Genetically engineered cell lines producing recombinant glycoproteins with mannose-terminated N-glycans

Abstract

The present invention provides a genetically engineered cell line that produces a recombinant glycoprotein having mannose-terminated N-glycans, and a method for producing said recombinant glycoprotein, or a method for producing said cell, clone, or cell line, wherein said cell line contains an insertion of less than 600 bp in the coding region of the chromosomal sequence encoding MGAT1.

Description

[Technical field]

[0001] The present application relates generally to genetically engineered cell lines that produce recombinant glycoproteins having mannose-terminated N-glycans, and methods for producing said recombinant glycoproteins or methods for making said cells, clones or cell lines. [Background technology]

[0002] One of the commercially useful glycoproteins is β-glucocerebrosidase, which catalyzes the hydrolysis of glucocerebroside to ceramide and glucose.Due to the lack of β-glucocerebrosidase in vivo, glucoceramide accumulates in peripheral macrophages, causing cell proliferation and leading to the failure and decline of the spleen, liver and bone marrow, which is an autosomal recessive lysosomal storage disease, Gaucher disease, and therefore exogenous administration of recombinantly expressed β-glucocerebrosidase is considered in the art to be an attractive method for treating Gaucher disease.

[0003] However, placenta-derived unmodified human β-glucocerebrosidase has the problem that it is poorly taken up by required cells, such as dendritic cells and macrophages, via mannose receptor-mediated endocytosis, due to the lack of exposed mannose residues. Therefore, there is a growing demand for convenient and effective production of recombinantly expressed glycoproteins with mannose-terminated N-glycans, such as β-glucocerebrosidase, to facilitate the uptake of glycoproteins into required cells via the mannose receptor, for example, by removing residues that block the mannose of the N-glycan (exposing mannose as the terminal residue of the N-glycan).

[0004] Therefore, there is a need for engineered cell lines that produce recombinant glycoproteins with mannose-terminated N-glycans, and it is desirable that these engineered cell lines stably produce proteins with terminal mannose residues while maintaining high protein productivity, high quality, and high enzymatic activity. Summary of the Invention

[0005] The inventors of the present invention have surprisingly discovered a genetically modified cell line that is deleted of the mannosyl(α-1,3-)-recombinant glycoprotein β-1,2-N-acetylglucosaminyltransferase 1 (MGAT1) gene by an insertion mutation. The cell line contains an insertion of less than 600 bp in the coding region of the chromosomal sequence encoding MGAT1 by selection by RCA. The recombinant glycoprotein produced by the cell line of the present invention has high protein productivity, high protein quality, and high enzyme activity.

[0006] In various aspects of the present invention, in one aspect, a cell line is provided that is deficient in mannosyl(α-1,3-)-glycoprotein β-1,2-N-acetylglucosaminyltransferase 1 (MGAT1), the cell line comprises an insertion of less than 600 bp in the coding region of the chromosomal sequence encoding MGAT1. In one embodiment, the cell line comprises an insertion of less than 400, 200, 100, 50, 30, 10 or 5 bp in the coding region of the chromosomal sequence encoding MGAT1. In one embodiment, the cell line comprises an insertion of 1 bp in the coding region of the chromosomal sequence encoding MGAT1. In one embodiment, the chromosomal sequence encoding MGAT1 is modified by a targeting endonuclease-mediated genome editing technique, the targeting endonuclease-mediated genome editing technique being, for example, clustered regularly interspaced short palindromic repeats (CRISPR), zinc finger nuclease (ZFN), or transcription activator-like effector nuclease (TALEN). In one embodiment, the cell is an animal cell, preferably a mammalian cell, more preferably a Chinese Hamster Ovary (CHO) cell. In one embodiment, the cell line expresses at least one glycoprotein that includes one or more terminal mannose residues. In one embodiment, the glycoprotein is an enzyme such as β-glucocerebrosidase.

[0007] Another aspect of the invention is a method for producing a recombinant glycoprotein, comprising the steps of: (1) culturing the genetically modified cell line; (2) recovering the glycoprotein.

[0008] Another aspect of the invention is a method for producing a recombinant glycoprotein having mannose-terminated N-glycans with one or more terminal mannose residues, comprising the steps of: (1) introducing a gene encoding the recombinant glycoprotein into a cell line; (2) mutating the mannosyl(α-1,3-)-recombinant glycoprotein β-1,2-N-acetylglucosaminyltransferase 1 (MGAT1) gene in the cell line obtained by step (1); (3) expressing the recombinant glycoprotein, The method includes the steps of: (a) mutating the MGAT1 gene by CRISPR; and (b) determining whether the sgRNA is a nucleotide sequence of SEQ ID NO:4 or a nucleic acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:4.

[0009] In one embodiment, the cell lines are selected by RCA. In one embodiment, the glycoprotein is an enzyme such as β-glucocerebrosidase. In one embodiment, the method further comprises selecting mutant cell lines in a batch refeeding assay. In one embodiment, the batch refeeding assay comprises refreshing the production medium daily. In one embodiment, the recombinant glycoprotein comprises an N-glycan having one or more terminal mannose residues, preferably 1-9 terminal mannose residues, more preferably 3-6 terminal mannose residues, and optionally 1-3 fucose residues that do not block the terminal mannose residues, the N-glycan being selected from the group consisting of Man3, Man4, Man4+1F, Man5, Man5+1F, and Man6. In one embodiment, the percentage of mannose-terminated N-glycans attached to the recombinant glycoprotein is greater than 80%, 85%, 86%, 87%, 88% or 89%. In one embodiment, the enzyme activity is greater than 0.5 U / mL. In one embodiment, the cell is an animal cell, preferably a mammalian cell, more preferably a Chinese Hamster Ovary (CHO) cell.

[0010] Another aspect of the invention includes a recombinant glycoprotein, such as β-glucocerebrosidase, produced by the cell line or method of the invention. Another aspect of the invention includes a method of treating a disease associated with β-glucocerebrosidase deficiency, such as Gaucher's disease, comprising administering to a subject in need thereof a recombinant glycoprotein, such as β-glucocerebrosidase, as disclosed in the present invention. Yet another aspect of the invention is a cell, clone or cell line produced by said method.

