Compositions comprising recombinant human hyaluronidase and variants thereof and related methods and uses

Abstract

The present disclosure provides compositions comprising a recombinant human hyaluronidase and variants thereof, wherein the variants are truncation variants or oxidation modification variants of the recombinant human hyaluronidase. Also provided are pharmaceutical formulations comprising the above compositions. Further provided are methods for detecting the above variants and uses of the above variants in quality control or quality assurance of recombinant human hyaluronidase products.

Description

Technical Field

[0001] This application relates to the field of biomedicine. Specifically, this disclosure provides compositions comprising recombinant human hyaluronidase and its variants, as well as related methods and applications. Background Technology

[0002] Recombinant human hyaluronidase (rHuPH20) has the unique function of breaking down hyaluronic acid. Hyaluronic acid is a polysaccharide widely found in human tissues, especially abundant in the skin, synovial fluid, and vitreous humor of the eye. Due to its excellent moisturizing and lubricating properties, hyaluronic acid has a wide range of applications in the medical and cosmetic fields. However, in some cases, such as cataract surgery or soft tissue injections, excessive hyaluronic acid can hinder drug diffusion and surgical procedures, requiring hyaluronidase to regulate its concentration.

[0003] Clinically, recombinant human hyaluronidase is mainly used to enhance local anesthesia, promote subcutaneous drug diffusion, assist in ophthalmic surgery, and treat adverse reactions caused by hyaluronic acid injections. Its use significantly improves the safety and efficiency of surgery and enhances the patient's treatment experience. Traditional hyaluronidase is extracted from animal testes, which is not only costly but may also cause immune reactions. The advent of recombinant human hyaluronidase has solved these problems.

[0004] As a drug, recombinant human hyaluronidase needs to maintain its necessary activity and efficacy. Quality control for drugs primarily involves controlling the content of the active ingredient and other related substances such as variants or impurities, especially ensuring that the content of these substances meets pharmaceutical requirements. Analysis and detection of variants or impurities of recombinant human hyaluronidase are of great significance for the development of effective products and formulations containing recombinant human hyaluronidase. Summary of the Invention

[0005] In a first aspect, this disclosure provides a composition comprising recombinant human hyaluronidase and a variant thereof, wherein the variant is recombinant human hyaluronidase in the R... 311 and S 312 The truncated variants are generated by the break between sites, wherein the content of the truncated variants is less than or equal to about 10% of the total amount of recombinant human hyaluronidase and its variants in the composition, as calculated based on the Lys-C enzyme digestion peptide mass fingerprint analysis method; and wherein the site numbering is based on the sequence shown in any one of SEQ ID NO: 1-4, for example based on the sequence shown in SEQ ID NO: 1.

[0006] In a specific implementation, based on the Lys-C enzyme digestion peptide mass fingerprint analysis method, the content of the truncated variant is less than or equal to about 10% of the total amount of recombinant human hyaluronidase and its variants in the composition.

[0007] In some embodiments, the content of recombinant human hyaluronidase is calculated based on Lys-C enzyme digestion peptide mass fingerprinting analysis, which is greater than or equal to about 90% of the total amount of recombinant human hyaluronidase and its variants in the composition.

[0008] In some embodiments, the content of the truncated variant is calculated based on the Lys-C enzyme cleavage peptide mass fingerprint analysis method to be greater than or equal to about 0.01%, greater than or equal to about 0.05%, greater than or equal to about 0.1%, greater than or equal to about 0.5%, greater than or equal to about 1%, greater than or equal to about 1.5%, greater than or equal to about 2%, greater than or equal to about 2.5%, or greater than or equal to about 3% of the total amount of recombinant human hyaluronidase and its variants in the composition.

[0009] In some embodiments, based on Lys-C enzyme cleavage peptide mass fingerprinting analysis, the content of the truncated variant is less than or equal to about 9%, less than or equal to about 8%, less than or equal to about 7%, less than or equal to about 6%, less than or equal to about 5%, less than or equal to about 4.5%, less than or equal to about 4%, less than or equal to about 3%, less than or equal to about 2%, less than or equal to about 1%, less than or equal to about 0.9%, or less than or equal to about 0.8%, less than or equal to about 0.7%, less than or equal to about 0.6%, less than or equal to about 0.5%, less than or equal to about 0.4%, less than or equal to about 0.3%, less than or equal to about 0.2%, or less than or equal to about 0.1%.

[0010] In some implementations, the content of the truncated variant can be any interval within the range defined by any two values ​​mentioned above, or any value within the interval.

[0011] In some embodiments, the composition further comprises another variant, which is recombinant human hyaluronidase at the M... 313 Oxidation-modified variants resulting from oxidation at position M and at position M 313 Oxidized variants resulting from oxidation at a site, wherein the sum of the contents of the oxidized variants, calculated based on Lys-C enzyme digestion peptide mass fingerprinting analysis, is less than or equal to about 15% of the total amount of recombinant human hyaluronidase and its variants in the composition; and wherein the site numbering is based on the sequence shown in any one of SEQ ID NO: 1-4, for example based on the sequence shown in SEQ ID NO: 1.

[0012] Preferably, based on the Lys-C enzyme digestion peptide mass fingerprint analysis method, the sum of the contents of the oxidatively modified variants is greater than or equal to about 0.01%, greater than or equal to about 0.05%, greater than or equal to about 0.1%, greater than or equal to about 0.5%, greater than or equal to about 1%, greater than or equal to about 1.5%, greater than or equal to about 2%, greater than or equal to about 2.5%, or greater than or equal to about 3%, greater than or equal to about 3.5%, greater than or equal to about 4%, greater than or equal to about 4.5%, or greater than or equal to about 5%, respectively.

[0013] In some embodiments, the recombinant human hyaluronidase comprises an amino acid sequence as shown in any one of SEQ ID NO: 1-4. In some embodiments, the full-length sequence of the recombinant human hyaluronidase is as shown in any one of SEQ ID NO: 1-4. In some embodiments, the recombinant human hyaluronidase is generated by truncating the N-terminus or C-terminus and / or modifying amino acid residues (including substitution, deletion, and insertion) based on the sequence shown in SEQ ID NO: 1.

[0014] In some specific implementations, the sequence of the truncated variant is as shown in SEQ ID NO: 5.

[0015] In some embodiments, the step of calculating the content of the truncated variant using the Lys-C enzyme-based peptide mass fingerprinting analysis method includes: The composition was digested with Lys-C enzyme and then subjected to peptide mass fingerprinting analysis by LC-MS / MS. After obtaining mass spectrometry data by LC-MS / MS analysis, the extractable ion current chromatograms (XICs) of the two charge forms with the strongest response of the truncated peptide and its corresponding native peptide were extracted. The peak areas of each were obtained by integration, and the proportion of truncated variants was calculated according to the following formula: .

[0016] In some specific implementations, the XIC peak area of ​​the truncated peptide is the peak area obtained by integrating the XIC plot of the truncated modified peptide (hereinafter referred to as "truncated peptide", the sequence of which is shown in SEQ ID NO:7) extracted from the mass spectrometry data of the sample, and the XIC peak area of ​​the native peptide is the peak area obtained by integrating the XIC plot of the untruncated modified peptide (hereinafter referred to as "native peptide", the sequence of which is shown in SEQ ID NO:6).

[0017] In some embodiments, the step of calculating the content of oxidative variants using the Lys-C enzyme-based peptide mass fingerprinting analysis method includes: After obtaining mass spectrometry data through LC-MS / MS analysis, the extractable ion current chromatograms (XICs) of the two charge forms with the strongest responses of the oxidized peptide and its corresponding native peptide were extracted. The peak areas of each were obtained by integration, and the content of the oxidized variant was calculated according to the following formula: .

