein sequence") as used herein is generally understood to include both the relevant amino acid sequence as well as the nucleic acid or nucleotide sequence encoding it, unless the context requires a more restrictive interpretation.

The terms "vector" and "nucleic acid construct" refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, in which additional DNA segments may be ligated into the viral genome. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and an episomal mammalian vector). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA technology are typically in the form of plasmids. However, other forms of expression vectors are also included, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

The specific embodiment is as follows:

the scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not noted in the examples and are carried out according to the techniques or conditions described in the literature in the art (for example, refer to J. Sam Brookfield et al, code Huang Peitang et al, molecular cloning Experimental guidelines, third edition, scientific Press) or according to the product specifications. The reagents or apparatus used are not manufacturer specific and are conventional products commercially available, for example, from Sigma company.

While embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the invention, the scope of which is defined by the claims and their equivalents.

Example 1 design and expression of anti-human PD-L1 nanobody/TGF-beta RII mutant fusion proteins

The humanized anti-human PD-L1 nanometer antibody hzF coding nucleotide sequence (source patent application number: CN 202010324761.8) (SEQ ID NO. 7) is connected with the C end of the humanized anti-human PD-L1 nanometer antibody hzF coding nucleotide sequence (SEQ ID NO. 8) through a connecting peptide coding nucleotide sequence (SEQ ID NO. 10) to obtain a PD-L1 nanometer antibody-TGF-beta RIIM coding nucleotide sequence hzF-TGF-beta RIIM (SEQ ID NO. 11), the amino acid sequence is shown as SEQ ID NO.12, and the humanized anti-human PD-L1 nanometer antibody hzF coding nucleotide sequence is cloned into a stable expression vector containing a Glutamine Synthetase (GS) screening gene through an enzyme cutting site, so that a eukaryotic expression vector hzF m 9-TGF-beta RIIM for stable transfection is constructed, and the plasmid containing a large amount of expressed hzF 2-TGF-beta RIIM fusion protein is transferred into escherichia coli for amplification and separation. The prepared plasmid, electrically transfected (Nucleofector IIb, lonza) suspension cultured CHO-K1 cells, through MSX pressurized screening, obtain cells hzF 2-TGF-beta RIIM stably expressing hzF-TGF-beta RIIM2 fusion protein. The cells are cultured, expressed and purified to obtain the hzF 2-TGF-beta RIIM mutant fusion protein hzF 2-TGF-beta RIIM.

Example 2: buffer system and prescription pH screening

The fusion protein hzF 2-TGF-beta RIIM obtained in example 1 is simply called a bispecific protein, the theoretical isoelectric point of the bispecific protein is 5.69, and degradation of the protein occurs due to poor protein stability under the condition of pH7.0 in the protein purification process, so that the pH range of the liquid composition of the bispecific protein examined in the example is 5.0-6.5. Based on this pH buffer range, several buffer systems, i.e., histidine buffer system, citric acid buffer system, acetic acid buffer system, were selected for examination in this example at a concentration of 20mM. The saccharide protectant comprises mannitol, sorbitol, sucrose, and trehalose, and the concentration of the composition is not more than 10%. The buffer system composition formulation was initially screened as shown in tables 1-3 below.

TABLE 1 His buffer System composition formulation prescreening (DSF)

Table 2 citric acid buffer System composition formulation prescreening (DSF)

TABLE 3 formulation prescreening for acetic acid buffer systems (DSF)

The DSF detection Tm reflects the conformational stability of the protein in the formulation of the composition, with higher Tm being better. As can be seen from tables 1, 2 and 3, the relative Tm of the bispecific protein was higher in 20mM citrate buffer system and 20mM histidine buffer system, followed by further screening with 20mM citrate buffer system and 20mM histidine buffer system. The addition of different saccharide components and NaCl components has no obvious influence on the Tm value of the composition, and the subsequent screening is needed. Under the histidine system, the Tm value of pH5.5 is relatively low, the main buffering pH range of the histidine buffer system is near pH6.0, and the histidine buffer system with pH 6.0-6.5 is continuously screened. The bispecific protein isoelectric point range is excluded under a citric acid buffer system, and the citric acid buffer system with pH of 5.5-6.0 is preferred for continuous screening.

Example 3: buffer System stability test

Based on the results of the initial screening of the compositions using DSF, 6MW5311 protein was formulated at target concentrations (50 mg/ml) in 20mM histidine and 20mM citric acid buffer systems of different pH values to be investigated, and the stability of the composition samples under simultaneous conditions of high temperature (40.+ -. 2 ℃), shaking (200 rpm) and light irradiation (4500 lx) was examined, and the formulation to be screened is shown in Table 4. Antibody protein purity assays included SEC, rSDS-PAGE and nrSDS-PAGE, and the results are shown in Table 5.

TABLE 4 buffer System and pH composition screening formulations

Table 5 buffer systems and pH composition screening purity analysis results

Through a 3-day forced degradation study, the protein purity analysis under the buffer conditions of different compositions can find that the bispecific protein has better stability in a citric acid buffer, and the protein stability under the condition of pH6.0 is better than that of pH5.5. Thus citric acid was chosen as a buffer for the bispecific protein composition and subsequent liquid composition development was performed at ph 6.0.

