Patent Description:
Lymphocytopoiesis and lymphocyte differentiation are regulated by cytokines. B lymphocyte stimulator (Blys, also known as B cell activating factor, BAFF) and a proliferation-inducing ligand (APRIL) are cytokines with an important regulatory effect on human immune response. They can promote the development and proliferation of B lymphocytes and increase the expression of immunoglobulin in the blood. BlyS and APRIL also have a key regulatory effect on the maturation of T lymphocytes, so they also have an important impact on cellular immunity.

Blys and APRIL regulate the immune response of lymphocytes through receptors on the surface of lymphocytes. They bind to cell membrane receptors, TACI (Transmenbrane Activator and CAML-interactor) and BCMA (B Cell Maturation Antigen). In addition, BLyS can also bind to another receptor, BAFF-R. B lymphocytes express TACI, BCMA and BAFF-R, and mature T lymphocytes express TACI. BlyS and APRIL regulate the activation, proliferation and development of lymphocytes through the signaling of these receptors, and produce an immune response. Furthermore, for lymphocyte tumors, BlyS and APRIL also have effects of promoting tumor cell division and inhibiting tumor cell apoptosis, thus accelerating the progress of tumors.

A number of studies have shown that the overexpression of BlyS and APRIL is one of the reasons for a variety of autoimmune diseases. These diseases include systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome and the like. Clinical studies have demonstrated that the concentration of BlyS is often positively correlated with the severity of autoimmune diseases. Therefore, inhibiting the production of BlyS and APRIL or reducing their concentration in the body becomes an effective way to treat autoimmune diseases. Meanwhile, since BlyS and APRIL can accelerate the process of B lymphocyte tumors, inhibition of BlyS and APRIL can also be used to treat B lymphocyte tumors, such as chronic lymphocytic leukemia, multiple myeloma, and B lymphocyte lymphoma.

Since TACI has a high affinity for BlyS and APRIL, soluble TACI (the extracellular part of TACI) is used to prevent the interaction between BlyS or APRIL and cell membrane receptors (TACI, BCMA and BAFF-R), so as to achieve the purpose of blocking the biological activity of BlyS and APRIL and treating autoimmune diseases or tumors. Multiple studies have shown that the fusion protein (TACI-Fc) with the extracellular part of TACI binding to IgG Fc fragment can effectively inhibit diseases related to BlyS and APRIL. For example, the clinical results of the TACI fusion protein Atacicept developed by ZymoGenetics and Merck Serono reveal that it has a therapeutic effect on SLE, rheumatoid arthritis, lymphoma and other diseases.

In addition, the patent <CIT> discloses a fusion protein consisting of truncated TACI and immunoglobulin Fc. The TACI portion of the fusion protein comprises the amino terminal region sequence starting from amino acid residue <NUM> in the extracellular region of TACI, the entire cysteine-rich region, and a partial sequence of the stalk region. The immunoglobulin Fc portion of the fusion protein comprises a hinge region, CH2 region and CH3 region. The TACI sequence and the Fc sequence are fused directly or via a linker sequence. Further, the TACI portion thereof is selected from positions <NUM>-<NUM> or <NUM>-<NUM> of the amino acid sequence of TACI, the linker sequence is 9Gly, and the immunoglobulin Fc fragment is selected from human or animal immunoglobulin Fc, which is selected from IgG, IgM, IgD, and IgA, with each immunoglobulin type including each subtype, such as IgG1. Patent <CIT> discloses the aforementioned TACI-Fc fusion protein useful in the treatment of autoimmune diseases, such as systemic lupus erythematosus. Patent application <CIT> proved the use of TACI-Fc fusion protein for the treatment of neuromyelitis optica spectrum disorder (NMOSD) and multiple sclerosis (MS). The NMOSD includes neuromyelitis optica, recurrent optic neuritis, longitudinally extending transverse myelitis, optic-spinal form of multiple sclerosis, long-term transverse myelitis, unilateral or bilateral optic neuritis, optic neuritis or myelitis accompanying with autoimmune disease, optic neuritis or myelitis accompanying with symptomatic or asymptomatic intracranial lesions.

Part of the formulations of other antibody drugs and fusion proteins that have been on the market are as follows:.