[0018] In some specific embodiments, the oxidized modified peptide is the peptide shown in SEQ ID NO:8 or SEQ ID NO:9. In specific embodiments, the XIC peak area of ​​the oxidized modified peptide is the sum of the peak area corresponding to the peptide shown in SEQ ID NO:8 and the peak area corresponding to the peptide shown in SEQ ID NO:9, and the XIC peak area of ​​the native peptide is the peak area obtained by integrating the XIC plot of the untruncation modified peptide (hereinafter referred to as "native peptide", the sequence of which is shown in SEQ ID NO:6).

[0019] In a second aspect, this disclosure provides a composition comprising recombinant human hyaluronidase and a variant thereof, wherein the variant is recombinant human hyaluronidase in the M... 310 Oxidation-modified variants resulting from oxidation at position M and at position M 313 Oxidized variants resulting from oxidation at a site, wherein the sum of the contents of the oxidized variants, calculated based on Lys-C enzyme digestion peptide mass fingerprinting analysis, is less than or equal to about 15% of the total amount of recombinant human hyaluronidase and its variants in the composition; and wherein the site numbering is based on the sequence shown in any one of SEQ ID NO: 1-4, for example based on the sequence shown in SEQ ID NO: 1.

[0020] In a specific implementation scheme, based on the Lys-C enzyme digestion peptide quality fingerprint analysis method, the sum of the contents of the oxidative modified variants is less than or equal to about 15% of the total amount of recombinant human hyaluronidase and its variants.

[0021] In some embodiments, the content of recombinant human hyaluronidase in the composition is calculated based on Lys-C enzyme digestion peptide mass fingerprinting analysis, for example, based on Lys-C enzyme digestion peptide mass fingerprinting analysis, to be greater than or equal to about 85% of the total amount of recombinant human hyaluronidase and its variants.

[0022] In some embodiments, based on the Lys-C enzyme digestion peptide mass fingerprint analysis, the sum of the contents of the oxidatively modified variants is greater than or equal to about 0.01%, greater than or equal to about 0.05%, greater than or equal to about 0.1%, greater than or equal to about 0.5%, greater than or equal to about 1%, greater than or equal to about 1.5%, greater than or equal to about 2%, greater than or equal to about 2.5%, or greater than or equal to about 3%, greater than or equal to about 3.5%, greater than or equal to about 4%, greater than or equal to about 4.5%, or greater than or equal to about 5%, of the total amount of recombinant human hyaluronidase and its variants.

[0023] In some embodiments, based on Lys-C enzyme digestion peptide mass fingerprint analysis, the sum of the contents of the oxidized modified variants is less than or equal to about 14%, less than or equal to about 13%, less than or equal to about 12%, less than or equal to about 11%, less than or equal to about 10%, less than or equal to about 9%, less than or equal to about 8%, less than or equal to about 7%, less than or equal to about 6%, less than or equal to about 5%, less than or equal to about 4%, less than or equal to about 3%, less than or equal to about 2%, less than or equal to about 1%, less than or equal to about 0.9%, or less than or equal to about 0.8%, less than or equal to about 0.7%, less than or equal to about 0.6%, less than or equal to about 0.5%, less than or equal to about 0.4%, less than or equal to about 0.3%, less than or equal to about 0.2%, or less than or equal to about 0.1%.

[0024] In some embodiments, the content of the oxidized variant can be any range within the range defined by any two values ​​mentioned above, or any value within the range.

[0025] In some embodiments, the composition further comprises another variant, which is recombinant human hyaluronidase in the R... 311 and S 312 The truncated variants are generated by breaks between sites, wherein the content of the truncated variants, calculated based on Lys-C enzyme cleavage peptide mass fingerprinting analysis, is less than or equal to about 10% of the total amount of recombinant human hyaluronidase and its variants in the composition; and wherein the site numbering is based on the sequence shown in any one of SEQ ID NO: 1-4. Preferably, the content of the truncated variants, calculated based on Lys-C enzyme cleavage peptide mass fingerprinting analysis, is greater than or equal to about 0.01%, greater than or equal to about 0.05%, greater than or equal to about 0.1%, greater than or equal to about 0.5%, greater than or equal to about 1%, greater than or equal to about 1.5%, greater than or equal to about 2%, greater than or equal to about 2.5%, or greater than or equal to about 3% of the total amount of recombinant human hyaluronidase and its variants in the composition.

[0026] In some embodiments, the recombinant human hyaluronidase comprises an amino acid sequence as shown in any one of SEQ ID NO: 1-4. In some embodiments, the full-length sequence of the recombinant human hyaluronidase is as shown in any one of SEQ ID NO: 1-4. In some embodiments, the recombinant human hyaluronidase is generated by truncating the N-terminus or C-terminus and / or modifying amino acid residues (including substitution, deletion, and insertion) based on the sequence shown in SEQ ID NO: 1.

[0027] In some embodiments, the oxidative modification variant is the oxidation of methionine (M) at position 310 of the sequence shown in any of SEQ ID NO: 1-4.

[0028] In some embodiments, the oxidative modification variant is the oxidation of methionine (M) at position 313 of the sequence shown in any of SEQ ID NO: 1-4.

[0029] In some embodiments, the step of calculating the content of oxidative variants using the Lys-C enzyme-based peptide mass fingerprinting analysis method includes: After obtaining mass spectrometry data through LC-MS / MS analysis, the extractable ion current chromatograms (XICs) of the two charge forms with the strongest responses of the oxidized peptide and its corresponding native peptide were extracted. The peak areas of each were obtained by integration, and the content of the oxidized variant was calculated according to the following formula: .

[0030] In some specific embodiments, the oxidized modified peptide is the peptide shown in SEQ ID NO:8 or SEQ ID NO:9. In specific embodiments, the XIC peak area of ​​the oxidized modified peptide is the sum of the peak area corresponding to the peptide shown in SEQ ID NO:8 and the peak area corresponding to the peptide shown in SEQ ID NO:9, and the XIC peak area of ​​the native peptide is the peak area obtained by integrating the XIC plot of the untruncation modified peptide (hereinafter referred to as "native peptide", the sequence of which is shown in SEQ ID NO:6).

[0031] In some embodiments, the step of calculating the content of the truncated variant using the Lys-C enzyme-based peptide mass fingerprinting analysis method includes: The composition was digested with Lys-C enzyme and then subjected to peptide mass fingerprinting analysis by LC-MS / MS. After obtaining mass spectrometry data by LC-MS / MS analysis, the extractable ion current chromatograms (XICs) of the two charge forms with the strongest response of the truncated peptide and its corresponding native peptide were extracted. The peak areas of each were obtained by integration, and the proportion of truncated variants was calculated according to the following formula: .

[0032] In some specific implementations, the XIC peak area of ​​the truncated peptide is the peak area obtained by integrating the XIC plot of the truncated modified peptide (hereinafter referred to as "truncated peptide", the sequence of which is shown in SEQ ID NO:7) extracted from the mass spectrometry data of the sample, and the XIC peak area of ​​the native peptide is the peak area obtained by integrating the XIC plot of the untruncated modified peptide (hereinafter referred to as "native peptide", the sequence of which is shown in SEQ ID NO:6).

[0033] Thirdly, this disclosure provides pharmaceutical formulations comprising the compositions described in the first and / or second aspects, and one or more pharmaceutically acceptable carriers. In some embodiments, the pharmaceutical formulations further comprise other pharmaceutically active ingredients; preferably, the pharmaceutically active ingredients are selected from the group consisting of immunoglobulins, recombinant proteins, synthetic peptides, RNA, DNA, and chemical drugs.

[0034] Fourthly, this disclosure provides a method for preparing the pharmaceutical formulation of the third aspect, comprising the following steps: (1) Preparing a composition comprising recombinant human hyaluronidase and variants thereof, wherein the variant is a truncated variant as defined in the first aspect above or an oxidatively modified variant as defined in the second aspect above, and (2) Evaluate the variant in the composition and confirm that the content of the variant does not significantly affect the activity of recombinant human hyaluronidase.

