Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
  • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2560/00—Chemical aspects of mass spectrometric analysis of biological material

Definitions

• Arylsulfatase A is a lysosomal enzyme, which catalyzes the hydrolysis of cerebroside sulfate (galactosylceramide-3-O-sulfate or sulfatide) to cerebroside and sulfate. Deficiency of this enzyme cumulates cerebroside sulfate and leads to destruction of myelin in the central and peripheral nervous systems resulting in a progressive demyelination disease known as metachromatic leukodystrophy (MLD). MLD patients can be treated by enzyme replacement therapy (ERT) using recombinant human arylsulfatase A (rhASA).
• ERT enzyme replacement therapy
• rhASA recombinant human arylsulfatase A
• the disclosure relates, at least in part, to methods of analyzing and/or preparing samples (e.g., protein samples) that contain one or more disulfide linkages.
• samples e.g., protein samples
• the disclosure provides methods that can be used to analyze, control, or monitor the production of proteins, e.g., ASA, e.g., rhASA.
• the proteins described herein can be glycosylated (e.g., fully or partially glycosylated) or non-glycosylated.
• the methods described herein can include one or more of enzymatic digestion, chromatography, and mass spectrometry, e.g., multi-enzyme digestion and/or liquid chromatography-mass spectrometry (LC-MS).
• the mass spectrometry utilizes collision induced dissociation (CID) and/or electron transfer dissociation (ETD).
• CID collision induced dissociation
• ETD electron transfer dissociation
• the mass spectrometry can utilize CID, ETD, and CID of the isolated charge-reduced ions (MS3), e.g., after ETD.
• MS3 isolated charge-reduced ions
• compounds and compositions that can be detected or prepared by the methods described herein are also disclosed.
• the methods described herein can be used with proteins, e.g., glycoproteins, e.g., ASA, e.g., rhASA.
• the analysis of proteins can be used to evaluate starting materials, processes, intermediates, and final products in the production of proteins.
• enzyme digestion e.g., multi- enzyme digestion
• LC-MS e.g., LC-MS utilizing CID and/or ETD
• the methods described herein are useful for analyzing, evaluating, or processing a protein preparation, e.g., a glycoprotein
• an ASA preparation e.g., an rhASA preparation
• the methods disclosed herein are useful, e.g., from a process standpoint, e.g., to monitor or ensure batch-to-batch consistency or quality, or to evaluate a sample with regard to a reference, e.g., a preselected value.
• a reference e.g., a preselected value.
• the presence, distribution, or amount of one or more subject entities e.g., a structure, species, or fraction described herein, can be used in these evaluations.
• the methods disclosed herein can be used where the presence, distribution, or amount, of one or more of the subject entities in the sample may possess or impinge on the biological activity. In some embodiments, the methods are useful from a structure- activity prospective, to evaluate or ensure biological equivalence.
• the disclosure provides a method of evaluating or processing a sample, e.g., a protein preparation, e.g., a glycoprotein preparation, e.g., an ASA preparation, e.g., an rhASA preparation.
• the method includes providing an evaluation of a parameter related to a subject entity, e.g., a structure, species, or fraction described herein, e.g., a subject entity depicted in Table 1, Table 2, or FIG. 1, thereby evaluating or processing the sample.
• a subject entity e.g., a structure, species, or fraction described herein, e.g., a subject entity depicted in Table 1, Table 2, or FIG.
• Such parameters can include, or are a function of, the presence, distribution, or amount of a subject entity, e.g., a structure, species, or fraction disclosed herein.
• the method includes providing a determination of whether a test value (e.g., a value correlated with absence or presence) determined for the parameter meets a preselected criteria, e.g., is present as described herein, present in the amount described herein, or distributed as described herein.
• the method includes providing a comparison of the test value determined for a parameter with a reference value, or values, to thereby evaluate or process the sample.
• the comparison includes determining if the test value has a preselected relationship with the reference value, e.g., determining if it meets the reference value.
• the value need not be a numerical value, but can be merely an indication of whether the subject entity is present.
• the method includes determining if a test value is equal to or greater than a reference value, if it is less than or equal to a reference value, or if it falls within a range (either inclusive or exclusive of one or both endpoints).