It can also be seen from the compositions of the adjuvant ingredients of the above antibody preparation and fusion protein that the composition of the fusion protein and antibody preparation has its own uniqueness. On the one hand, due to the low stability and complicated structure of monoclonal antibody drugs, it is extremely challenging to manufacture and store such drugs. On account of the heterogeneous structure of antibodies, especially the complementarity determining regions (CDRs) and Fc glycosylation, the development of different monoclonal antibody formulations needs to be carried out individually based on case. In the development of antibody formulations, there are challenges in antibody conformation, colloid or chemical structure, such as oxidation, isomerization, deamidation, aggregation, denaturation and fragmentation. When exposed to different temperature, humidity, pH and stress conditions, the stereostructure of mAb may change, especially in the hypervariable regions (HVRs). Poor products may exhibit reduced activity, and more importantly, increased immunogenicity can endanger the patient. Therefore, it is of vital importance to choose the best adjuvants to protect the antibody (Reference <NUM>: <NPL>). On the other hand, the easy aggregation and poor stability of fusion protein products cause adverse immune responses or interfere with the purification process, which has always been a problem to be solved in this field. Moreover, the factors that affect the aggregation of fusion proteins are also complicated, which can be divided into external factors and internal factors. The external factors mainly include temperature, physical pressure and solvent factors (pH, ionic strength, concentration, metal ions, etc.); and the internal factors mainly include the structural characteristics of the fusion protein, sensitive residues and unpaired cysteine, etc.. These factors will affect the aggregation of the fusion protein, thereby affecting its stability and storage time. Therefore, the selection of adjuvants for fusion proteins requires a lot of experimental exploration (Reference <NUM>: <NPL>). Patent document <CIT> discloses stable formulations comprising TACI-Fc fusion protein.

Therefore, the purpose of the present disclosure is to obtain a formulation combination of TACI-Fc fusion protein through a wide range of screening and concentration range research on the available adjuvants for biological formulations to achieve the following technical effects: the TACI-Fc fusion protein can dissolve well before and after lyophilization, with the insoluble microparticles and visible foreign matters meeting the standards of injection for human use, and yet remain stable for a long time during the lyophilization and storage process, which is not prone to polymerization or degradation after redissolution, maintaining a good biological activity.

The present disclosure relates to an aqueous liquid pharmaceutical formulation of TACI-Fc fusion protein, comprising TACI-Fc fusion protein, a non-reducing sugar, and an amino acid; wherein the non-reducing sugar is <NUM>-<NUM> mmol/L of mannitol and <NUM>-<NUM> mmol/ L of sucrose, the amino acid is <NUM>-<NUM> mmol/L of arginine hydrochloride and <NUM>-<NUM> mmol/L of histidine hydrochloride, the concentration of TACI-Fc fusion protein is <NUM>-<NUM>/ml; and the TACI-Fc fusion protein has an amino acid sequence as shown in SEQ ID NO.

In some embodiments, the histidine hydrochloride concentration is about <NUM> mmol/L; and the arginine hydrochloride concentration is <NUM>-<NUM> mmol/L, <NUM>-<NUM> mmol/L, or about <NUM> mmol/L, or about <NUM> mmol/L.

In some embodiments, the concentration of sucrose is about <NUM> mmol/L.

In some embodiments, the concentration of mannitol is <NUM>-120mmol/L, or about <NUM> mmol/L, or about <NUM> mmol/L.

According to the claims, the TACI-Fc fusion protein has an amino acid sequence as shown in SEQ ID NO. The protein comprises amino acids <NUM>-<NUM> of TACI and an optimized Fc fragment that reduces ADCC and CDC effects.

In the TACI-Fc fusion protein shown in SEQ ID NO. <NUM>, in order to avoid antibody-dependent cell-mediated toxicity (ADCC) effect generated by membrane-bound BLyS or a proliferation-inducing ligand (APRIL), the Fc fragment derived from IgG1 was sequenced optimized, wherein amino acids <NUM>-<NUM> in the CH2 region of the Fc fragment were mutated from leucine (L)-leucine (L)-glycine (G)-glycine (G) to alanine (A)-glutamic acid (E)-glycine (G)-alanine (A), to reduce the affinity of Fcy receptors. In addition, the CH2 region of the Fc sequence was also mutated (amino acid residues <NUM>~<NUM> were mutated from alanine (A)-proline (P) to serine (S)-serine (S)) to reduce complement binding or fixation, thereby reducing complement-dependent cytotoxicity (CDC) effects.