[0011] Another aspect of the present invention is a pharmaceutical formulation comprising a therapeutically effective amount of a recombinant enzyme, a buffer, an osmolality regulator, and a surfactant. In one embodiment, the enzyme is β-glucocerebrosidase with a content of 50-150 U / ml. In one embodiment, the buffer is a citrate buffer with a content of 2-10 g / mL. In one embodiment, the osmolality regulator is sucrose with a content of 40-120 g / L. In one embodiment, the surfactant is polysorbate 80 with a content of 0.1-1 g / L. In one embodiment, the pH of the formulation is 5-7.

[0012] The above is a summary, and therefore includes only a brief and general outline of the essentials, omitting details. Thus, those skilled in the art will appreciate that the above summary is illustrative only and is not intended to be limiting. Other aspects, features and advantages of the methods, compositions and / or devices described herein and / or other subject matter will become apparent under the teachings of the present invention. The above summary is intended to introduce in a concise manner a selection of concepts that are further described in the detailed description below. The above summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. Additionally, all references, patents, and published patent applications cited in this application are incorporated herein by reference in their entirety. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 shows a plasmid containing Cas9 and sgRNA5. [Diagram 2] FIG. 2 is a glycan chromatogram of a monoclone in which the MGAT1 gene was mutated. [Diagram 3] FIG. 3 is a glycan chromatogram of a control pool containing wild-type MGAT1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Although the present invention may be embodied in various different forms, disclosed herein are specific and exemplary embodiments illustrating the principles of the present invention. It should be emphasized that the present invention is not limited to these specific and exemplary embodiments. It should be noted that any section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0015] (definition) Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. More specifically, the singular forms "a," "an," and "the" as used herein and in the claims include plural referents unless the context clearly dictates otherwise. Thus, for example, "a protein" includes a plurality of proteins, and "a cell" includes mixtures of cells and the like. In this application, "or" means "and / or" unless otherwise specified. Additionally, the term "comprising," as well as other forms such as "comprises" and "comprised," are not limiting. Furthermore, the scope of the specification and claims includes the two endpoints and all points therebetween.

[0016] As used herein, a "gene" refers to a DNA region (including exons and introns) that encodes a gene product and all DNA regions that control the production of the gene product, whether or not regulatory sequences flank the coding and / or transcribed sequence. Thus, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational control sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions.

[0017] In the context of the present invention, MGAT1, also called N-acetylglucosaminyltransferase I (GlcAc-T I or GnT I) or N-glycosyl-oligosaccharide-glycoprotein N-acetylglucosaminyltransferase I, catalyzes the transfer of N-acetylglucosamine (GlcNAc) to the oligomannose core (i.e., Man5GlcNAc2 (Man5) moiety) in growing N-glycans. Malfunction of MGAT1 affects the nature of N-glycans, resulting in glycoproteins with N-glycans at terminal mannose residues, such as β-glucocerebrosidase. In the context of the present invention, MGAT1 is used interchangeably with GnT I.

[0018] The term "monoclone" as used herein refers to a group of cells produced from a single ancestor cell by repeated cell replication. In the more specific context of the present invention, a monoclone expressing a recombinant glycoprotein such as β-glucocerebrosidase can be generated by cloning a single cell derived from a pool expressing a recombinant glycoprotein such as β-glucocerebrosidase.

[0019] The term "mutation" as used herein refers to a deletion mutation or an insertion mutation. In the more specific context of the present invention, an insertion mutation refers to the insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more base pairs in a gene encoding a target protein or in a regulatory nucleic acid sequence operably linked to the gene encoding the protein, such as a promoter or enhancer. In a specific embodiment, the mutation of the mannosyl (α-1,3-)-recombinant glycoprotein β-1,2-N-acetylglucosaminyltransferase 1 (MGAT1) gene in a cell line is performed by insertion mutation. A deletion mutation refers to the deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more base pairs in a gene encoding a target protein or in a regulatory nucleic acid sequence operably linked to the gene encoding the protein, such as a promoter or enhancer. Those skilled in the art know that knock out includes introduction of deletion, substitution or insertion mutations into a gene encoding a target protein or into a regulatory nucleic acid sequence operably linked to the gene encoding the protein, such as a promoter or enhancer, and that the mutations cause a reduction in expression levels or functional defects, such as inactivation of the protein.

[0020] The term "pool" as used herein refers to a mixed cell population. In the more specific context of the present invention, a pool expressing a recombinant glycoprotein such as β-glucocerebrosidase can be generated by transfecting a gene encoding the recombinant glycoprotein such as β-glucocerebrosidase into a host cell.

[0021] The term "recombination" refers to the process of exchange of genetic information between two polynucleotides. In the present invention, "homologous recombination" refers to a specialized form of such exchange that occurs, for example, during repair of double-strand breaks in cells. This process requires sequence similarity between two polynucleotides and uses a "donor" or "exchange" molecule to template repair of a "target" molecule (i.e., the molecule that underwent the double-strand break), and is variously known as "non-crossover gene conversion" or "short tract gene conversion", to result in the transfer of genetic information from the donor to the target. Without being bound to any particular theory, such transfer may involve mismatch correction of the heteroduplex DNA formed between the cleaved target and the donor, and / or "synthesis-dependent strand annealing", in which the donor is used to resynthesize the genetic information that will become part of the target, and / or related processes. Such specific homologous recombination often results in an alteration of the sequence of the target molecule, such that some or all of the sequence of the donor polynucleotide becomes integrated into the target polynucleotide.

[0022] The term "sequence identity" as used in the present invention refers to the degree to which sequences are identical between nucleotides or between amino acids in a comparison window.Therefore, "percentage of sequence identity" is calculated by comparing two optimally aligned sequences in a comparison window, determining the number of positions where identical nucleic acid bases (e.g., A, T, C, G, I, U) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occur in both sequences to determine the number of matching positions, and dividing the number of matching positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to obtain the percentage of sequence identity. In the present invention, the percentage of sequence identity is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0023] The term "pharmaceutical formulation" refers to a formulation that is in a form that effectively potentiates the biological activity of the active ingredient and does not contain additional ingredients that have a toxicity that cannot be tolerated by the subject to whom the formulation is administered.