• a test value is equal to or greater than a reference value, if it is less than or equal to a reference value, or if it falls within a range (either inclusive or exclusive of one or both endpoints).
• a test value is equal to or greater than a reference value, if it is less than or equal to a reference value, or if it falls within a range (either inclusive or exclusive of one or both endpoints).
• a range either inclusive or exclusive of one or both endpoints.
• test value or an indication of whether the preselected relationship is met, can be memorialized, e.g., in a computer readable record.
• a decision or step is taken, e.g., the sample is classified, selected, accepted or discarded, released or withheld, processed into a drug product, shipped, moved to a different location, formulated, labeled, packaged, released into commerce, or sold or offered for sale, depending on whether the preselected relationship is met. For example, based on the result of the determination, or upon comparison to a reference standard, the batch from which the sample is taken can be processed, e.g., as described herein.
• the subject entities described herein include, but are not limited to, a structure, a species, or a fraction in a sample, e.g., a protein preparation.
• a structure can be, e.g., a particular residue, or group of resides, e.g., an unpaired cysteine, a disulfide linkage (e.g. , to form a single disulfide, a nested disulfide, or a cystine knot), or a combination thereof, existing in a protein, polypeptide, or peptide.
• the structure comprises an unpaired cysteine, a disulfide linkage (e.g., to form a single disulfide, a nested disulfide, or a cystine knot), or a combination thereof, as depicted in Table 1, Table 2, or FIG. 1.
• the structure comprises one or more of the unpaired cysteine residues at C20, C51, and C276 in ASA.
• C51 is converted to formylglycine. In some embodiments, C51 is not converted to
• the structure comprises a disulfide linkage between C282 and C396 in ASA. In some embodiments, the structure comprises one or two of the disulfide linkages between C138 and C154, and between C143 and C150, in ASA. In some embodiments, the structure comprises one, two, or three of the disulfide linkages between C470 and C482, between C471 and C484, and between C475 and C481, in ASA.
• the structure comprises one or more, e.g., two, three, four, five, or all, of the disulfide linkages between C282 and C396, between C138 and C154, between C143 and C150, between C470 and C482, between C471 and C484, and between C475 and C481, in ASA.
• the structure is detected or determined by a method described herein, e.g., enzyme digestion (e.g., multi-enzyme digestion) and/or LC-MS (e.g., LC-MS utilizing CID and/or ETD, e.g., CIO, ETD, and CID-MS3).
• a species can be, e.g., a peptide with one or more, e.g., two, three, four, five, six, or more unpaired cysteine residues; a peptide with one or more, e.g., two, three, four, five, six, or more, disulfide linkages (e.g.
• disulfide-linked peptides with one or more, e.g., two, three, four, five, six, or more, unpaired cysteine residues and one or more, e.g., two, three, four, five, six, or more, disulfide linkages (e.g. , to form a single disulfide, a nested disulfide, or a cystine knot).
• disulfide linkage is located within a peptide.
• the disulfide linkage is located between two peptides.
• the species e.g., the peptide or disulfide-linked peptides
• the species is a peptide with an unpaired cysteine residue, disulfide-linked peptides with one disulfide linkage, disulfide-linked peptides with two disulfide linkages, or a peptide with three disulfide linkages, as depicted in Table 1.
• the species is GCYGHPSSTTPNL (19-31).
• the species is
• YVPVSLC(fgly)TPSRAAL 45-58.
• the species is
• the species is RMSRGGCSGL (270-279). In some embodiments, the species is LRCGKGTTYEGGVRE (282-294) and FTQGSAHSDTTADPACHASSSL (381-402) linked with a disulfide linkage between C282 and C396. In some embodiments, the species is CGK (282-284) and
• AHFFTQGSAHSDTTADPACHASSSLTAHEPPLLYDLSK (378-415) linked with a disulfide linkage between C282 and C396.
• the species is
• the species is PALQICCHPGCTPRPACCHCPDP with the disulfide linkages between C470 and C482, between C471 and C484, and between C475 and C481.
• the species is a peptide or disulfide-linked peptide having the theoretical or observed mass disclosed in Table 1.