The sequence of SEQ ID NO:<NUM> is as follows:
<IMG>.

In the liquid formulation, two TACI-Fc fusion protein monomers may form a double-stranded structure due to the formation of an interchain disulfide bond in Fc hinge region.

In some embodiments, the concentration of the TACI-Fc fusion protein is about <NUM>/ml to <NUM>/ml.

In some embodiments, the non-reducing sugar is <NUM>-<NUM> mmol/L of mannitol and <NUM>-<NUM> mmol/L of sucrose, the amino acid is <NUM>-<NUM> mmol/L of arginine hydrochloride and <NUM>-<NUM> mmol/L of histidine hydrochloride, and the concentration of the TACI-Fc fusion protein is <NUM>-<NUM>/ml; and the concentration of the histidine hydrochloride is more preferably about <NUM> mmol/L.

In some embodiments, it comprises about <NUM>% to about <NUM>% (w/v, g/<NUM>) of the TACI-Fc fusion protein, preferably about <NUM>-<NUM>% (w/v, g/<NUM>) of the TACI-Fc fusion protein.

In some embodiments, the non-reducing sugar is about <NUM> mmol/L of mannitol and about <NUM> mmol/L of sucrose, the amino acid is about <NUM> mmol/L of arginine hydrochloride and about <NUM> mmol/L of histidine hydrochloride, and the concentration of the TACI-Fc fusion protein is about <NUM>/ml.

In some embodiments, the formulation has a pH of <NUM> to <NUM>, preferably <NUM> to <NUM>, <NUM> to <NUM>, or about <NUM>. The pH value of the solution is adjusted by NaOH or hydrochloric acid.

In another aspect, the present disclosure relates to a lyophilized pharmaceutical formulation obtained by lyophilization of the aqueous liquid pharmaceutical formulation.

In some embodiments of the lyophilized pharmaceutical formulation, the aqueous liquid pharmaceutical formulation comprises about <NUM> mmol/L of mannitol, about <NUM> mmol/L of sucrose, about <NUM> mmol/L of arginine hydrochloride, about <NUM> mmol/L of histidine hydrochloride, and about <NUM>/ml of the TACI-Fc fusion protein, and the aqueous liquid pharmaceutical formulation has a pH of <NUM> to <NUM>.

In some embodiments of the lyophilized pharmaceutical formulation, the non-reducing sugars in the aqueous liquid pharmaceutical formulation are mannitol and sucrose at concentrations of about <NUM>/ml and about <NUM>/ml, respectively; and the amino acids in the aqueous liquid pharmaceutical formulation are arginine hydrochloride and histidine hydrochloride at concentrations of about <NUM>/ml and about <NUM>/ml, respectively.

The present disclosure further relates to the pharmaceutical formulation for use in the treatment of an autoimmune disease, and the autoimmune disease includes systemic lupus erythematosus, rheumatoid arthritis, neuromyelitis optica spectrum disorder (NMOSD), multiple sclerosis (MS) and Sjogren's syndrome, wherein the neuromyelitis optica spectrum disorder comprises neuromyelitis optica, recurrent optic neuritis, longitudinally extending transverse myelitis, optic-spinal form of multiple sclerosis, long-term transverse myelitis, unilateral or bilateral optic neuritis, optic neuritis or myelitis accompanying with autoimmune disease, optic neuritis or myelitis accompanying with symptomatic or asymptomatic intracranial lesions, and the lymphoma includes chronic lymphocytic leukemia, multiple myeloma and B lymphocyte lymphoma.

The present disclosure also relates to a method for preparing a TACI-Fc fusion protein pharmaceutical formulation, comprising: (<NUM>) preparing the formulation as described above; and (<NUM>) evaluating the stability of the TACI-Fc fusion protein in the formulation.