[0024] The term "stable" as used in the context of a pharmaceutical formulation means that the protein retains physical and / or chemical stability and / or biological activity upon storage. Stability can be measured at a selected temperature and for a selected time. Preferably, the formulation is stable at room temperature (about 25°C) or 40°C for at least 3 months, and / or at about 2-8°C for at least 1 year.

[0025] The term "buffer" refers to a buffer solution that resists changes in pH due to the action of its acid-base conjugate components. Buffers in the present invention have a pH in the range of about 4 to 8, preferably about 4.5 to 7, and most preferably about 5.0 to 6.5. Examples of buffers that adjust the pH to this range include acetate, succinate, glucose, histidine, citrate and other organic acid buffers.

[0026] The term "osmotic modifier" refers to a reagent added to a formulation to obtain a desired level of osmolality. Osmolality can be expressed as the concentration of osmotically active particles dissolved in 1 kg of solution. Examples of osmotic modifiers include sugars and / or sugar alcohols.

[0027] The term "surfactant" refers to a reagent having surface activity. Examples of surfactants that may be used in the present invention include polysorbates and poloxamers.

[0028] The term "reconstitution" refers to dissolving a lyophilized protein formulation in a diluent such that the protein is dispersed in the diluent. The reconstituted formulation is suitable for administration.

[0029] Generally, the terms and techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, and hybridization described herein are well known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally performed by conventional methods known in the art and described in the various general and specific references cited and discussed herein. The terms and experimental procedures and techniques of analytical chemistry, synthetic organic chemistry, and medicinal chemistry and drug discovery chemistry described herein are well known and commonly used in the art. It should be noted that any section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0030] (cell line) One aspect of the present invention includes a cell line deficient in mannosyl(α-1,3-)-recombinant glycoprotein β-1,2-N-acetylglucosaminyltransferase 1 (MGAT1), the cell line comprising an insertion of less than 600 bp in the coding region of the chromosomal sequence encoding MGAT1.

[0031] In one embodiment, the MGAT1-deficient cell line includes an insertion of about 1 to 599 bp in the chromosomal sequence encoding MGAT1. In another embodiment, the MGAT1-deficient cell line includes an insertion of about 1 to 50 bp, about 50 to 100 bp, about 100 to 200 bp, about 200 to 300 bp, about 300 to 400 bp, or less than about 600 bp in the coding region of MGAT1. In an exemplary embodiment, the MGAT1-deficient cell line includes an insertion of about 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 10 bp, 25 bp, or 50 bp in the chromosomal sequence encoding MGAT1, resulting in functional deficiency of MGAT1. An insertion of about 1 to 599 bp in the chromosomal sequence encoding MGAT1 results in expression deficiency or inactivation of MGAT1. The sequence encoding MGAT1 undergoes a shift in the reading frame, preventing the production of the protein product. The chromosomal sequence encoding MGAT1 can be mutated by genome editing technology mediated by a targeting endonuclease, which will be described later.

[0032] In one embodiment, the MGAT1-deficient cell line comprises a deletion of about 1 to 1300 bp in the chromosomal sequence encoding MGAT1. In another embodiment, the MGAT1-deficient cell line comprises a deletion of about 1 to 50 bp, about 50 to 100 bp, about 100 to 200 bp, about 200 to 300 bp, about 300 to 400 bp, about 400 to 600 bp, or more than about 600 bp in the coding region of MGAT1. In an exemplary embodiment, the MGAT1-deficient cell line comprises a deletion of about 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 10 bp, 25 bp, or 50 bp in the chromosomal sequence encoding MGAT1, resulting in functional deficiency of MGAT1. A deletion of about 1 to 599 bp in the chromosomal sequence encoding MGAT1 results in expression deficiency or inactivation of MGAT1. The sequence encoding MGAT1 undergoes a shift in the reading frame, preventing production of the protein product. The chromosomal sequence encoding MGAT1 can be mutated by genome editing techniques mediated by targeted endonucleases, which will be described later.

[0033] In some embodiments, cells that can be used for the production of recombinant glycoproteins are, in principle, all cells known to those skilled in the art that are capable of expressing recombinant glycoproteins. The cells may be animal cells, in particular mammalian cells. Examples of mammalian cells include CHO (Chinese Hamster Ovary) cells, preferably CHO-K1 cells, hybridomas, BHK (Baby Hamster Kidney) cells, myeloma cells, e.g. HEK-293 cells, human lymphoblastoid line cells, human cells such as E1-immortalized HER cells, and mouse cells, e.g. NSO cells. In one embodiment, the cells are animal cells, preferably mammalian cells, more preferably Chinese Hamster Ovary (CHO) cells. In an exemplary embodiment, the cell line is of a type that is widely used for the production of recombinant proteins, such as glycoproteins. In an exemplary embodiment, the cell line is a CHO cell line. Numerous CHO cell lines are available from the ATCC. Suitable CHO cell lines include, but are not limited to, CHO-K1 cells and their derivatives.

[0034] In one embodiment, the cell line expresses at least one glycoprotein having one or more terminal mannose residues, hi one embodiment, the glycoprotein is β-glucocerebrosidase.

[0035] (Genome editing technology via targeted endonucleases) In one embodiment, the chromosomal sequence encoding MGAT1 is modified by a targeted endonuclease-mediated genome editing technique, such as clustered regularly interspaced short palindromic repeats (CRISPR), zinc finger nuclease (ZFN) or transcription activator-like effector nuclease (TALEN).

[0036] The CRISPR system, which forms an adaptive immune system in bacteria, has been modified for genome engineering. The CRISPR system for engineering contains two components: a guide RNA (gRNA or sgRNA) and a CRISPR-associated endonuclease (Cas endonuclease). The gRNA is a short synthetic RNA that consists of a scaffold sequence required for Cas binding and a user-defined spacer of about 20 nucleotides that defines the genome target to be modified. In the present invention, gRNA and sgRNA can be used interchangeably. Therefore, the genome target of the Cas protein can be changed simply by changing the target sequence present in the gRNA.