• the species is a peptide or disulfide-linked peptide prepared by a method described herein, e.g. , multi-enzyme digestion.
• the species is a peptide or disulfide-linked peptide is detected by a method described herein, e.g. , LC-MS.
• the species is a fragment ion having the theoretical or observed mass disclosed in Table 2.
• the species, e.g., the peptide or disulfide-linked peptides comprises a glycosylation site.
• the structure is detected or determined by a method described herein, e.g., enzyme digestion (e.g., multi-enzyme digestion) and/or LC-MS (e.g., LC-MS utilizing CID and/or ETD, e.g., CID, ETD, and CID-MS3).
• enzyme digestion e.g., multi-enzyme digestion
• LC-MS e.g., LC-MS utilizing CID and/or ETD, e.g., CID, ETD, and CID-MS3
• a fraction can be, e.g., a part, portion, or subset of a sample, e.g., protein preparation, e.g., ASA preparation, e.g., rhASA preparation, having one or more of the structures described herein, or comprising one or more of the species described herein.
• a sample e.g., protein preparation, e.g., ASA preparation, e.g., rhASA preparation, having one or more of the structures described herein, or comprising one or more of the species described herein.
• the method includes determining the presence
• the unpaired cysteine residues and disulfide linkages e.g. , to form a single disulfide, a nested disulfide, or a cystine knot
• the unpaired cysteine residues and disulfide linkages e.g., the unpaired cysteine residues and disulfide linkages
• a sample e.g., a protein preparation, e.g., a glycoprotein preparation, e.g., an ASA preparation, e.g., an rhASA preparation.
• the sample is evaluated for the presence, distribution, or amount of each of the unpaired cysteine residues and disulfide linkages (e.g. , to form a single disulfide, a nested disulfide, or a cystine knot) depicted in Table 1.
• one or more structures or species from a subset of the structures or species depicted in Table 1 are evaluated.
• the sample comprises one or more of the compounds depicted in Table 1.
• the method includes determining the presence, amount, or distribution of one or more unpaired cysteine residues in a protein preparation, e.g., a glycoprotein preparation, e.g., an ASA preparation, e.g., an rhASA preparation, e.g., the presence, amount, or distribution of one or more of unpaired cysteine residues at positions 20, 51, and 276 of ASA, e.g., by peptide digestion, e.g., pepsin digestion, e.g., at a pH less than 8, e.g., at a pH less than 6.8, e.g., at pH 2.
• the method includes determining the presence, amount, or distribution of one or more single disulfides in a protein preparation, e.g., a glycoprotein preparation, e.g., an ASA preparation, e.g., an rhASA preparation, e.g., the presence, amount, or distribution of a single disulfide, e.g., between positions 282 and 396 of ASA, e.g., by peptide digestion, e.g., pepsin digestion (e.g., at a pH less than 8, e.g., at a pH less than 6.8, e.g., at pH 2), e.g., trypsin digestion (e.g., at a pH equal to or less than 8, e.g., at pH 6.8), e.g., Lys-C digestion (e.g., at a pH equal to or less than 8, e.g., at pH 6.8), or a combination thereof.
• peptide digestion e.g
• the peptide digestion includes pepsin, e.g., at a pH less than 8, e.g., at a pH less than 6.8, e.g., at pH 2.
• the peptide digestion includes trypsin and Lys-C, e.g., at a pH equal to or less than 8, e.g., at pH 6.8.
• the method includes determining the presence, amount, or distribution of one or more nested disulfides in a protein preparation, e.g., a glycoprotein preparation, e.g., an ASA preparation, e.g., an rhASA preparation, e.g., the presence, amount, or distribution of one or more of nested disulfides, e.g., including the disulfide bonds between positions 138 and 154 and between positions 143 and 150 of ASA, e.g., by peptide digestion, e.g., PNGase F digestion (e.g., at a pH equal to or less than 8, e.g., at pH 6.8), e.g., Asp-N digestion (e.g., at a pH equal to or less than 8, e.g., at pH 6.8), e.g., Lys-C digestion (e.g., at a pH equal to or less than 8, e.g., at pH 6.8),
• the peptide digestion includes PNGase F, Asp-N, Lys-C, and trypsin, e.g., at a pH equal to or less than 8, e.g., at pH 6.8.