In another aspect, the present disclosure also relates to a method for preparing the pharmaceutical formulation as described above, comprising the following steps: obtaining the protein stock solution, pre-freezing, primary drying, secondary drying, and subpackaging.

In certain embodiments, the step of obtaining the protein stock solution includes:.

In certain embodiments, the step of obtaining the protein stock solution further includes: upon passing the test, the purified protein is mixed with "<NUM>× formulation buffer" for ultrafiltration and concentration to obtain the protein stock solution.

In certain embodiments, the obtained protein stock solution needs to be accurately diluted to the required protein concentration with a protein-free formulation buffer to obtain a semi-finished protein solution, which is then divided into vials for vacuum lyophilization. The main technical effect achieved by the present disclosure is that, the TACI-Fc protein after highly purified has a purity of more than <NUM>% of non-reducing SDS-PAGE, and the CHO cell host protein, CHO cell host DNA, bacterial endotoxin and other indicators meet the requirements of "<NPL>on) standard. The pharmaceutical formulation comprising the TACI-Fc fusion protein obtained in the present disclosure has reached excellent standards in terms of moisture content, number of particles, number of visible foreign matters and insoluble microparticles, and the pharmaceutical formulation has long-term storage stability.

Total RNA was extracted from human peripheral blood mononuclear cells with Qiagen's RNA extraction kit. Then cDNA was synthesized from RNA by reverse transcriptase, before polymerase chain reaction was performed with the primers (Forward: AGCCGTGTGGACCAGGAGG ; Reverse: GAGCTCTGGTGGAAGGTTCACTG) to amplify the desired TACI fragments. The immunoglobulin Fc fragment was obtained by PCR amplification from a cloned sequence-optimized IgG1 Fc plasmid with reduced ADCC and CDC effects. Finally, the TACI and Fc sequences were fused by PCR to construct a DNA sequence of the TACI-Fc fusion protein (its amino acid sequence is shown in SEQ ID NO. Later, by using TA cloning kit, the PCR products of TACI and Fc were cloned into pCR2. <NUM> plasmid respectively, which was then transfected into E. After that, white colonies were picked and added with LB medium for overnight culture. Then the plasmid was extracted with Qiangen's plasmid extraction kit, and the TACI and Fc sequences were identified by restriction enzyme digestion and sequencing. Finally, TACI and IgG Fc cDNA were ligated together by the splicing PCR method. The TACI-Fc fragment was inserted into an expression plasmid. Subsequently, the recombinant plasmid was transfected into E. coli, then positive colonies were picked followed by plasmid extraction, and the target sequences were identified by restriction enzyme digestion and sequencing. Then the plasmid was transfected into CHO cells, and the genetically recombined CHO cells were cultured under standard conditions. When the nutrients in the medium were exhausted and the cells no longer grew, the culture mixture was collected. Then the cells were separated by centrifugation or filtration, and the supernatant containing TACI-Fc protein was collected and loaded onto Protein A affinity chromatography column for the first purification. The eluted target protein was then concentrated by ultrafiltration to a target protein concentration of <NUM>-<NUM>/ml and loaded onto a composite packing chromatography column for the second purification. The target protein was collected and then loaded onto the third column for the third purification in a target protein penetration mode. The purified protein that has passed the test on various indicators was mixed with "<NUM>× formulation buffer" in a volume ratio of <NUM>:<NUM> and then concentrated by ultrafiltration to obtain a protein at a concentration of about <NUM>/ml, which was the protein stock solution and could be stored at -<NUM> for a long time.

Through a large amount of information analysis and experimental screening in the early stage, sucrose, mannitol, glycerol, histidine, arginine, polysorbate <NUM> (i.e. Tween <NUM>) and the like were preliminarily identified as candidate adjuvants for further screening, and the following tests were further carried out.

After overnight culture of TACI-Fc protein in arginine-free buffer (for example, <NUM> mmol/L NaH<NUM>PO<NUM>-Na<NUM>HPO<NUM> <NUM> mmol/L NaCl, pH5. <NUM>) at a protein concentration of above <NUM>/ml in a <NUM> refrigerator, there will appear flocculent precipitate at the bottom of the container, and the inspection of visible foreign matters and insoluble microparticles cannot be performed due to the floating precipitate when shaken gently. The higher the protein concentration, the more serious the protein precipitates. Preliminary experimental studies show that adding arginine hydrochloride to the protein solution can make the precipitate disappear quickly.