[0037] CRISPR can be used to generate knockout (e.g., mutant) cells or animals by co-expressing an endonuclease such as Cas9 or Cpf1 with a gRNA specific for the gene of interest. The genomic target can be any DNA sequence of about 20 nucleotides, as long as it meets two criteria: (1) the sequence is unique relative to the rest of the genome, and (2) the target is located immediately adjacent to a protospacer adjacent motif (PAM). This PAM sequence is essential for target binding, but the exact sequence is determined by the Cas protein. A commonly used Cas protein is Streptococcus pyogenes Cas9 (SpCas9). When expressed, the Cas9 protein and gRNA form a ribonucleoprotein complex through interactions between the gRNA scaffold and a positively charged groove exposed on the surface of Cas9. Cas9 undergoes a conformational change upon gRNA binding, shifting the molecule from an inactive, non-DNA-binding conformation to an activated, DNA-binding conformation. Importantly, the spacer region of the gRNA is free to interact with the target DNA.

[0038] Cas9 cleaves a given locus only if the gRNA spacer sequence shares sufficient homology with the target DNA. Once the Cas9-gRNA complex is bound to the putative DNA target, the seed sequence (8-10 bases at the 3' end of the gRNA target sequence) begins to anneal to the target DNA. If the seed and target DNA sequences match, the gRNA continues to anneal to the target DNA in the 3' to 5' direction. The zipper-like annealing mechanism of Cas9 could explain why mismatches between the target sequence within the 3' seed sequence completely abolish target cleavage, while mismatches toward the 5' end distal to the PAM often still allow target cleavage.

[0039] The Cas9 nuclease has two functional endonuclease domains, RuvC and HNH. Upon target binding, Cas9 undergoes a second conformational change that positions the nuclease domains to cleave opposite strands of the target DNA. The end result of Cas9-mediated DNA cleavage is the formation of a double-strand break (DSB) in the target DNA, approximately 3–4 nucleotides upstream of the PAM sequence.

[0040] The resulting DSBs are then repaired by one of two repair pathways: (1) the efficient but error-prone non-homologous end joining (NHEJ) pathway, and (2) the less efficient but high fidelity homology-directed repair (HDR) pathway. The NHEJ repair pathway is the most active repair mechanism and frequently results in small nucleotide insertions or deletions (indels) at the DSB site. The randomness of NHEJ-mediated DSB repair has important practical implications, as cell populations expressing Cas9 and gRNAs result in diverse mutations. In most cases, NHEJ generates small indels in the target DNA, resulting in amino acid deletions, insertions or frameshift mutations, which create premature stop codons in the open reading frame (ORF) of the target gene. The ideal end result is a loss-of-function mutation in the target gene.

[0041] In an embodiment, the knockout mutation of MGAT1 gene by CRISPR-based means can be carried out by any feasible protocol, such as, for example, that taught in Yang 2014 (Yang, et al., CRISPR / Cas9-Directed Genome Editing of Cultured Cells. Current Protocols in Molecular Biology, 2014, 107(1):31.1.1-31.1.17). In a more specific embodiment, MGAT1 gene is mutated by CRISPR-based means, wherein sgRNA comprises or consists of SEQ ID NO:4 or comprises or consists of the nucleic acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:4.

[0042] Methods for Producing Recombinant Glycoproteins, Cells, Clones or Cell Lines Another aspect of the invention is a method for producing a recombinant glycoprotein having mannose-terminated N-glycans with one or more terminal mannose residues, comprising the steps of: (1) introducing a gene encoding the recombinant glycoprotein into a cell line; (2) mutating the mannosyl(α-1,3-)-recombinant glycoprotein β-1,2-N-acetylglucosaminyltransferase 1 (MGAT1) gene in the cell line obtained by step (1); (3) expressing the recombinant glycoprotein, The method includes the steps of: (a) mutating the MGAT1 gene by CRISPR; and (b) determining whether the sgRNA is a nucleotide sequence of SEQ ID NO:4 or a nucleic acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:4.

[0043] Another aspect of the invention is a method of producing a cell, clone or cell line that produces a recombinant glycoprotein having mannose-terminated N-glycans, comprising the steps of: (1) introducing a gene encoding the recombinant glycoprotein into a cell line; (2) mutating the mannosyl(α-1,3-)-recombinant glycoprotein β-1,2-N-acetylglucosaminyltransferase 1 (MGAT1) gene in the cell line obtained by step (1); The method includes the steps of: (a) mutating the MGAT1 gene by CRISPR; and (b) determining whether the sgRNA is a nucleotide sequence of SEQ ID NO:4 or a nucleic acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:4.

[0044] The plasmids usable in the present invention may be any commercially available plasmids for the intended purpose. Those skilled in the art can easily know which plasmids are suitable for which purpose, and understand that variations in the plasmids used do not substantially change the technical effect of the method or product of the present invention.

[0045] In one embodiment, a plasmid for introducing a gene into a cell such as a CHO cell can be prepared manually by incorporating elements such as an origin of replication (ori), a promoter, a multiple cloning site for transferring a gene of interest, and one or more selection marker-encoding genes into a single circular sequence. Non-limiting examples of plasmids conventionally used for introducing a gene into a cell such as a CHO cell include pMX241 (Addgene Cat. No. 23017).

[0046] In one embodiment, the CRISPR-based plasmid can be prepared manually by incorporating elements such as an origin of replication (ori), a promoter, a sgRNA scaffold for receiving a purposefully designed sgRNA, a gene encoding a CRISPR-associated (Cas) endonuclease, and one or more selection marker encoding genes into a single circular sequence. Non-limiting examples of conventionally available knockout vectors using CRISPR include pSpCas9(BB)-2A-Puro(PX459)V2.0 (Addgene Cat.No.62988) and the like.

[0047] In the present invention, the selection marker may be any one commercially available in the art, including, but not limited to, neomycin, puromycin, hygromycin, zeocin, blasticidin, etc.

[0048] In one embodiment, cell lines are selected by RCA. In one embodiment, transfected cells are cultured with castor bean agglutinin-I (RCA-I). MGAT1-deficient cell lines can be further enriched by culturing with castor bean agglutinin-I (RCA-I). RCA-I is a cytotoxic lectin that does not bind to terminal mannose residues, allowing the selection of cells that lack MGAT1 activity (because they produce glycoproteins with terminal mannose residues).