[0035] For example, the content of the truncated variant should be less than or equal to about 10% of the total amount of recombinant human hyaluronidase and its variants in the composition, and the content of the oxidatively modified variant should be less than or equal to about 15% of the total amount of recombinant human hyaluronidase and its variants in the composition.

[0036] In some embodiments, step (2) above includes performing peptide mass fingerprinting analysis on the composition to obtain XIC spectra of variant peptides and native peptides, and calculating the content of the variant based on the corresponding peak areas.

[0037] In some embodiments, the method further includes combining the composition following step (2) with a pharmaceutically acceptable carrier.

[0038] Fifthly, this disclosure provides a method for detecting a variant of recombinant human hyaluronidase in a composition or pharmaceutical formulation containing recombinant human hyaluronidase, wherein the variant is a variant of recombinant human hyaluronidase in step R. 311 and S 312 The method comprises: a truncated variant resulting from a break between positions, wherein the above-mentioned site numbering is based on the sequence shown in any one of SEQ ID NO: 1-4; The composition or pharmaceutical preparation is enzymatically digested and then subjected to peptide mass fingerprint analysis, for example, using LC-MS / MS to obtain XIC spectra of truncated peptides and native peptides, and the content of the truncated variant is calculated based on the corresponding peak areas.

[0039] Sixthly, this disclosure provides a method for detecting a recombinant human hyaluronidase variant in a composition or pharmaceutical formulation containing recombinant human hyaluronidase, wherein the variant is a variant that is present in the Mth... 310 Oxidation-modified variants and M resulting from oxidation at the site. 313 Oxidation-modified variants resulting from oxidation at a site, wherein the site numbering is based on the sequence shown in any one of SEQ ID NO: 1-4, the method comprising: The composition or pharmaceutical preparation is enzymatically digested and then subjected to peptide mass fingerprint analysis, for example, using LC-MS / MS to obtain XIC spectra of oxidized modified peptides and native peptides, and the content of the oxidized modified variants is calculated based on the corresponding peak areas. In a seventh aspect, this disclosure provides a method for quality inspection or quality control of products containing recombinant human hyaluronidase, comprising detecting the content of a recombinant human hyaluronidase variant in the product. In some embodiments, the variant is recombinant human hyaluronidase at step R... 311 and S 312 A truncated variant resulting from a break between positions. In some embodiments, the variant is recombinant human hyaluronidase at position M. 310 Oxidation-modified variants and M resulting from oxidation at the site. 313 Oxidation-modified variants resulting from oxidation at the site. The site numbers above are based on the sequences shown in any one of SEQ ID NO: 1-4.

[0040] In some embodiments, the product containing recombinant human hyaluronidase is a composition described in the first or second aspect or a pharmaceutical preparation described in the third aspect.

[0041] In some embodiments, if the content of the truncated variant is detected to be less than or equal to about 10% of the total amount of recombinant human hyaluronidase and its variants in the composition, it indicates that the recombinant human hyaluronidase meets pharmaceutical requirements.

[0042] In some embodiments, if the content of the oxidized variant is detected to be less than or equal to about 15% of the total amount of recombinant human hyaluronidase and its variants in the composition, it indicates that the recombinant human hyaluronidase meets pharmaceutical requirements.

[0043] Eighthly, this disclosure provides the use of recombinant human hyaluronidase variants in the quality inspection or quality control of products containing recombinant human hyaluronidase. In some embodiments, the variant is recombinant human hyaluronidase in the R... 311 and S 312 A truncated variant resulting from a break between positions. In some embodiments, the variant is recombinant human hyaluronidase at position M. 310 Oxidation-modified variants and M resulting from oxidation at the site. 313 Oxidation-modified variants resulting from oxidation at the site. The site numbers above are based on the sequences shown in any one of SEQ ID NO: 1-4.

[0044] In some embodiments, the product containing recombinant human hyaluronidase is a composition described in the first or second aspect or a pharmaceutical preparation described in the third aspect. Attached Figure Description

[0045] Figure 1 The unconvolution plot of the non-reducing intact molecular weight analysis of recombinant human hyaluronic acid cleavage of N-glycans is shown. Note: "Dea" indicates deamidation modification; "-Y" indicates C-terminal truncation of Y; "R" indicates deamidation modification. 311 / S 312 "For the occurrence of R" 311 / S 312 Fracture modification; the number in parentheses indicates the number of modifications that occur in this component.

[0046] Figure 2 The results showed that after digestion of recombinant human hyaluronidase samples with Lys-C enzyme, F 280 -R 311 The mass spectrum results of peptide detection, where A is the first-order mass spectrum result and B is the second-order mass spectrum result.

[0047] Figure 3 The standard curves plotted against the average absorbance of recombinant human hyaluronidase with different activities are shown. The formula for the step curve is: y = 0.412 - 0.008x, R 2 =0.989.

[0048] Figure 4 Typical RP-HPLC component collection chromatograms are shown, which are typical component collection chromatograms of recombinant human hyaluronidase sample main peak and impurities before the main peak collected simultaneously using the Aglient 1260 liquid chromatography automatic collection module.

[0049] Figure 5The results show the unconvolution chromatograms of the impurity peaks separated and collected by RP-HPLC from the recombinant human hyaluronidase sample, as well as the unconvolution chromatograms of the N-glycans in the original sample. In the figure, A represents the unconvolution chromatogram of the N-glycans in the impurity peaks, and B represents the unconvolution chromatogram of the N-glycans in the original sample. Note: "Dea" indicates deamidation modification; "-Y" indicates C-terminal truncation of Y; "-FY" indicates C-terminal truncation of FY; "-IFY" indicates C-terminal truncation of IFY; Oxidation indicates oxidation modification; the numbers in parentheses indicate the number of modifications in that component.

[0050] Figure 6 Impurity peaks collected by RP-HPLC were separated from sample M. 310 With M 313 Secondary mass spectrometry analysis results of oxidatively modified peptides, where A represents M. 310 Secondary mass spectrometry analysis results of oxidatively modified peptides, B represents M. 313 Secondary mass spectrometry analysis results of oxidatively modified peptides.

[0051] Figure 7 The RP-HPLC localization confirmation chromatogram of the collected components is shown.

[0052] Figure 8 Typical RP-HPLC chromatograms of the illuminated sample and the control sample are shown.

[0053] Sequence Description SEQ ID NO:1 is the amino acid sequence of human hyaluronidase with 447 amino acid residues: LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY SEQ ID NO:2 is the amino acid sequence of human hyaluronidase with one amino acid (i.e., tyrosine (Y)) deleted at the C-terminus of the polypeptide sequence shown in SEQ ID NO:1: LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIF SEQ ID NO:3 is the amino acid sequence of human hyaluronidase with two amino acids (tyrosine (Y) and phenylalanine (F)) deleted from the C-terminus of the polypeptide sequence shown in SEQ ID NO:1. LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVID WEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNEST ALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIIN VTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQI SEQ ID NO:4 is the amino acid sequence of human hyaluronidase with three amino acids (tyrosine (Y), phenylalanine (F), and isoleucine (I)) deleted from the C-terminus of the polypeptide sequence shown in SEQ ID NO:1. LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVID WEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNEST ALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIIN VTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQ SEQ ID NO:5 is the Rth sequence in the above SEQ ID NO:4 sequence. 311 and S 312 An exemplary truncated variant sequence resulting from a break between amino acid residues. This R... 311 and S 312 Cleavages between amino acid residues can occur in polypeptides including, but not limited to, those shown in SEQ ID NO: 1-4, and in polypeptide sequences derived therefrom by N-terminal or C-terminal truncation and / or modification of amino acid residues (including substitution, deletion, and insertion). 311 and S 312 Even after a break occurs between molecules, they can still pass through C. 25 =C 316 Disulfide bonds linking them together (indicated by underscores in the sequence). The structure and amino acid sequence of the truncated variant (break sites are indicated by " / ") are exemplarily shown here using the sequence SEQ ID NO:4: LNFRAPPVIPNVPFLWAWNAPSEF CLGKFDEPLDMSLFSFIGSPINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVET IKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMR 311 / SMKS C LLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQ SEQ ID NO:6 is the sequence of the native peptide used in calculating the variant content in Examples 4 and 5, corresponding to amino acids 280-314 of any one of the sequences SEQ ID NO:1-4 above: FLSQDELVYTFGETVALGASGIVIWGTLSIM R 311 S 312 MK SEQ ID NO:7 is the sequence of the truncated peptide used in calculating the content of the truncated variant in Example 4, corresponding to amino acids 280-311 of any of the sequences SEQ ID NO:1-4 above: FLSQDELVYTFGETVALGASGIVIWGTLSIM R 311 SEQ ID NO:8 is the sequence of the oxidized peptide used in calculating the content of the oxidized variant in Example 5, corresponding to amino acids 280-314 of any one of the sequences SEQ ID NO:1-4 above, wherein the methionine at position 310 is oxidized (Oxi). FLSQDELVYTFGETVALGASGIVIWGTLSI M 310 (Oxi) R SEQ ID NO:9 is the sequence of the oxidized peptide used in calculating the content of the oxidized variant in Example 5, corresponding to amino acids 280-314 of any one of the sequences SEQ ID NO:1-4 above, wherein methionine at position 313 is oxidized (Oxi). FLSQDELVYTFGETVALGASGIVIWGTLSIMRS M 313 (Oxi) K Detailed Implementation