Further experiments: <NUM>) <NUM> of purified TACI-Fc protein was dialyzed into <NUM> mmol/L of histidine (pH <NUM>) buffer with a protein concentration of <NUM>/ml; <NUM>) The protein solution was divided into <NUM> groups, <NUM> portions per group, <NUM> portions in total, with the protein content of <NUM> portions in each group of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> respectively, and each of the <NUM> groups was added with <NUM> mol/L arginine hydrochloride (pH <NUM>) solution until the final concentration of arginine hydrochloride was <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> mmol/L respectively, and then concentrated with a centrifugal ultrafiltration tube with a pore size of <NUM> KD to a final volume of <NUM>. Whether there was obvious precipitation of the protein was observed. If there was obvious precipitation, no further procedure would be required, and "-" was directly marked in the table. If the precipitation was not obvious, the protein solution would be filtered to a sterile <NUM> vial with a <NUM> sterile syringe filter in the super clean bench. Each vial was sealed with a sterile rubber stopper, put on an aluminum cap, and stored in a <NUM> refrigerator for <NUM> hours to observe whether there would be any precipitation.

Inspection standard: Observing under the YB-II clarity detector. Completely clear or no more than <NUM> white spots is marked as "-". If there is precipitation, it is marked as "+", "++" and "+++", according to the amount of precipitation. More "+" signs indicate more precipitation. The experimental results are shown in Table <NUM> that the optimal concentration of arginine hydrochloride is between <NUM> mmol/L and <NUM> mmol/L.

Through the similar steps as in Example <NUM>, the moisture content and appearance of the lyophilized powder prepared with different concentrations of TACI-Fc fusion protein in the presence of different concentrations of mannitol were investigated. Experiments show that when the mannitol content is less than <NUM> mmol/L, it will affect the moisture content and appearance of the lyophilized powder.

The stability of lyophilized powders of pharmaceutical formulations containing different concentrations of sucrose under the conditions of <NUM>, <NUM> and <NUM> was investigated through experiments, which showed that when the concentration of sucrose in the lyophilized stock solution is lower than <NUM> mmol/L, the stock solution will lose the activity of the TACI-Fc fusion protein during lyophilization, and fail to play an effective lyophilization protective effect.

On the basis of the above experiments, a variety of formulas of the formulation were designed for further investigation.

Preparation of histidine hydrochloride solution:
According to the formulas in Table <NUM>, <NUM>× formulation buffers of formula No. <NUM>-<NUM> were prepared. Taking formula No. <NUM> in Table <NUM> as an example:.

Other formulas were prepared according to the above steps according to the amount of each formula.

Preparation of dialysis buffer: <NUM> mmol/L of histidine hydrochloride, pH <NUM>.

The pH value of the above solutions were all adjusted with <NUM> mol/L sodium hydroxide or <NUM> mol/L hydrochloric acid, and the solutions were used after ultrafiltration with an ultrafiltration membrane with a pore size not greater than 10KD to remove endotoxins.

The protein stock solution was taken out from the -<NUM> refrigerator, thawed, diluted accurately with "<NUM>× formulation buffer" to a protein concentration of <NUM>/ml, and subpackaged into a sterile, pyrogen-free standard <NUM> lyophilized vial, <NUM> per vial, and then subjected to freeze drying in vacuum.

After lyophilization, the vials were sealed with a rubber stopper in a vacuum state, taken out of the lyophilizer and put on an aluminum cap.

Acceptance standards for the appearance of lyophilized powders are: uniform color, even and dense pores, and the volume and shape before and after lyophilization remain basically unchanged, showing a block or spongy mass structure.

The experimental results are shown in Table <NUM> that the appearance of the lyophilized powder of formula <NUM>, formula <NUM>, formula <NUM> and formula <NUM> does not meet the requirements and is eliminated. The appearance of lyophilized powder of other formulas meets the requirements.

Test standard for moisture content assay:.

The moisture content of the lyophilized powder was measured by SF-<NUM> type micro-moisture analyzer (Karl Fischer Coulometric Method), and the acceptance standard is not more than <NUM>%.