[0049] In one embodiment, the cell is an animal cell, particularly a mammalian cell, more preferably a Chinese Hamster Ovary (CHO) cell. In an exemplary embodiment, the cell line is of a type commonly used for the production of recombinant proteins, such as glycoproteins. In an exemplary embodiment, the cell line is a CHO cell line. Numerous CHO cell lines are available from the ATCC. Suitable CHO cell lines include, but are not limited to, CHO-K1 cells and their derivatives.

[0050] (recombinant glycoprotein) Another aspect of the invention includes recombinant glycoproteins, such as β-glucocerebrosidase, having mannose-terminated N-glycans produced by the methods of the invention.

[0051] In one embodiment, the recombinant glycoprotein is an enzyme such as β-glucocerebrosidase. In one embodiment, the recombinant glycoprotein has an N-glycan that includes one or more terminal mannose residues. In some embodiments, the recombinant glycoprotein has an N-glycan that includes 2-9 terminal mannose residues (also referred to as Man2-Man9), e.g., 3-6 terminal mannose residues (also referred to as Man3-Man6), and optionally 1-3 fucose residues (also referred to as 1F-3F) that do not block the terminal mannose residues, e.g., 1 fucose residue (also referred to as 1F). Exemplary N-glycans of the present invention are selected from the group consisting of Man3, Man4, Man4+1F, Man5, Man5+1F, Man6, etc. In one embodiment, the percentage of mannose-terminated N-glycans attached to the recombinant glycoprotein is greater than 80%, 85%, 86%, 87%, 88%, or 89%.

[0052] Enzyme activity was measured using p-nitrophenyl-bD-glucopyranoside (Sigma-N7006) as substrate. The released product was 4-nitrophenol (Sigma-35836-1G) detected by measuring absorbance at 405 nm with a plate reader (MOLECULAR DEVICE, i3X). A reference standard curve was assayed in parallel to quantify the concentration of 4-nitrophenol. Enzyme activity is defined as the amount of enzyme used to catalyze the release of 1 nmole of 4-nitrophenol from p-nitrophenyl-bD-glucopyranoside per mL per hour at 37° C. In one embodiment, the enzyme activity of β-glucocerebrosidase produced by the cell line of the present invention is greater than about 0.2 U / mL, about 0.3 U / m, about 0.4 U / mL, about 0.5 U / mL, about 0.6 U / mL.

[0053] The recombinant glycoprotein is purified to a purity of greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, and greater than about 90%, as detected by Caliper-SDS and SEC-HPLC assays, respectively.

[0054] (Treatment method) Another aspect of the present invention is a method for treating a disease associated with β-glucocerebrosidase deficiency, such as Gaucher's disease, comprising administering to a subject in need thereof a recombinant glycoprotein having a mannose-terminated N-glycan, such as the β-glucocerebrosidase disclosed in the present invention, or using a recombinant glycoprotein having a mannose-terminated N-glycan, such as the β-glucocerebrosidase disclosed in the present invention, in the manufacture of a composition for the treatment of a disease associated with β-glucocerebrosidase deficiency, such as Gaucher's disease.

[0055] The advantages that can be achieved by the method and product of the present invention can be summarized as follows:

[0056] First, by mutating the MGAT1 gene to express a recombinant glycoprotein such as β-glucocerebrosidase in cells such as CHO cells, the present invention has high purity, high quality and high enzyme activity.

[0057] Second, the present invention developed a new plasmid containing a specific sgRNA (e.g., sgRNA_5 shown in SEQ ID NO:4) with higher mutation efficiency.

[0058] Third, in the present invention, the MGAT1-deficient cell line can be further enriched by culturing with castor bean agglutinin-I (RCA-I) in the production process of the MGAT1-deficient cell line.

[0059] Fourth, in the present invention, a gene encoding a recombinant glycoprotein is introduced into a cell before the mutation of the MGAT1 gene in the cell, and the merit of doing so is that the requirements for product quality attributes (PQA) and productivity of the expressed recombinant glycoprotein for clone screening are clear, so that the recombinant glycoprotein with mannose-terminated N-glycan can be produced in less time, at lower cost, and with higher work efficiency than the method of the prior art. In contrast, since there is no clearly defined standard for PQA and productivity for screening the optimal host cell, it becomes difficult to introduce a gene encoding a recombinant glycoprotein into the mutant host cell after screening the best host cell with MGAT1 gene mutation. Note that, in the method of the present invention, mutation and cloning are carried out in the same process.

[0060] (Pharmaceutical preparations) Another aspect of the present invention includes a pharmaceutical preparation of β-glucocerebrosidase. The preparation includes a therapeutically effective amount of β-glucocerebrosidase, a buffer, an osmotic pressure regulator, and a surfactant. In one embodiment, the content of β-glucocerebrosidase is 50 to 150 U / ml, 75 to 125 U / ml, 80 to 120 U / ml, 90 to 110 U / ml, or 100 U / ml. In one embodiment, the buffer is a citrate buffer having a content of 2 to 10 g / mL, 3 to 9 g / mL, 4 to 8 g / mL, 5 to 7 g / mL, or 5 to 6 g / mL. In one embodiment, the osmotic pressure regulator is sucrose having a content of 40 to 120 g / L, 50 to 110 g / L, 60 to 100 g / L, 70 to 90 g / L, or 80 g / L. In one embodiment, the surfactant is polysorbate 80 having a content of 0.1-1 g / L, 0.1-0.9 g / L, 0.1-0.8 g / L, 0.1-0.7 g / L, 0.1-0.6 g / L, 0.1-0.5 g / L, 0.1-0.4 g / L, 0.1-0.3 g / L, or 0.2 g / L. In one embodiment, the pH of the formulation is 5-7.

[0061] The pharmaceutical formulation according to the present invention provides a stable formulation of β-glucocerebrosidase with extended shelf life and improved stability. The formulation is stable for at least 12 weeks at 2-8° C., or at least 12 weeks at room temperature (about 25° C.), or at least 4 weeks at 40° C. The formulation is capable of retaining its physical and / or chemical and / or biological activity under different storage conditions.