[0054] Hyaluronidase is a general term for enzymes that hydrolyze hyaluronic acid. It is an enzyme that can reduce the activity of hyaluronic acid in the body, thereby increasing the permeability of tissues. Many pathological processes are often accompanied by changes in hyaluronidase and hyaluronic acid, which can alter the distribution of some drugs and physiologically active substances in the body.

[0055] The term "recombinant human hyaluronidase" as used herein refers to human hyaluronidase expressed through genetic engineering, also known as hyaluronidase. Exemplary recombinant human hyaluronidases may be selected from the polypeptides of rHuPH20 shown in any of SEQ ID NO:1-4. Hyaluronidases may also include those containing chemical or post-translational modifications and those not containing chemical or post-translational modifications. Such modifications include, but are not limited to, PEGylation, albuminization, glycosylation, farnesylation, carboxylation, hydroxylation, phosphorylation, and other polypeptide modifications known in the art.

[0056] Both hyaluronidase and recombinant human hyaluronidase have the function of breaking down hyaluronic acid and are widely used in the medical and cosmetic fields. Recombinant human hyaluronidase is produced using genetic engineering technology, offering higher safety and production efficiency, and avoiding the limitations of traditional extraction methods. Recombinant human hyaluronidase is produced by inserting the human hyaluronidase gene into a suitable expression vector, followed by expression in host cells (such as mammalian cells or bacteria). It has been found that during the recombinant expression of human hyaluronidase, a deletion of 1-3 amino acids at the C-terminus may occur, and this deletion does not affect enzyme activity. For example, SEQ ID NO:2 lacks tyrosine (Y) at the C-terminus of SEQ ID NO:1; SEQ ID NO:3 lacks tyrosine (Y) and phenylalanine (F) at the C-terminus of SEQ ID NO:1; and SEQ ID NO:4 lacks tyrosine (Y), phenylalanine (F), and isoleucine (I) at the C-terminus of SEQ ID NO:1. Besides the recombinant human hyaluronidase shown in SEQ ID NO:1, the sequences shown in SEQ ID NO:2-4 are also common expression forms of recombinant human hyaluronidase. These forms can coexist in recombinant human hyaluronidase samples or preparations. In addition, existing technologies have conducted various N-terminal and / or C-terminal truncation and amino acid residue modifications (including substitution, deletion and insertion) based on the polypeptide sequence of recombinant human hyaluronidase shown in SEQ ID NO:1. For example, truncation to positions 1-432 of the sequence shown in SEQ ID NO:1 can still retain hyaluronidase activity (see, for example, Chinese patents CN104244968B, CN112203642B, and Gregory I Frost; Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration; Expert Opinion on Drug Delivery, 2007, 4(4), 427-440). Therefore, the “recombinant human hyaluronidase” referred to in this article includes rHuPH20 polypeptides having the amino acid sequence shown in any one of SEQ ID NO:1-4, or having been truncated at the N-terminus or C-terminus based on the amino acid sequence shown in SEQ ID NO:1, or having been modified (including substitution, deletion, and insertion) of amino acid residues based on the amino acid sequence shown in SEQ ID NO:1.

[0057] The inventors of this application have unexpectedly discovered that other variants of human hyaluronidase, such as truncated variants or oxidatively modified variants, are also generated during the recombinant expression of human hyaluronidase.

[0058] In some embodiments, the variant is recombinant human hyaluronidase at the Rth amino acid sequence corresponding to any of the amino acid sequences shown in SEQ ID NO:1-4. 311 and S 312 Truncation variants are produced by the breakage between amino acid residues at position R. 311 and S 312 After a break occurs between amino acid residues, the polypeptide fragments on both sides of the break site can still pass through the C1 amino acid sequence corresponding to any of the amino acid sequences shown in SEQ ID NO:1-4. 25 and C 316 Disulfide bonds (C16) between amino acid residues 25 =C 316 The amino acid sequence of an exemplary recombinant human hyaluronidase truncated variant is shown in SEQ ID NO:5. The inventors also discovered that a content of less than or equal to approximately 10% of the total amount of recombinant human hyaluronidase and its variants does not significantly affect the activity of the recombinant human hyaluronidase sample. Therefore, if the variant is assessed or detected to be no higher than approximately 10% during the preparation of recombinant human hyaluronidase, there is no need to perform a process to remove the variant or impurities, which simplifies the production process and saves production costs to some extent.

[0059] In some embodiments, the variant is recombinant human hyaluronidase at sequence M corresponding to any one of SEQ ID NO: 1-4. 310 Oxidation-modified variants and M resulting from oxidation at the site. 313 Oxidation-modified variants resulting from oxidation at the site.

[0060] In some embodiments, the oxidative modification variant is the oxidation of methionine (M) at position 310 of the sequence corresponding to any one of SEQ ID NO: 1-4. In some embodiments, the oxidative modification variant is the oxidation of methionine (M) at position 313 of the sequence corresponding to any one of SEQ ID NO: 1-4. In some embodiments, the oxidative modification variant is an oxidative modification variant resulting from the oxidation of methionine (M) at position 313 of the sequence corresponding to any one of SEQ ID NO: 1-4. The side chain structure of methionine is -CH2-CH2-S-CH3, wherein the sulfur atom (S) is oxidized to generate sulfoxide, thus becoming -CH2-CH2-SO-CH3. Oxidation of methionine can lead to changes in the secondary, tertiary, or quaternary structure of proteins, thereby affecting their function. For example, the activity of recombinant human hyaluronic acid may be reduced or lost due to oxidation.

[0061] The inventors also discovered that the content of the aforementioned oxidized variants being less than or equal to approximately 15% of the total amount of recombinant human hyaluronidase and its variants does not significantly affect the activity of the recombinant human hyaluronidase sample. Therefore, if the oxidized variants are assessed or detected to be no higher than approximately 15% during the preparation of recombinant human hyaluronidase, there is no need to perform operations to remove variants or impurities, which simplifies the production process and saves production costs to some extent.

[0062] In some embodiments, the compositions described herein comprise recombinant human hyaluronidase and its truncated variants (e.g., as shown in SEQ ID NO:5).

[0063] In some embodiments, the compositions described herein comprise recombinant human hyaluronidase and its oxidatively modified variants (e.g., the Mth variant corresponding to the sequence shown in any one of SEQ ID NO: 1-4). 310 Oxidation-modified variants and M resulting from oxidation at the site. 313 Oxidation-modified variants resulting from oxidation at the site.