Example 4: saccharide protectant screening

After finishing the screening of the buffer system and pH of the bispecific protein composition, further screening the saccharide protecting agent comprising mannitol, sucrose and trehalose, preparing the bispecific protein with target concentration (50 mg/mL) in a 20mM citric acid buffer system containing different saccharide protecting agents and having pH of 6.0 to be inspected, inspecting the stability of the composition sample under the conditions of high temperature (40+/-2 ℃), oscillation (200 rpm) and illumination (4500 lx) and freezing and thawing 5 times (-70+/-15 ℃) for 12 hours or more, taking out the room temperature (10-30 ℃) and standing for not less than 1 hour to complete thawing, wherein the stability is 1 time under the freezing and thawing conditions. The formulations to be screened are shown in Table 6, and the purity of the proteins SEC and rCE-SDS is examined, and the results are shown in Table 7.

Table 6 saccharide protectant screening formulations

TABLE 7 results of analysis of saccharide protectant screening protein purity

As can be seen from the detection result, the protection effect of sucrose on the bispecific protein is better in a 20mM citric acid buffer system with pH of 6.0. The antibody protein is placed for 12 days under the conditions of high temperature, vibration and illumination and repeatedly frozen and thawed for 5 times, the purity of rCE-SDS and SEC in a prescription containing sucrose is less changed, and the sucrose is selected as a saccharide protecting agent of the bispecific protein liquid composition.

Example 5: naCl content and PS80 content screening

To ensure that the osmolality of the composition meets the isotonicity requirement (250 to 350 mOsm/kg), this example examines the stability of the composition at 30 to 40mM NaCl,0.02%, 0.05% and 0.1% PS80. Bispecific protein was filled at 0.8 mL/vial in 2mL penicillin vials and the stability of the samples was examined under high temperature (40±2 ℃), shaking (100 rpm) and light (4500 lx) conditions. The formulation to be screened is shown in Table 8. The protein SEC purity and appearance were focused on and the results are shown in tables 9 and 10.

Table 8 screening formulations for NaCl and PS80 content

TABLE 9 results of SEC analysis of screening proteins for NaCl and PS80 contents

Description: the sample failed for 10 days of oscillation for prescription No. 4, and the prescription was not examined any further.

TABLE 10 screening appearance results for NaCl content and PS80 content

From the above detection results, it can be found that the PS80 added in the range of 0.05% to 0.1% and the NaCl added or not, the bispecific protein showed good high temperature stability, and the protein SEC purity did not significantly decrease after 15 days of high temperature action. At a low PS80 content (0.02%), significant protein particles precipitated after 10 days of shaking of the composition, and foreign matter was visually unacceptable, whereas shaking was not affected at 0.05% and 0.1% PS80 content, and therefore, the PS80 content was selected to be 0.05% or higher. In case the stability of the composition is met, the PS80 content preferably meets the required lower concentration, i.e. 0.05%.

Example 6: other additive content screening

According to the light sensitivity characteristic of the bispecific protein, the addition of diethylenetriamine pentaacetic acid (DTPA) (metal ion complex) and methionine (Met, antioxidant) is tried based on the existing composition formula, and the usage amount is referred to the large molecular antibody like products already on the market. Bispecific proteins were formulated into the formulations to be screened and filled into 2mL penicillin bottles at 0.8 mL/bottle, and the effect on protein stability in different composition formulations under conditions of high temperature (40±2 ℃), shaking (100 rpm) and light (4500 lx) was examined. The formulation to be screened is shown in Table 11, and the results of the protein SEC purity change are shown in Table 12.

Table 11 other additive screening formulations

TABLE 12 results of SEC analysis of other additives screening proteins

From the above detection results, it can be found that, if MET and DTPA are added or not, the bispecific protein shows good high temperature stability, and the protein SEC purity is not significantly reduced after 15 days of high temperature action. The composition formulation of the examples of the present invention opts not to add MET and DTPA in cases where the stability of the composition is MET.

Meanwhile, the bispecific protein is found to be sensitive to illumination, the oxidation reaction caused by the illumination is presumed to accelerate the change of the protein purity, the illumination also causes the change of the color of the protein sample, the color change is more obvious along with the increase of the PS80 content, and the change of the protein purity caused by the illumination is needed to be taken as the investigation direction. The addition of NaCl mainly adjusts the osmotic molar concentration, and the influence of the addition or the non-addition of NaCl on illumination is still to be further confirmed.

Considering that the bispecific protein composition does not receive strong light directly for a long time in practical application, the present example continues to examine the color change condition within 10 days of illumination under the condition of adding components of NaCl, sucrose, MET and PS80, and the results are shown in table 13.