[0062] (Summary of sequence listing) This application is accompanied by a sequence listing containing a large number of nucleic acid and amino acid sequences as follows:

[0063] SEQ ID NO:1 is the amino acid sequence of human β-glucocerebrosidase (NP_000148.2) ( https: / / www.ncbi.nlm.nih.gov / protein / NP_000148.2 ).

[0064] SEQ ID NO:2 is the nucleic acid sequence of human β-glucocerebrosidase (NM_000157.3) ( https: / / www.ncbi.nlm.nih.gov / nuccore / NM_000157.3 ).

[0065] SEQ ID NO:3 is the genomic nucleic acid sequence of the MGAT1 gene in CHO-K1 cells.

[0066] SEQ ID NO: 4 is the nucleic acid sequence of sgRNA_5.

[0067] (abbreviation) Cas:CRISPR related CEX: Cation exchange CHO cells: Chinese hamster ovary cells CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats DSB: double strand break FACS: Fluorescence-activated cell sorting GnT I:N-acetyl-glucosaminyltransferase I gRNA: guide RNA HDR: Homologous recombination repair HILIC: Hydrophilic Interaction Liquid Chromatography LDC: limiting dilution cloning MGAT1: Mannosyl(α-1,3-)-recombinant glycoprotein β-1,2-N-acetylglucosaminyltransferase NHEJ: Non-homologous end joining ORF: Open reading frame ori: origin of replication PAM: protospacer adjacent motif PCR: polymerase chain reaction Qp: Specific productivity RT: Response vs. retrieval time sgRNA: single-stranded guide RNA SpCas9: Streptococcus pyogenes Cas9 TALEN: Transcription Activator-Like Effector Nucleases UPLC: Ultra-High Performance Liquid Chromatography VCD: variable cell concentration VIA: Survival rate ZFN: zinc finger nuclease 2-AB: 2-aminobenzamide

[0068] [Example] The invention having been generally described will be better understood with reference to the following examples, which are for illustrative purposes only and are not intended to limit the invention, and are not intended to represent that the following experiments are all or the only experiments performed.

[0069] [Example I] (Creation of CHO cells stably expressing β-glucocerebrosidase) Plasmids containing the nucleic acid sequence shown in SEQ ID NO:2 (NM_000157.3, available at https: / / www.ncbi.nlm.nih.gov / nuccore / NM_000157.3) for encoding β-glucocerebrosidase (https: / / www.ncbi.nlm.nih.gov / protein / NP_000148.2, SEQ ID NO:1), which has been deposited in GenBank under accession number NP_000148.2, and the two antibiotics, which have been commercially available from Thermo Fisher Scientific Inc., were transfected by lipofection into CHO-K1 cells cultured in BM001H medium, respectively.

[0070] Enzyme activity was measured using p-nitrophenyl-bD-glucopyranoside (Sigma-N7006) as substrate. The released product was 4-nitrophenol (Sigma-35836-1G) detected by measuring absorbance at 405 nm with a plate reader (MOLECULAR DEVICE, i3X). A reference standard curve was assayed in parallel to quantify the concentration of 4-nitrophenol. Enzyme activity is defined as the amount of enzyme used to catalyze the release of 1 nmole of 4-nitrophenol from p-nitrophenyl-bD-glucopyranoside per mL per hour at 37°C.

[0071] A stable pool with relatively high enzyme activity and good viability in batch refeed culture was selected and used for cloning, and the results are shown in Table 1.

[0072] [Table 1]

[0073] [Example II] Mutations in the MGAT1 gene in pools stably expressing β-glucocerebrosidase A plasmid containing Cas9 and gRNA targeting MGAT1 was constructed. After designing gRNA targeting MGAT1, it was ligated into an in-house vector containing Cas9 protein as shown in Figure 1. sgRNA5 as shown in SEQ ID NO:4 (sgRNA_5, TGACAATGGCAAGGAGAGA) was selected for further use.

[0074] The sgRNA_5 plasmid shown in FIG. 1 was transfected into the pool prepared by the method of Example I by commercially available lipofection.

[0075] RCAI (Castor Agglutinin) was also applied for clone screening because it was highly toxic to wild-type CHO-K1 cells and all mutants that survived from RCAI selection contained dysfunctional MGAT1. Once clones were recovered in 96-well plates, they were split into two plates, one plate A was cultured in normal medium and the other plate B was cultured in medium with RCA selection. Clones recovered from plate B in medium with RCAI were selected and screened by enzyme activity assay. After enzyme activity screening, monoclones with relatively high enzyme activity and confirmed monoclonality from single cell images were selected from plate A cultured in normal medium and propagated from plate A.

[0076] The monoclones were expanded sequentially from 96-well plates to 24-well plates and then transferred to spin tubes. Clones with poor growth during expansion were discarded. Meanwhile, MGAT1 mutation analysis was performed on all monoclones by sequencing, and clones with MGAT1 mutation were selected for batch refeed screening. At the time of inoculation, the cell culture was cultured at 5 × 10 in spin tubes. 5The cells were inoculated by dilution into fresh production medium at a concentration of 100000 cells / mL. The majority of the medium was refreshed daily with fresh production medium from day 3. When the VCD reached plateau, a bleeding process was performed daily, in which the VCD was adjusted to maintain the peak VCD the next day. Glucose was also added to the culture depending on the consumption. Since the majority of the medium was refreshed frequently, the cells could be cultivated under optimal conditions, and VIA is usually better maintained compared to the conventional fed-batch process. As a result, batch refeeding is more suitable for the production of fragile biopharmaceuticals, e.g., fusion proteins that are sensitive to endogenous proteases or enzymes in this case. The batch refeed supernatants of the clones were subjected to enzyme activity analysis. The enzyme activity of the clones was also measured using p-nitrophenyl-bD-glucopyranoside (Sigma-N7006) as a substrate. The released product was 4-nitrophenol (Sigma-35836-1G) detected by measuring absorbance at 405 nm with a plate reader (MOLECULAR DEVICE, i3X). A reference standard curve was assayed in parallel to quantify the concentration of 4-nitrophenol. Enzyme activity is defined as the amount of enzyme used to catalyze the release of 1 nmole of 4-nitrophenol from p-nitrophenyl-bD-glucopyranoside per mL per hour at 37°C. A clone prepared according to the method disclosed in Chinese patent application CN108588127A was used as a control.