[0064] In some implementations, the aforementioned truncated variants or oxidatively modified variants in recombinant human hyaluronidase products can be reduced or removed by methods commonly used in the art, such as ion exchange chromatography, hydrophobic chromatography, complex chromatography, or reversed-phase HPLC.

[0065] In some implementations, products containing recombinant human hyaluronidase, such as compositions or pharmaceutical formulations containing recombinant human hyaluronidase, are subjected to peptide mass fingerprinting analysis after enzymatic digestion to detect variants in the recombinant human hyaluronidase product.

[0066] In the specific implementation plan, the above-mentioned enzymatic digestion is performed using Lys-C enzyme. Lys-C enzyme (Lysylendopeptidase) is an endonuclease that specifically cleaves the carboxyl side of Lys (lysine) residues in proteins. It is commonly used in protein digestion analysis to study protein structure and function.

[0067] In some implementations, liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis is used to detect variants in recombinant human hyaluronidase products. For example, LC-MS / MS is used to perform peptide mass fingerprinting analysis on the test sample. LC-MS / MS is a highly sensitive and selective analytical technique that combines the physical separation capabilities of liquid chromatography (LC) with the precise detection capabilities of tandem mass spectrometry (MS / MS). It allows for efficient separation of components in complex samples via chromatographic columns, while high-resolution mass spectrometry can provide information on component composition, modification sites, and modification types through mass spectrum analysis. This technique combines the high separation capabilities of liquid chromatography (LC) with the high sensitivity and specificity of mass spectrometry (MS), making it a core tool for drug analysis, metabolic research, and quality control.

[0068] In a specific implementation plan, after obtaining mass spectrometry data through LC-MS / MS analysis, the extractable ion current chromatograms (XICs) of the two charge forms with the strongest responses of the truncated peptide and its corresponding native peptide are extracted. The peak areas of each are integrated to obtain their respective peak areas, and the content of the truncated variant is calculated according to the following formula: .

[0069] In some embodiments, the truncated peptide is the peptide shown in SEQ ID NO:7, and the native peptide is the peptide shown in SEQ ID NO:6.

[0070] In a specific implementation plan, after obtaining mass spectrometry data through LC-MS / MS analysis, the extractable ion current chromatograms (XICs) of the two charge forms with the strongest responses of the oxidized peptide and its corresponding native peptide are extracted, the peak areas of each are integrated, and the content of the oxidized variant is calculated according to the following formula: .

[0071] In some embodiments, the oxidized peptide is the peptide shown in SEQ ID NO:8 or SEQ ID NO:9. In a specific embodiment, the XIC peak area of ​​the oxidized peptide is the sum of the peak area corresponding to the peptide shown in SEQ ID NO:8 and the peak area corresponding to the peptide shown in SEQ ID NO:9. In some embodiments, the native peptide is the peptide shown in SEQ ID NO:6.

[0072] In some embodiments, the content of the truncated variant (e.g., as shown in SEQ ID NO:5) in the compositions disclosed herein comprising recombinant human hyaluronidase and its truncated variants is about 0.01% to about 10% of the total amount of recombinant human hyaluronidase and its variants, or any range within the above range or any value within the range.

[0073] In some embodiments, the oxidized variants (e.g., sequence M shown in any one of SEQ ID NO: 1-4) of the compositions disclosed herein comprising recombinant human hyaluronidase and its oxidized variants are described. 310 and M 313 The content of the oxidized variants (resulting from oxidation of one of the sites) is about 0.01% to about 15% of the total amount of recombinant human hyaluronidase and its variants, or any interval or any value within the range described above.

[0074] In this specification and claims, the term "about" means a range of values ​​that a person skilled in the art would consider equivalent to the listed values ​​(e.g., having the same function or result), such as + / - 10% of the listed values. For example, regarding the limitation that the content of the truncated variant is less than or equal to about 10% of the total amount of recombinant human hyaluronidase and its variants, a person skilled in the art would understand that if the content of the truncated variant is 11%, it is also within the scope of protection claimed in this application.

[0075] In a specific embodiment, the content of the aforementioned truncated variant is 3%-11% of the total amount of recombinant human hyaluronidase and its variants, for example, 3.26%-10.96%. In another specific embodiment, the content of the aforementioned oxidatively modified variant is 5%-16% of the total amount of recombinant human hyaluronidase and its variants, for example, 5.31%-15.42%.

[0076] This document also discloses pharmaceutical formulations for preparing the disclosed compositions by mixing the compositions with an optional pharmaceutically acceptable carrier and storing them as lyophilized formulations or aqueous solutions. The pharmaceutically acceptable carrier is non-toxic to the recipient at the dosage and concentration used. In some embodiments, the pharmaceutically acceptable carrier includes water; buffers such as phosphates, citrates, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives; hydrophilic polymers such as polyvinylpyrrolidone; chelating agents such as EDTA, etc. In some embodiments, the formulation for in vivo administration must be sterile. This can be easily achieved by using sterile filter membrane filtration.

[0077] In this specification and claims, the words “comprising,” “including,” and “containing” mean “including but not limited to” and are not intended to exclude other parts, additives, components, or steps.

[0078] It should be understood that the features, characteristics, components or steps described in a particular aspect, embodiment or example of this application may be applied to any other aspect, embodiment or example described herein, unless there is any contradiction.

[0079] The foregoing disclosure generally describes the present invention, and the following embodiments further illustrate the invention. These embodiments are described merely to illustrate the invention and not to limit its scope. Although specific terms and values ​​are used herein, they are also understood to be exemplary and do not limit the scope of the invention. Unless specifically indicated, the experimental methods and techniques described herein are methods and techniques well known to those skilled in the art.

[0080] Example Example 1: Construction and screening of cell lines expressing recombinant human hyaluronidase The nucleic acid sequence (amino acid sequence as shown in SEQ ID NO: 1) encoding the disclosed recombinant human hyaluronidase was synthesized by a gene synthesis company and ligated to the multiple cloning insertion site of the expression vector. After construction was completed and sequenced to verify its correctness, the plasmid was extracted.

[0081] The recombinant expression plasmid was introduced into CHOZN via electroporation. ® GS - / - In blank cells, transfected cells were seeded into 96-well plates at a density of 3000-10000 cells / well. After seeding, the 96-well plates were placed in a CO2 incubator for static culture at 36.5℃ and 5% CO2. Once the cells had filled the wells, expression levels were assessed using dot-blot technology, and pools with higher expression levels were selected for expansion into 24-well plates. Through stepwise expansion and selection using 96-well plates, 24-well plates, shake tubes, and shake flasks, high-yield cell pools were selected for fed-batch culture in shake flasks. Based on the combined cell count and yield results, high-yield cell pools were selected for single-clone cell line selection.

[0082] Using a limiting dilution method, cells with higher expression levels were seeded into 96-well plates at an average density of <0.5 cells / well. After seeding, the 96-well plates were placed in a CO2 incubator for static culture at 36.5℃, 5% CO2, and 80% humidity. Once the cells had filled the wells, the cells were progressively expanded and screened using 96-well plates, 24-well plates, shake tubes, and shake flasks. High-yielding monoclonal cell lines were ultimately selected for fed-batch culture in shake flasks. Based on the combined results of cell growth, expression levels, and quality, a monoclonal cell line expressing recombinant human hyaluronidase was finally obtained.