TABLE 13 development of bispecific protein composition additional Components (20 mM citric acid, pH 6.0)

From the above results, it was found that the addition of Met was beneficial for the formulation light stability of bispecific proteins, but the potential effect of Met on other qualities of proteins was temporarily unknown, irrespective of the addition of antioxidant Met in the formulation recipe. The results show that NaCl has a potential effect on the light stability of the formulation of the bispecific protein formulation, no NaCl is added as an osmolality regulator for the formulation, and the osmolality is adjusted by increasing the sucrose usage to 7%. Less auxiliary materials are added under the condition that the stability of the protein preparation is ensured as much as possible in the formula of the composition, and MET and NaCl are not added in the formula of the composition in the embodiment of the invention.

Example 6: prescription screening preliminary stability validation

The principle of composition development is that less auxiliary materials are added under the condition of ensuring the stability of the protein composition, and other auxiliary materials are not added according to the earlier research results.

A composition formulation containing 7% sucrose and 0.05% PS80 in 20mM citric acid, pH6.0 was selected for preliminary stability studies, protein concentration was selected at 50mg/mL, and the composition formulation to be confirmed is shown in Table 14.

Table 14 formulation of bispecific protein to be validated composition

The conditions of the composition formulation for confirming and examining the influencing factors include high temperature, oscillation, illumination and repeated freezing and thawing, and the detection indexes include insoluble particles, concentration (UV 280), protein purity (SEC, rCE-SDS) and binding activity (double targets), and the detection results are shown in tables 15-19.

TABLE 15 results of analysis of insoluble microparticles for compositions formulations to be validated for bispecific proteins

TABLE 16 results of analysis of the formulation concentration (UV 280) of the bispecific protein to be validated compositions

Table 17 results of SEC analysis of bispecific protein compositions to be validated

TABLE 18 results of rCE-SDS analysis of the compositions to be confirmed for bispecific proteins

TABLE 19 results of composition formulation binding Activity to be confirmed by bispecific protein (double target) analysis

In summary, the composition formulation containing 7% sucrose and 0.05% PS80 in 20mM citric acid, protein concentration 50mg/mL, was subjected to bispecific protein highlight irradiation for 5 days at pH6.0, and protein SEC purity was slightly reduced, so that the composition was required to be preserved in a dark place. Meanwhile, the purity of the protein SEC and rCE-SDS is found to be reduced when the protein SEC and rCE-SDS are placed at a high temperature (40+/-2 ℃) for 20 days, and the preservation temperature is strictly controlled in the actual application process of the finished product.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Claims

1. A pharmaceutical composition comprising:

30-70mg/ml antibody;

15-25mM acidic buffer;

5-10% (w/v) stabilizer; and

0.02-0.1% (w/v) surfactant.

2. The pharmaceutical composition of claim 1, wherein the antibody is directed against PD-L1 and/or TGF- β.

3. The pharmaceutical composition of claim 1, wherein the antibody to PD-L1 is a single domain antibody.

4. The pharmaceutical composition of claim 3, wherein the antibodies to PD-L1 and TGF- β are fusion proteins.

5. A pharmaceutical composition according to claim 3, wherein the antibody to TGF- β is a TGF- βrii mutant having a mutation at one or more amino acid residue positions selected from the group consisting of: bit 6, bit 12, bit 20.

6. The pharmaceutical composition of claim 6, wherein the TGF- βrii mutant has one or more mutations relative to wild-type TGF- βrii selected from the group consisting of: Q6N, D12T, G20T.

7. The pharmaceutical composition of claim 3, wherein the antibody CDR1-CDR3 directed against PD-L1 comprises an amino acid sequence having 75% or more identity to the amino acid sequence shown in SEQ ID NO. 1-3, respectively,

optionally, the amino acid sequence of the heavy chain variable region of the antibody against PD-L1 has an amino acid sequence having more than 75% identity to the amino acid sequence shown in SEQ ID NO. 4.

8. The pharmaceutical composition of claim 7, wherein the antibody CDR1 for PD-L1 comprises the amino acid sequence of SEQ ID NO. 5,

optionally, the amino acid sequence of the heavy chain variable region of the antibody against PD-L1 comprises the amino acid sequence shown in SEQ ID NO. 6.

9. The pharmaceutical composition of claim 1, wherein the acidic buffer is at least one of a citric acid/sodium citrate buffer, an acetic acid/acetate buffer, and a histidine/histidine salt buffer, preferably a citric acid/sodium citrate buffer,

optionally, the stabilizer is a saccharide,

optionally, the saccharide is at least one of mannitol, trehalose and sucrose, preferably sucrose,

optionally, the surfactant is polysorbate 80 or polysorbate 20,

optionally, the pharmaceutical composition has a pH of 5.0-6.5, preferably 5.7-6.3,

optionally, the concentration of the antibody is 40-60mg/ml,

optionally, the acidic buffer is at a concentration of 8-12mM,

optionally, the stabilizer is present at a concentration of 6-8% (w/v),

optionally, the surfactant concentration is 0.02-0.1% (w/v).

10. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is selected from one of the following combinations:

11. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is in the form of an injection, preferably a liquid injection.