• the peptide digestion further includes pepsin digestion, e.g., at a pH less than 8, e.g., at a pH less than 6.8, e.g., at pH 2.
• the method includes determining the presence, amount, or distribution of one or more cystine knots in a protein preparation, e.g., a glycoprotein preparation, e.g., an ASA preparation, e.g., an rhASA preparation, e.g., the presence, amount, or distribution of one or more of cystine knots, e.g., including the disulfide bonds between positions 470 and 482, between positions 471 and 484, and between positions 475 and 481 of ASA, e.g., by peptide digestion, e.g., pepsin digestion (e.g., at a pH less than 8, e.g., at a pH less than 6.8, e.g., at pH 2).
• peptide digestion e.g., pepsin digestion (e.g., at a pH less than 8, e.g., at a pH less than 6.8, e.g., at pH 2).
• the evaluation of the presence, distribution, or amount of a subject entity can show if the subject entity or a protein preparation, e.g., a glycoprotein preparation, e.g., an ASA preparation, e.g., an rhASA preparation, meets a reference standard.
• a protein preparation e.g., a glycoprotein preparation, e.g., an ASA preparation, e.g., an rhASA preparation.
• the methods disclosed herein can be used to determine if a test batch of a protein, e.g., a glycoprotein, e.g., ASA, e.g., rhASA, can be expected to have one or more of the properties of the protein.
• properties can include a property listed on the product insert of a protein, e.g., a glycoprotein, e.g., ASA, e.g., rhASA, a property appearing in a compendium, e.g., the US Pharmacopeia, or a property required by a regulatory agency, e.g., the FDA, for commercial use.
• a determination made by a method disclosed herein can be a direct or indirect measure of such a property.
• a direct measurement can be where a desired property is a preselected level of a subject entity, e.g., a structure, species, or fraction, measured.
• the measured subject entity is correlated with, or is a proxy for a desired property, e.g., a property described herein.
• Exemplary properties for rhASA can include, but not limited to, a preselected level of specific arylsulfatase activity, e.g., between about 10 and about 500 U/mg, e.g., between about 50 and about 140 U/mg, between about 50 and about 100 U/mg, or between about 100 and about 140 U/mg; a preselected value for disulfide linkage formation, e.g., at least about 50%, e.g., at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, of the rhASA molecules in a preparation have one or more of the unpaired cysteine residues, disulfide linkages, and/or cystine knots, as described herein; a preselected value for average molecular weight; a preselected value for glycosylation; and a set of preselected values for molecular weight distribution, e.g., based on glycosylation
• the sample is processed or evaluated by enzyme digestion, e.g., multi-enzyme digestion.
• the method includes providing a sample, e.g., a protein preparation, e.g., a glycoprotein preparation, e.g., an ASA preparation, e.g., an rhASA preparation; and subjecting the sample to processing or evaluation, e.g., by enzyme digestion, e.g., multi-enzyme digestion.
• the protein can be digested to peptides, disulfide-linked peptides, or a combination thereof, e.g., by one or more endoproteinases, e.g., in the absence or presence of one or more endoglycosidases.
• the peptide comprises at least one unpaired cysteine residue.
• the disulfide-linked peptides comprise one or more, e.g., two, three, four, five, or six, disulfide linkages.
• the disulfide-linked peptides comprise at least one unpaired cysteine residue.
• the peptide comprises a formylglycine residue.
• the disulfide-linked peptides comprise a formylglycine residue.
• the size of the peptide or disulfide-linked peptides is between about 0.2 kDa and about 20 kDa, e.g., between about 0.5 kDa and about 10 kDa, between about 0.5 kDa and about 2 kDa, between about 0.5 kDa and about 1 kDa, between about 1 kDa and about 2 kDa, and between about 1 kDa and about 5 kDa.
• the size of the peptide or disulfide-linked peptide is adjusted, e.g., to ensure sufficiently high-charge states that results in a low mass-to-charge ratio, e.g., m/z ⁇ 900, e.g., for effective ETD fragmentation.