[0077] The best clones with higher β-glucocerebrosidase enzyme activity and Qp were selected and their supernatants were purified by cation exchange chromatography and hydrophobic interaction chromatography. The purified enzymes were subjected to SEC, Caliper-SDS and N-glycan analysis.

[0078] The final clone was selected based on enzyme activity, Qp, cell culture performance, monoclonality, low copy number, and product quality including SEC, mannose level, and Caliper-SDS (non-reducing). The final clone showed a single nucleic acid insertion in the functional region of the original MGAT1 sequence, causing the dysfunction of the gene (Table 2).

[0079] [Table 2]

[0080] The final clones showed higher enzyme activity and better productivity than the control clones (Table 3). The average enzyme activity of the final clones was 0.54 U / mL, whereas that of the control clones was 0.26 U / mL. The average Qp of the final clones was 0.020 U / mL / cell / day, which was higher than that of the control clones.

[0081] [Table 3]

[0082] The non-reduced and reduced purity of β-glucocerebrosidase produced by the final clone was detected by Caliper-SDS assay using Caliper LC90 CE-SDS gel technology, PerkinElmer. We designed an automated chip-based fluorescence detection method that can analyze TIFF0007671780000004.tif11158 protein. As shown in the data, the purity of non-reduced β-glucocerebrosidase was 87.7%, while the purity of the control clone was 70.6% (Table 4).

[0083] [Table 4]

[0084] A specific SEC method was developed to analyze monomers and aggregates of the final clones. An Agilent AdvanceBio SEC column (300 Å, 7.8 mm × 300 mm, 2.7 μm) was used to separate the monomers and aggregates by applying an isocratic elution gradient. As shown in Table 5, the percentage of β-glucocerebrosidase monomers expressed from the final clones was higher than that of the control clones.

[0085] [Table 5]

[0086] The effect of MGAT1 gene mutation on the N-linked glycosylation of β-glucocerebrosidase was determined. N-glycan profiling was analyzed by UPLC method. N-oligosaccharides were released by fast peptide N-glycosidase F (PNGase F) enzyme and the released oligosaccharides were labeled with 2-AB. HILIC-based UPLC was applied for separation and quantification of N-glycans. A pool containing wild-type MGAT1 gene was used as a control.

[0087] As shown in Table 6 and Figure 2, of all the N-glycans attached to β-glucocerebrosidase expressed from the final clone, 89.3% were mannose-terminated N-glycans, i.e., N-linked Man3, Man4, Man4+1F, Man5, Man5+1F, Man6, etc. Compared with the control pool (Figure 3), the proportion of N-linked Man5 in the final clone was significantly increased, resulting in much higher Man levels. These data revealed that the mutation of MAGT1 gene in CHO cells expressing β-glucocerebrosidase significantly increased the proportion of mannose-terminated N-glycans attached to the recombinant β-glucocerebrosidase.

[0088] [Table 6]

[0089] [Example III] (Preparation of Formulations) The β-glucocerebrosidase produced from the final clone of Example II was formulated as shown in Table 7.

[0090] [Table 7]

[0091] The ingredients used in the formulation include citrate buffer 20 mM, sucrose 8% (w / v), 0.02% (w / v) PS80, pH 6.0.

[0092] Preparation of 1 kg of PS80 stock solution (10% w / w) The ingredients were weighed out at 100g PS80 and 900g water. The weighed ingredients were placed in a container. The solution was mixed until all the PS80 was visually dissolved.

[0093] Preparation of 1 kg of formulation buffer The ingredients were weighed out as follows: 0.64 g citric acid monohydrate, 4.81 g trisodium citrate dihydrate, 77.52 g sucrose, 1.94 g 10% (w / w) PS80 stock solution, and 915.09 g water. All weighed excipients were transferred to a 1 L container. Water was added until the total weight was 1000 g. The solution was mixed until all excipients were visually completely dissolved.

[0094] (Dilute and mix with 1L DS) Add formulation buffer to DS (e.g., enzyme specific activity 44.5U / mg and enzyme activity 4.1mg / mL) to complete mixing of protein (100U / mL). After pouring 0.55L of DS into a container, 0.45L of formulation buffer was also poured into the same container. Then, the diluted DS was mixed uniformly.

[0095] [Example IV] (Formulation Research) Table 8 shows the key performance study of the formulation. The study revealed that the formulation is stable for at least 12 weeks at 2-8° C., or at least 12 weeks at room temperature (about 25° C.), or at least 4 weeks at 40° C. The formulation can retain its physical and / or chemical and / or biological activity under different storage conditions.

[0096] [Table 8a]

[0097] [Table 8B]

[0098] [Table 8C]

[0099] (exterior) After cleaning the outer wall of the glass vial, the neck of the glass vial was brought close to the YB-2 light box, 25 cm away from the end of the gobo. The appearance of the samples, including color, transparency, and visible particles, were inspected against a black and white background at illumination levels of 2000 to 3750 lx.

[0100] (Osmotic pressure) The osmolality of 20 μL of undiluted sample was measured by Advanced 2020. Before and after the measurement, the osmometer was calibrated with a 290 mOsm reference solution.

[0101] (pH) The pH meter was calibrated with three different standard buffer solutions (pH 4.01, 7.00 and 9.21) before use, and then the pH was measured for each sample with a loading volume of 50 μL.

[0102] (moisture) Moisture content was measured with a Mettler Toledo C30D Karl Fischer coulometer. Room temperature was approximately 15-30°C, with air humidity below 50%. The analyte was added first and the instrument was balanced until the relative shift was less than 15 μg / min. The lyophilized powder was then opened, weighed, and added to the instrument until the instrument gave a moisture content reading. To avoid absorption of moisture from the air by the lyophilized powder, the steps from opening to weighing the powder must be performed quickly.

[0103] (reconfiguration time) The flip-off aluminum cap was removed from the glass vial, and ultrapure water was added from the wall of the vial with a sterile injector to avoid direct impact with the lyophilized sample. The vial was rotated slightly to allow the ultrapure water to penetrate completely, and then allowed to stand. The time it took for the ultrapure water to be injected until the lyophilized sample was completely dissolved was recorded.