[0083] Example 2: Preparation of recombinant human hyaluronidase using cell pool cells In this embodiment, fed-batch culture was performed in a bioreactor. The cell pool cells prepared in Example 1 were thawed in a 37°C water bath and then cultured in basal medium. The cells were passaged every 2-4 days until an appropriate volume was reached in the basal medium, after which they were inoculated into the bioreactor at a rate of 0.3 × 10⁻⁶ cells / day. 6 -1×10 6 Cells were seeded at a density of [number] cells / ml and cultured at a temperature of 36.5±1℃, a pH of 6.8-7.2, and a stirring rate of 250 rpm. Feeding was performed intermittently from day 4±1, with the feed volume being 3-7% of the initial culture volume. On day 6±2, the culture temperature was adjusted to 32.5±1℃ and maintained until the end of the culture. Samples were taken periodically during culture, and cell viability and density were monitored using CountStar. The recombinant human hyaluronidase content in the cell culture medium was detected by RP-HPLC. The recombinant human hyaluronidase activity in the cell culture medium was detected by turbidimetric assay.

[0084] The harvested cell culture medium is subjected to deep filtration and sterile filtration to obtain a clear cell culture medium; the clear cell culture medium is then purified by steps including anion exchange chromatography, affinity chromatography, and hydrophobic chromatography to prepare a stock solution.

[0085] Example 3: Production of recombinant human hyaluronidase using a monoclonal cell line In this embodiment, fed-batch culture was performed in a bioreactor. The monoclonal cell lines prepared in Example 1 were thawed in a 37°C water bath and then cultured in basal medium. Subculture was performed every 2-4 days. After reaching an appropriate volume in basal medium, the cells were inoculated into the bioreactor at a rate of 0.3-1 × 10⁻⁶ cells / day. 6 Cells were seeded at a density of [number] cells / ml and cultured at a temperature of 36.5±1℃, a pH of 6.8-7.2, and a stirring rate of 250 rpm. Feed was added intermittently from day 2-4, with the feed volume being 3-7% of the initial culture volume. At day 6±2, the culture temperature was adjusted to 32.5±1℃ and maintained until the end of the culture. Samples were taken periodically during culture, and CountStar was used to count the viable cells and monitor changes in cell density and viability. The content of recombinant human hyaluronidase in the cell culture medium was detected by RP-HPLC, and the activity of recombinant human hyaluronidase in the cell culture medium was detected by turbidimetric assay.

[0086] The harvested cell culture medium is subjected to deep filtration and sterile filtration to obtain a clear cell culture medium; the clear cell culture medium is then purified by steps including anion exchange chromatography, affinity chromatography, and hydrophobic chromatography to prepare a stock solution.

[0087] Example 4 Recombinant human hyaluronidase R311 / S 312 Analysis of truncated variants resulting from truncation The recombinant human hyaluronidase sample prepared in Example 2 was analyzed for its intact N-glycan cleavage molecular weight. Because the sample was treated with PNGase F, the asparagine (N) linked to the N-glycan was converted to aspartic acid (D) during N-glycan cleavage, i.e., deamidation modification (Dea). Additionally, the C-terminus of the hyaluronidase is prone to C-terminal truncation (-Y). Therefore, in the intact N-glycan cleavage molecular weight analysis, Dea(6) modified components and Dea(6),-Y modified components were observed. Besides these two components, a corresponding +18 Da component, namely Dea(6),R, was also found. 311 / S 312 Fracture and Dea(6), -Y,R 311 / S 312 Breakdown of modified components. Hyaluronidase generation R 311 / S 312 During the breakage modification, the disulfide bond connection pattern in the SEQ ID NO:5 sequence indicates that the two parts after the breakage are still connected by C. 25 =C 316 The disulfide bonds are connected, therefore it is connected to the unaffected R 311 / S 312 Compared to the fragmented component, the molecular weight is approximately 18 Da, a result consistent with the non-reduced intact molecular weight of the cleaved N-glycan (see [link to original text]). Figure 1 The sample was further validated by LC-MS / MS analysis after digestion with lysine endonuclease (Lys-C). The recombinant human hyaluronidase sample should produce F when digested with Lys-C. 280 -K 314 Peptides, in theory, will not produce F. 280 -R 311 Peptide fragments. However, significant F was detected in this sample. 280 -R 311 peptide signal, this F 280 -R 311 Peptide fragments can be confirmed using both primary and secondary mass spectra (see [link]). Figure 2 Based on the combined results of non-reduced intact molecular weight analysis of N-glycans and peptide mapping analysis, the recombinant human hyaluronidase sample should contain R. 311 / S 312 Cut off and modify impurities.

[0088] Urokinase, also known as urokinase plasminogen activator (uPA), is a serine protease encoded by the PLAU gene. Urokinase was initially isolated from urine, but it is also present in blood and the extracellular matrix of many tissues. Literature reports that urokinase exhibits good enzymatic activity against the SGR / X motif, with higher activity when X is S (the enzyme activity sequence corresponding to X is S>R,K,A). R was found in the analysis of recombinant human hyaluronidase samples. 311 / S 312 The variant resulting from the truncation site (one of its possible sequences is shown in SEQ ID NO:5) has a truncation site similar to the cleavage site of uPA. Enzymatic digestion attempts were made using uPA enzyme. By optimizing experimental conditions to enable uPA enzyme to digest this site, it can be used to prepare samples with this truncation site.

[0089] The recombinant human hyaluronidase sample prepared in Example 3 was digested with uPA for R... 311 / S 312 Preparation of truncated variants (one of whose exemplary sequences is shown in SEQ ID NO:5). The preparation process is as follows: uPA enzyme was added to a PBS buffer system at pH 7.4 at protein:uPA enzyme (m / m) ratios of 20:1, 10:1, and 5:1, with a sample without uPA enzyme added as a control. All samples were mixed and incubated overnight at 37°C to prepare R variants with different ratios. 311 / S 312 Truncation of variant samples.

[0090] R 311 / S 312 Calculation of the cutoff ratio of the truncated variant samples: Different ratios of R obtained using uPA enzyme were compared. 311 / S 312 The truncated variant samples were digested with Lys-C enzyme in a 100mM Tris-HCl buffer system at pH 7.4, with a protein:Lys-C enzyme (m / v) ratio of 20:1. After mixing, the samples were incubated at 37°C for 6 hours. The digested samples were then analyzed by LC-MS / MS. After detection, the mass spectrometry data were processed using data processing software to determine the mass-to-charge ratio and retention time of the truncated and untruncated peptides. XIC spectra of the truncated peptide (hereinafter referred to as "truncated peptide," its sequence is shown in SEQ ID NO:7) and the untruncated peptide (hereinafter referred to as "native peptide," its sequence is shown in SEQ ID NO:6) were extracted from the mass spectrometry data of the samples. The peak areas of each component were integrated, and the Rc concentration in each sample was calculated using the following formula. 311 / S 312Content of truncated variants resulting from truncation:

[0091] Different R values ​​were obtained by adding different proportions of uPA enzyme for digestion. 311 / S 312 The R-value of recombinant human hyaluronidase samples with a truncated ratio can be calculated by digesting them with Lys-C enzyme and based on the peak area of ​​the peptides. 311 / S 312 The proportion of truncated variants generated by the cutoff point is shown in the table below (Table 1).

[0092] Table 1. R in samples digested with uPA enzyme at different ratios 311 / S 312 Truncated modification ratio results

[0093] The activity of recombinant human hyaluronidase samples containing different proportions of truncated variants was determined. According to Chapter 1207, "Hyaluronidase Assay," Part IV, of the 2020 edition of the Chinese Pharmacopoeia, the activity of this product was determined using the turbidimetric method. The principle is that hyaluronic acid solution can form a stable colloidal solution with acidified serum, producing turbidity. Hyaluronidase can enzymatically hydrolyze the substrate hyaluronic acid, thereby reducing the turbidity of the solution. A standard curve was plotted by measuring the absorbance of different concentrations of standards to determine the activity of this product.