• the protein is digested by a single enzyme. In some embodiments, the protein is digested by a mixture of two or more, e.g., three, four, five, or six different enzymes. In some embodiments, the protein is digested sequentially by two or more, e.g., three, four, five, or six, different enzymes. In some embodiments, the enzyme is selected on the basis of the size or sizes of the peptides or disulfide-linked peptides that can be generated by the digestion.
• the enzyme is selected on the basis of the structures, e.g., an unpaired cysteine residue, a single disulfide linkage, multiple disulfide linkages, or a cystine knot, of the peptides or disulfide-linked peptides that can be generated by the digestion.
• the enzyme is selected to reduce the complexity of mass spectra, e.g., when the protein is glycosylated, e.g., having N-linked glycosylation.
• the enzyme is an endoproteinase.
• the enzyme is an endoglycosidase.
• the enzyme is selected from the group consisting of Lys-C, trypsin, Asp-N, pepsin, and PNGase F. In some embodiments, the enzyme is pepsin. In some embodiments, the mixture of enzymes comprises two or more, e.g., three, four, or all, of Lys-C, trypsin, Asp-N, pepsin, and PNGase F. In some embodiments, the mixture of enzymes comprises Lys-C and trypsin. In some
• the mixture of enzymes comprises Lys-C, trypsin, Asp-N, and PNGase F.
• the digestion pH is optimized to minimize the disulfide scrambling.
• the disulfide scrambling is reduced by at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99%, e.g., at pH 2, compared to the disulfide scrambling at a digestion pH that is not optimized, e.g., at pH 6.8 or 8.
• the disulfide scrambling can be measured by a method described herein, e.g. , LC-MS.
• the sample is digested at a pH between about 2 and about 10, e.g., between about 2 and about 8, between about 2 and about 6, between about 2 and about 4, between about 4 and about 10, between about 6 and about 10, or between about 8 and about 10.
• a pH between about 2 and about 10, e.g., between about 2 and about 8, between about 2 and about 6, between about 2 and about 4, between about 4 and about 10, between about 6 and about 10, or between about 8 and about 10.
• the sample is digested at a pH about 2. In some embodiments, the sample is digested at a pH about 6.8. In some embodiments, the sample is digested at a pH about 8.
• the sample is processed or analyzed by LC-MS, e.g., performed on a digested protein preparation, e.g., a digested rhASA preparation.
• the method includes providing a sample, e.g., a protein preparation, e.g., a glycoprotein preparation, e.g., an ASA preparation, e.g., an rhASA preparation; and subjecting the sample to processing or analysis, e.g., by LC-MS, and optionally, evaluating the presence, distribution, or amount of a selected subject entity, e.g., a structure, species, or fraction described herein.
• a selected subject entity e.g., a structure, species, or fraction described herein.
• the LC-MS comprises a chromatography column having about 160 A, 180 A, 200 A, 220 A, or 240 A pore size, and/or about 3 ⁇ , 5 ⁇ , 7 ⁇ , or 9 ⁇ particle size.
• the LC-MS comprises a survey spectrum from m/z 100 to m/z 5000, e.g., from m/z 300 to m/z 2000.
• the LC-MS comprises tandem mass spectrometry (MS/MS).
• the LC- MS comprises CID, e.g., as CID-MS2.
• the LC-MS comprises ETD, e.g., as ETD-MS2.
• the LC-MS comprises both CID and ETD.
• the CID and ETD are performed on the same precursor ion.
• a second CID is performed, e.g., after ETD, e.g., as CID-MS3.
• one or more steps of LC-MS are repeated, e.g., to gain additional disulfide linkage information.
• the method includes determining if a contaminant fraction is present.
• contaminants include, e.g., contaminants associated with the manufacturing process, e.g., misfolded proteins, e.g., proteins having one or more undesired unpaired cysteines and disulfide linkages.
• the method includes identifying the distribution of one or more of the fractions, e.g., proteins (e.g., glycoproteins, e.g., ASA, e.g., rhASA) with a desired structure, e.g., a desired unpaired cysteine residue, disulfide linkage, and/or cystine knot, relative to a fraction of proteins with an undesired structure, e.g., an undesired unpaired cysteine residue and/or disulfide linkage (e.g. , to form a single disulfide, a nested disulfide, or a cystine knot), in a preparation.