[0104] (protein concentration) After the samples were mixed homogeneously, protein concentration was determined by UV280 measurements using a NanoDrop 2000 spectrophotometer. The extinction coefficient was 1.703AU*mL*mg -1 *cm -1 All measurements were repeated twice with a loading volume of 2.5 μL each time and the average values ​​were taken.

[0105] (CE-SDS(NR&R)) CE-SDS, driven by a high-voltage direct current electric field, is a capillary electrophoresis method that can separate samples by molecular size. The support was a continuous gel packed into a capillary, forming a molecular sieve within the capillary and acting as a separation channel. For CE-SDS, sample preparation prior to electrophoresis included heat denaturation of the sample in the presence of SDS at a specific concentration, which masked the intrinsic charge of the sample and gave all species a similar charge-to-size ratio. When a constant electric field was applied, the samples migrated towards the anode at different speeds depending on their size.

[0106] (CE-SDS-NR) The CE_NR method for WBP108 was performed under the following experimental conditions: 50 μg of sample was transferred to the diluent solution PB-CA for a total volume of 25 μL, then 75 μL of 1% SDS sample buffer and 5 μL of alkylation reagent were added for a final volume of 105 μL, and the solution was mixed thoroughly. The samples were incubated at 60° C. for 10 min in a heating block and then cooled at room temperature for at least 3 min. 90 μL of the prepared sample was transferred to the insert and the insert was placed in a vial. The vial was capped and then placed in the sample vial holder for sample analysis. The injection time was 40 s.

[0107] (CE-SDS-R) The CE_R method was performed under the following experimental conditions: 50 μg of sample was transferred to the diluent solution PB-CA to a total volume of 25 μL, then 75 μL of 1% SDS sample buffer and 5 μL of reducing reagent were added to a final volume of 105 μL, and the solution was mixed thoroughly. The samples were incubated at 60°C in a heat block for 10 min, then cooled at room temperature for at least 3 min. 90 μL of the prepared sample was transferred to the insert, and the insert was placed in a vial. After the vial was capped, the vial was placed in the sample vial holder for sample analysis. The injection time was 40 s.

[0108] (RP-HPLC) Reversed-phase chromatography (RPC) is an elution chromatography that uses a non-polar reversed-phase support as the stationary phase and an aqueous solution of a polar organic solvent as the mobile phase to separate and purify solutes based on the difference in polarity (hydrophobicity) of the solutes. The solutes are distributed on the surface of the stationary phase due to hydrophobic interactions. However, the surface of the RPC stationary phase is completely covered with non-polar groups and is strongly hydrophobic. Therefore, it is necessary to use a polar organic solvent (e.g., methanol, acetonitrile, etc.) or its aqueous solution for elution and separation.

[0109] The RP method was performed on an HPLC system under the following experimental conditions: Waters BioResolve™ PR mAb Polyphenyl, 450A, 2.7um, 2.1mm*150mm, 1 / pk is used as the column; 0.1% TFA in water and 0.1% TFA in ACN solution are applied as the mobile phase; 280nm is selected as the wavelength of the UV detector; the column temperature is set to 45°C; the sample is placed at 5°C; the isocratic flow rate is 0.3mL / min; the injection volume is 10μg and the run time is 27min.

[0110] (SEC-HPLC) Size Exclusion Chromatography (SEC), also known as gel filtration, separates molecules according to differences in size (hydrodynamic radius) as they pass through a column packed with SEC resin. SEC resin consists of a porous matrix of spherical particles designed not to interact with the molecules being separated. After being applied to the column, molecules larger than the pores are unable to diffuse into the beads and therefore elute first. Molecules smaller than the pore size can penetrate the pores to varying degrees based on their size.

[0111] The SEC method was performed by HPLC system under the following experimental conditions: Agilent AdvanceBio SEC (300Å, 7.8mm*300mm, 2.7μm) is used as the column; 55mM sodium citrate + 200mM L-arginine (pH5.5±0.1) is applied as the mobile phase; 280nm is selected as the wavelength of UV detector; the column temperature is set at 45℃; the sample is placed at 5℃; the isocratic flow rate is 0.6mL / min; the injection volume is 30μg, and the run time is 40min. With the exceptions shown below, this SEC method is applied to all studies in this report.

[0112] (DLS) Scattering occurs when the laser light passes through the particles due to the Brownian motion of the particles in the solution. The hydrodynamic radius of the particles can be calculated and obtained by measuring the diffusion coefficient of the particles in the solution with DLS. DLS analysis was performed on a Malvern ZEN3600. 70 μL of sample was added to a disposable cuvette in a biosafety hood for the experiment at 25 °C.

[0113] (HIAC) Sub-visible particles were monitored by the HIAC system. Each sample was run in four consecutive runs (1 mL / run). The results of the first run were discarded and the average of the remaining three runs was recorded. Finally, the data were analyzed using a soft TIFF0007671780000012.tif12161 was displayed as a number.

[0114] (Enzyme activity) The diluted enzyme was mixed with 20 mM p-NPG for 1 h at 37 °C. The reaction was stopped by adding 1 M glycine. The absorbance at 405 nm was measured to generate a signal proportional to the activity of the enzyme in the sample. A standard curve of the enzyme reaction product P-NP was plotted and regressed according to a linear regression model using SoftMax software. The OD values ​​of the experimental samples were compared to the standard curve to calculate the enzyme activity of the experimental samples.

[0115] Those skilled in the art will further appreciate that the present invention may be embodied in other specific forms without departing from its spirit or central attributes. It is to be understood that the above description of the present invention discloses only exemplary embodiments thereof, and that other variations are contemplated within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments described in detail herein. Rather, reference should be made to the following claims as indicating the scope and content of the present invention.

Claims

[Claim 1] 1. A pharmaceutical formulation comprising a therapeutically effective amount of β-glucocerebrosidase, a buffer, a osmolality adjusting agent, and a surfactant, 1. A pharmaceutical formulation comprising: β-glucocerebrosidase at a content of 100 U / mL; the buffer solution is a citrate buffer solution at a content of 5-6 g / L; the osmotic pressure regulator is a sucrose solution at a content of 80 g / L; the surfactant is a polysorbate 80 solution at a content of 0.2 g / L; and the pH of the formulation is 5.5-6.5.

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