The experimental results show that the lyophilized powders of formulas <NUM>, <NUM>, and <NUM> are very easy to absorb water, and the moisture content does not meet the requirements. Formulas <NUM>-<NUM> meet the requirements of the moisture content test.

The lyophilized powder was redissoluted with sterile water for injection, each with a volume of <NUM>. Before redissolution, it was relieved from the vacuum, and then the water was slowly added along the inner wall of the vial.

YB-II clarity detector was used for visible foreign matters inspection. In accordance with "<NPL> and the relevant provisions of "<NPL>], <NUM> vials were inspected for each formula, and the average content of visible foreign matters in each vial was calculated.

Acceptance standards for visible foreign matters inspection: Among the <NUM> test samples (vials) inspected, no foreign matter such as glass shards, fibers, color spots, color blocks shall be detected, and no more than <NUM> other visible foreign matters (white spots, fine protein floccules or protein particles) shall be detected.

The inspection of insoluble microparticles was carried out using ZWJ-<NUM> insoluble microparticle detector. In accordance with "<NPL>, the number of insoluble microparticles above <NUM> microns and above <NUM> microns in each milliliter of protein solution was examined. <NUM> vials were inspected for each formula, and the average content of insoluble microparticles in each vial was calculated.

Acceptance standards for insoluble microparticles inspection: each test container shall contain no more than <NUM>,<NUM> microparticles equal to or above <NUM> and no more than <NUM> microparticles equal to or above <NUM>.

According to the data obtained in Table <NUM>, it can be seen that formulas <NUM> and <NUM> do not meet the requirements for inspection of visible foreign matters and insoluble microparticles.

Formulas <NUM>, <NUM>, <NUM>, and <NUM> were eliminated due to the appearance inspection of lyophilized powder, formulas <NUM> and <NUM> were eliminated due to the moisture content inspection, and formulas <NUM> and <NUM> were eliminated due to the inspection of visible foreign matters and insoluble microparticles. Stability investigation was performed on the remaining formulas <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>. The lyophilized powder of each formula was stored at <NUM>, <NUM> and <NUM> respectively, and was sampled at different times for inspections such as SDS-PAGE, reverse phase HPLC, biological activity by ligand binding method, moisture content, appearance, pH, visible foreign matters and insoluble microparticles. The results are shown in Tables <NUM>, <NUM>, and <NUM>.

The results show that the formula with a sucrose concentration of <NUM> mmol/L had poor stability, and the formula <NUM> and formula <NUM> with a sucrose concentration of <NUM> mmol/L had better long-term storage stability. In all aspects, formulas <NUM>-<NUM> meet the requirements in terms of moisture content, visible foreign matters, appearance, insoluble microparticles, and stability.

Based on the above experimental data, it can be seen that on the one hand, the choice of specific adjuvants in a class of substances such as non-reducing sugars and amino acids has an unpredictable effect on the final formulation. For the fusion protein of the present disclosure, a large number of experiments are needed to test various properties in order to finally obtain a good combination of components. For example, according to experimental evidence, although formulas <NUM>-<NUM> all contain histidine hydrochloride, mannitol, arginine hydrochloride and sucrose, due to the difference in the content of these components, the final inspection results of moisture content, visible foreign matters, appearance, insoluble microparticles and stability varied considerably, and only formulas <NUM> and <NUM> succeeded to pass the above tests. As such, the choice of the types and contents of the adjuvant components of pharmaceutical formulations is of critical significance and the predictability is poor.

Claim 1:
An aqueous liquid pharmaceutical formulation of TACI-Fc fusion protein, comprising TACI-Fc fusion protein, a non-reducing sugar, and an amino acid; wherein the non-reducing sugar is <NUM>-<NUM> mmol/L of mannitol and <NUM>-<NUM> mmol/L of sucrose, the amino acid is <NUM>-<NUM> mmol/L of arginine hydrochloride and <NUM>-<NUM> mmol/L of histidine hydrochloride, the concentration of TACI-Fc fusion protein is <NUM>-<NUM>/ml; and the TACI-Fc fusion protein has an amino acid sequence as shown in SEQ ID NO. <NUM>.