• proteins e.g., glycoproteins, e.g., ASA, e.g., rhASA
• a desired structure e.g., a desired unpaired cysteine residue, disulfide linkage, and/or cystine knot
• an undesired structure e.g., an undesi
• the method includes determining if one or more of the fractions, e.g., proteins (e.g., glycoproteins, e.g., ASA, e.g., rhASA) with a desired structure, e.g., a desired unpaired cysteine residue, disulfide linkage, and/or cystine knot, are present at a higher intensity than one or more fractions of proteins with an undesired structure, e.g., an undesired unpaired cysteine residue, disulfide linkage, and/or cystine knot, in a preparation.
• proteins e.g., glycoproteins, e.g., ASA, e.g., rhASA
• a desired structure e.g., a desired unpaired cysteine residue, disulfide linkage, and/or cystine knot
• the method includes evaluating a subject entity, e.g., a subject entity described herein, to determine the amount of that entity in a sample.
• the amount can be expressed, e.g., in terms of % (e.g., weight % or mole %) of the subject entity in the sample.
• the amount of the subject entity is evaluated to determine if it is present in a preselected amount or range, e.g., at least about 50%, e.g., at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.
• a percentage of an unpaired cysteine residue, disulfide linkage, or cystine knot, in a preparation has been determined.
• the method includes determining or confirming that the percent of the unpaired cysteine residue or disulfide linkage (e.g. , to form a single disulfide, a nested disulfide, or a cystine knot) is in that range.
• the method includes determining the presence, amount, or distribution of one or more formylglycine residues and/or one or more cycteine residues that can be converted to formylglycine form, e.g., the presence, amount, or distribution of Cys 51 and/or formylglycine at position 51 of ASA in a sample, e.g., to determine if formylglycine is present at position 51 of ASA in a preselected amount or range, e.g., at least about 50%, e.g., at least about 60%, at least about 70%, at least about 80%, at least about 90%, e.g., about 50% to about 90%, e.g., about 60% to about 80%, about 65% to about 75%, e.g., about 70%.
• the method further includes classifying, selecting, accepting or discarding, releasing or withholding, processing into drug product, shipping, moving to a different location, formulating, labeling, packaging, releasing into commerce, selling or offering to sell the preparation based, e.g., on the result of the determination or upon comparison to a reference standard.
• the disclosure features, a method of evaluating or processing a protein preparation, e.g., a glycoprotein preparation, e.g., an ASA preparation, e.g., an rhASA preparation.
• the method includes: providing an evaluation of a parameter related to a subject entity, e.g., a subject entity described herein, resolved by enzyme digestion, e.g., multi-enzyme digestion; providing an evaluation of a parameter related to a subject entity resolved by LC-MS; and, optionally, providing a determination of whether a test value (e.g., a value correlated to presence or absence, distribution, or amount) determined for the parameter for Table 1, or Table 2, each meets a preselected criterion for that subject entity, e.g., is present or is present with a certain distribution, or amount as described herein, e.g., as depicted in Table 1, or Table 2, thereby evaluating or processing the preparation.
• the method includes providing two or more determinations, e.g.,
• the method includes providing a comparison of the value determined for a parameter with a reference value or values, to thereby evaluate the sample.
• the comparison includes determining if the test value has a preselected relationship with the reference value, e.g., determining if it meets the reference value.
• the disclosure features a method of evaluating or processing a sample, e.g., a protein preparation, e.g., a glycoprotein preparation, e.g., an ASA preparation, e.g., an rhASA preparation, that includes making a determination about a sample, e.g., a protein preparation, e.g., a glycoprotein preparation, e.g., an ASA preparation, e.g., an rhASA preparation, based upon a method or analysis described herein.
• a sample e.g., a protein preparation, e.g., a glycoprotein preparation, e.g., an ASA preparation, e.g., an rhASA preparation
• the method further includes classifying, selecting, accepting or discarding, releasing or withholding, processing into drug product, shipping, moving to a different location, formulating, labeling, packaging, releasing into commerce, selling or offering to sell the protein preparation, e.g., the glycoprotein preparation, e.g., the ASA preparation, e.g., the rhASA preparation, based, e.g., on the analysis.