[0094] Using this product's reference standard (calibrated with the national standard for hyaluronidase) as the standard, the standard was serially diluted to create a standard curve. The activity of the enzyme-digested samples was then determined based on the standard curve. The specific procedures are as follows: The reference sample was diluted to 40, 32, 24, 26, 8, 4, and 0 IU / ml as standard curve points. Simultaneously, the enzyme-digested samples were diluted 5000 and 10000 times as test samples. The test samples, standards, and reaction plates containing potassium hyaluronic acid solution were preheated at 37°C for 10 min. The test samples and standards were then mixed with the potassium hyaluronic acid working solution, and the standards and test samples underwent enzymatic digestion of the potassium hyaluronic acid solution at 37°C. After incubation at 37°C for 6–8 min, acidified serum solution was added to terminate the reaction, resulting in solutions of different turbidities. Standard curves were plotted by measuring the absorbance of standards with different activities at 640 nm (see [reference]). Figure 3 The activity value is calculated based on the absorbance of the test sample.

[0095] Table 2. Absorbance data at various points on the standard curve

[0096] Substitute the absorbance value of the test sample into Figure 3 The standard curve was calculated, and the enzyme digestion sample activity results are shown in Table 3.

[0097] Table 3

[0098] The detected value is lower than the limit of quantitation of this method. The results showed that R in recombinant human hyaluronidase 311 / S 312 The content of truncated variants resulting from the breakage of the enzyme can affect the activity of recombinant human hyaluronidase samples. As the content of these truncated variants gradually increases, the activity of the recombinant human hyaluronidase sample gradually decreases. Therefore, the content of these truncated variants needs to be controlled in recombinant human hyaluronidase products.

[0099] To ensure the activity of recombinant human hyaluronidase products and to prevent the aforementioned truncated variants from significantly affecting enzyme activity (i.e., not less than 70% of the initial activity), it is necessary to control the proportion or content of the truncated variants, for example, to keep them no more than about 10% of the total amount of recombinant human hyaluronidase and its variants.

[0100] Example 5 Recombinant human hyaluronidase M 310 Or M 313 Analysis of oxidation-modified variants resulting from oxidation During RP-HPLC analysis of the recombinant human hyaluronidase sample prepared in Example 3, an impurity peak was observed before the main peak (see [link to RP-HPLC analysis]). Figure 4 The impurity peak was separated and collected, and the collected impurity peak and the original liquid sample were subjected to molecular weight analysis of the non-reduced intact N-glycans (see [reference]). Figure 5 The results showed that the main component of the original sample was a Dea(6),-IFY modified component. The molecular weight of the main component of the impurity peak sample (50691 Da) was approximately 16 Da lower than that of the main component of the original sample (50675 Da). This was likely due to the oxidative modification of the recombinant human hyaluronidase, i.e., the Dea(6),Oxidation,-IFY modified component. Further confirmation was obtained by analyzing the peptide spectra of the impurity peak sample and the original sample (see [link to relevant documentation]). Figure 6 The results showed that the impurity peak in the sample was significantly different from that in the original sample. 310 Or M 313 The site showed obvious oxidation modification, further confirming that the impurity peak component was mainly M. 310 Or M 313 Oxidation occurred at the site. Peptide phantom data analysis showed that M in the recombinant human hyaluronidase sample... 310 Or M 313The site-truncation oxidative modification ratio is approximately 5% of the total amount of recombinant human hyaluronidase and its variants.

[0101] The main peak and impurities preceding the main peak of the recombinant human hyaluronidase sample prepared in Example 3 were simultaneously collected using an Aglient 1260 liquid chromatography automated collection module. Each injection consisted of 500 μg, for a total of 27 injections. The collected main peak and impurity components from each injection were transferred to their respective centrifuge tubes, approximately 300 μl per tube. The tubes were concentrated and evaporated using a centrifuge for an appropriate time (approximately 5 hours) until all liquid in the centrifuge tubes was evaporated to dryness. 150 μl of the stock solution excipient buffer solution was added to the bottom of each centrifuge tube to fully dissolve the proteins. 20 μl of each sample was then used for fraction localization and purity analysis. Typical chromatograms of the collected components are shown below. Figure 7 Table 4 shows the results of protein concentration determination of the collected components by RP-HPLC.

[0102] Table 4 Results of protein concentration determination of RP-HPLC collected components

[0103] The protein content of the collected components was determined using a Lunatic micro-spectroscopy analyzer, followed by RP-HPLC localization and purity analysis using a Waterse2695 liquid chromatograph. The localization confirmation chromatograms are shown below. Figure 7 The purity analysis results are shown in Table 5.

[0104] Table 5 Results of RP-HPLC purity analysis of collected components

[0105] Impurity peaks and main peaks collected by liquid chromatography, along with the unpurified stock solution, were digested with Trypsin at a protein:Trypsin enzyme ratio of 10:1 in a 50 mM NH4HCO3 buffer system. After mixing, the mixture was incubated at 37°C for 60 min. The digested samples were then analyzed by LC-MS / MS. After detection, the mass spectrometry data were processed using data processing software to determine the M... 310 Or M 313 Information such as mass-to-charge ratio and retention time of oxidized and native peptides was obtained. XIC spectra of oxidized and native peptides were extracted from the mass spectrometry data of the samples, and the peak areas of each component were obtained by integration. The Mo value of each sample was calculated using the following formula. 310 Or M 313 Content of oxidative modified variants:

[0106] Calculate M 310The native peptide sequence at the site oxidation modification ratio is FLSQDELVYTFGETVALGASGIVIWGTLSIMR 311 As shown in SEQ ID NO:7. Calculate M. 313 When the site oxidation ratio is high, due to Trypsin M 313 The site is located in the peptide segment SM 313 The K sequence is too short to be retained on the chromatographic column, therefore, in the case of M... 313 The native peptide sequence used in calculating the site oxidation modification ratio is FLSQDELVYTFGETVALGASGIVIWGTLSIMR. 311 S 312 Calculation of the oxidative modification ratio of MK peptides (as shown in SEQ ID NO:6).

[0107] M 310 The site-modified peptide sequence is FLSQDELVYTFGETVALGASGIVIWGTLSIM(Oxi)R (SEQ ID NO:8); M 313 The site-oxidized peptide sequence is FLSQDELVYTFGETVALGASGIVIWGTLSIMRSM(Oxi)K (SEQ ID NO:9).

[0108] The peak area of ​​the XIC peak of the above-mentioned oxidized modified peptide is the sum of the peak area corresponding to the peptide shown in SEQ ID NO:8 and the peak area corresponding to the peptide shown in SEQ ID NO:9.

[0109] Post-translational modification analysis was performed on impurities collected before the peak in reverse-phase samples and the original sample. The results showed that M... 310 Or M 313 The oxidation ratio was significantly higher than that of the original sample, indicating that the impurity peak before the main peak was mainly composed of M. 310 Or M 313 This is caused by oxidation at the site. M in each sample 310 Or M 313 The oxidation ratios are shown in the table below (Table 6).

[0110] Table 6. RP-HPLC Collected Components and Original Solution M 310 Or M 313 Oxidation results

[0111] The stock solution samples were tested for light stability in a stability testing chamber at 25°C and 5000 lux for 1-5 days. Then, the purity of the light stability testing samples and the control sample (untested sample) was analyzed by RP-HPLC using a Waters e2695 liquid chromatograph. Typical chromatograms are shown below. Figure 8.

[0112] The photostability test samples and control samples were subjected to Lys-C enzyme digestion in a 100mM Tris-HCl buffer system at pH 7.4, with Lys-C enzyme added at a ratio of 10:1 (protein:Lys-C enzyme (m / v)). After mixing, the samples were incubated at 37°C for 60 min. The digested samples were then analyzed by LC-MS / MS. After the analysis, the M value was calculated using the method described above based on the acquired mass spectrometry data. 310 Or M 313 Oxidation ratio.

[0113] The results showed that M in the samples tested for light stability 310 Or M 313 The oxidation ratio was significantly higher than that of the control sample, and the M of each sample was higher. 310 Or M 313 The sum of the oxidation ratios is shown in the table below (Table 7).

[0114] Table 7. M in the samples tested for light stability and the control samples 310 Or M 313 Oxidation modification results

[0115] The activity of recombinant human hyaluronidase was measured in light-exposed samples and light-protected samples (control samples), and the measurement method was as described in Example 3.

[0116] The activity results of the light-exposed sample and the control sample are shown in Table 8 below.

[0117] Table 8

[0118] The results showed that the impurity peak preceding the main peak in RP-HPLC analysis was M. 310 Or M 313 Oxidative modification leads to M 310 Or M 313 Oxidized variants resulting from oxidation can affect the activity of the product. As the content of oxidized variants gradually increases, the activity of recombinant human hyaluronidase samples gradually decreases.

[0119] To ensure the quality of recombinant human hyaluronidase products, the activity of recombinant human hyaluronidase should not be less than 80,000 IU / ml. Therefore, it is necessary to control the proportion or content of the above-mentioned oxidative modification variants, for example, to control it to not exceed about 15% of the total amount of recombinant human hyaluronidase and its variants.

[0120] Various changes and equivalent substitutions may be made to the embodiments disclosed in this application without departing from the spirit and scope of this application. Unless the context otherwise requires, any feature, step, or embodiment of the embodiments disclosed in this application may be used in combination with any other feature or embodiment.

Claims

1. A composition comprising recombinant human hyaluronidase and a variant thereof, wherein said variant is recombinant human hyaluronidase in the M... 310 Oxidation-modified variants resulting from oxidation at position M and the Mth 313 Oxidized variants resulting from oxidation at the site, wherein the sum of the contents of the two oxidized variants, calculated based on Lys-C enzyme cleavage peptide mass fingerprinting analysis, is less than or equal to approximately 15% of the total amount of recombinant human hyaluronidase and its variants in the composition; and, The above site numbers are based on the sequence shown in SEQ ID NO:

1.

2. The composition of claim 1, wherein the content of the recombinant human hyaluronidase is greater than or equal to about 85% of the total amount of recombinant human hyaluronidase and its variants in the composition, as calculated based on the Lys-C enzyme digestion peptide mass fingerprint analysis method.

3. The composition of claim 1 or 2, wherein, based on Lys-C enzyme digestion peptide mass fingerprint analysis, the sum of the contents of the two oxidatively modified variants is greater than or equal to about 0.01%, greater than or equal to about 0.05%, greater than or equal to about 0.1%, greater than or equal to about 0.5%, greater than or equal to about 1%, greater than or equal to about 1.5%, greater than or equal to about 2%, greater than or equal to about 2.5%, or greater than or equal to about 3%, greater than or equal to about 3.5%, greater than or equal to about 4%, greater than or equal to about 4.5%, or greater than or equal to about 5%.

4. The composition according to any one of claims 1-3, further comprising recombinant human hyaluronidase in the R... 311 and S 312 Truncated variants resulting from breaks between sites, wherein the content of said truncated variants, calculated based on Lys-C enzyme cleavage peptide mass fingerprinting analysis, is less than or equal to approximately 10% of the total amount of recombinant human hyaluronidase and its variants in the composition; and, The above site numbers are based on the sequence shown in SEQ ID NO:

1.

5. The composition of claim 4, wherein, based on Lys-C enzyme-digested peptide mass fingerprinting analysis, the content of the truncated variant is greater than or equal to about 0.01%, greater than or equal to about 0.05%, greater than or equal to about 0.1%, greater than or equal to about 0.5%, greater than or equal to about 1%, greater than or equal to about 1.5%, greater than or equal to about 2%, greater than or equal to about 2.5%, or greater than or equal to about 3% of the total amount of recombinant human hyaluronidase and its variants in the composition.

6. A composition comprising recombinant human hyaluronidase and a variant thereof, wherein said variant is recombinant human hyaluronidase in the R... 311 and S 312 Truncated variants resulting from breaks between sites, wherein the content of said truncated variants, calculated based on Lys-C enzyme cleavage peptide mass fingerprinting analysis, is less than or equal to approximately 10% of the total amount of recombinant human hyaluronidase and its variants in the composition; and, The above site numbers are based on the sequence shown in SEQ ID NO:

1.

7. The composition of claim 6, wherein the content of the recombinant human hyaluronidase is greater than or equal to about 90% of the total amount of recombinant human hyaluronidase and its variants in the composition, as calculated based on Lys-C enzyme digestion peptide mass fingerprint analysis.

8. The composition of claim 6 or 7, wherein, based on Lys-C enzyme cleavage peptide mass fingerprinting analysis, the content of the truncated variant is greater than or equal to about 0.01%, greater than or equal to about 0.05%, greater than or equal to about 0.1%, greater than or equal to about 0.5%, greater than or equal to about 1%, greater than or equal to about 1.5%, greater than or equal to about 2%, greater than or equal to about 2.5%, or greater than or equal to about 3% of the total amount of recombinant human hyaluronidase and its variants in the composition.

9. A pharmaceutical formulation comprising the composition of any one of claims 1-5, and / or the composition of any one of claims 6-8, and one or more pharmaceutically acceptable carriers.

10. The pharmaceutical preparation of claim 9, further comprising other active pharmaceutical ingredients, preferably selected from the group consisting of immunoglobulins, recombinant proteins, synthetic polypeptides, RNA, DNA, and chemical drugs.

11. The composition according to any one of claims 1-5, or the pharmaceutical formulation according to claim 9, wherein the Lys-C enzyme-based peptide mass fingerprinting analysis method comprises: The composition or pharmaceutical preparation was digested with Lys-C enzyme and then subjected to peptide mass fingerprint analysis by LC-MS / MS. After obtaining mass spectrometry data by LC-MS / MS analysis, the extracted ion current chromatograms (XICs) of the two charge forms with the strongest response of the oxidized peptide and its corresponding native peptide were extracted. The peak areas of each were integrated, and the content of the oxidized variant was calculated according to the following formula: 。 12. The composition according to any one of claims 4-5 or 6-9, or the pharmaceutical formulation according to claim 9, wherein the Lys-C enzyme-based peptide mass fingerprinting analysis method comprises: The composition or pharmaceutical preparation was digested with Lys-C enzyme and then subjected to peptide mass fingerprinting analysis using LC-MS / MS. After obtaining mass spectrometry data through LC-MS / MS analysis, the extracted ion current chromatograms (XICs) of the two charge forms with the strongest response of the truncated peptide and its corresponding native peptide were extracted. The peak areas of each were obtained by integration, and the proportion of truncated variants was calculated according to the following formula: 。 13. A method for quality inspection or quality control of products containing recombinant human hyaluronidase, comprising detecting the content of a recombinant human hyaluronidase variant in the product, wherein the variant is 1) recombinant human hyaluronidase in the R... 311 and S 312 2) Truncation variants resulting from breaks between positions, or recombinant human hyaluronidase at position M. 310 Oxidation-modified variants resulting from oxidation at position M and the Mth 313 Oxidation-modified variants resulting from oxidation of the site, wherein the site numbering is based on the sequence shown in SEQ ID NO:

1.

14. The method of claim 13, wherein if the detected content of the truncated variant is less than or equal to about 10%, the product meets pharmaceutical requirements.

15. The method of claim 13, wherein if the sum of the contents of the two oxidatively modified variants detected is less than or equal to about 15%, the product is deemed to meet pharmaceutical requirements.

16. Application of recombinant human hyaluronidase variants in quality inspection or quality control of products containing recombinant human hyaluronidase, wherein the recombinant human hyaluronidase variant is 1) recombinant human hyaluronidase in the R... 311 and S 312 truncated variants resulting from breaks between positions, or 2) recombinant human hyaluronidase at position M 310 Oxidation-modified variants resulting from oxidation at position M and the Mth 313 Oxidation-modified variants resulting from oxidation of the site, wherein the site numbering is based on the sequence shown in SEQ ID NO: 1.

Images