• the party making the evaluation does not practice the method or analysis described herein, but merely relies on results which are obtained by a method or analysis described herein.
• the disclosure features methods of making a preparation, e.g., a standard preparation of known concentration, by providing a compound described herein, e.g., a compound depicted in Table 1, or Table 2, and combining it with a solvent.
• the standard is at least about 50, 60, 70, 80, 90, 95, 99, 99.5, or 99.9 % of the total amount of the structures or species in the sample. The percentage can be determined, e.g., by dry weight, chain, or molarity.
• the disclosure features an isolated, enriched, or purified fraction of a sample, e.g., a protein preparation, e.g., glycoprotein preparation, e.g., an ASA preparation, e.g., an rhASA preparation.
• a sample e.g., a protein preparation, e.g., glycoprotein preparation, e.g., an ASA preparation, e.g., an rhASA preparation.
• the sample is digested, e.g., by one or more enzymes.
• the fraction has one or more structures or species depicted in Table 1, or Table 2.
• the disclosure features a method of analyzing a process, e.g., a manufacturing process, of a protein, e.g., a glycoprotein, e.g., ASA, e.g., rhASA, e.g., rhASA made by a selected process.
• a process e.g., a manufacturing process
• a protein e.g., a glycoprotein, e.g., ASA, e.g., rhASA, e.g., rhASA made by a selected process.
• the method includes: providing a protein preparation, e.g., a glycoprotein preparation, e.g., an ASA preparation, e.g., an rhASA preparation; analyzing the protein preparation, using, e.g., a method described herein, e.g., to identify and/or quantify one or more subject entities, e.g., one or more of the subject entities disclosed herein, thereby allowing analysis, e.g., qualitative and/or quantitative analysis, of one or more of the subject entities in the protein preparation.
• the method further includes comparing the presence, distribution, or amount of one or more of the subject entities with a reference value, to thereby analyze the process, e.g., the manufacturing process.
• the method further includes maintaining the process, e.g., the manufacturing process, based, at least in part, upon the analysis. In one embodiment, the method further includes altering the process, e.g., the manufacturing process, based, at least in part, upon the analysis.
• the absence or presence of a subject entity, e.g., a subject entity described herein can indicate whether the process, e.g., the manufacturing process, needs to be altered.
• the party making the evaluation does not practice the method or analysis described herein, but merely relies on results which are obtained by a method or analysis described herein.
• the method includes comparing two or more protein preparations, e.g., glycoprotein preparations, e.g., ASA preparations, e.g., rhASA preparations, e.g., by a method of monitoring or controlling batch-to-batch variation or to compare a preparation to a reference standard.
• protein preparations e.g., glycoprotein preparations, e.g., ASA preparations, e.g., rhASA preparations
• the method includes: providing a first protein preparation, e.g., glycoprotein preparation, e.g., ASA preparation, e.g., rhASA preparation; providing the presence, amount, or distribution of one or more subject entities, e.g., one or more of the subject entities described herein, in the first sample; optionally, providing a second protein preparation, e.g., glycoprotein preparation, e.g., ASA preparation, e.g., rhASA preparation; providing the presence, distribution, or amount of one or more subject entities in the second preparation; and comparing the presence, distribution, or amount of the one or more subject entities of the first protein preparation with the one or more subject entities of the second protein preparation.
• the subject entity is analyzed by a method described herein.
• the method can further include making a decision, e.g., to classify, select, accept or discard, release or withhold, process into drug product, move to a different location, ship, formulate, label, package, release into commerce, sell or offer to sell the preparation, e.g., the protein preparation, e.g., the glycoprotein preparation, e.g., the ASA preparation, e.g., the rhASA preparation, based, at least in part, upon the determination, and optionally, carrying out the decision.
• a decision e.g., to classify, select, accept or discard, release or withhold, process into drug product, move to a different location, ship, formulate, label, package, release into commerce, sell or offer to sell the preparation, e.g., the protein preparation, e.g., the glycoprotein preparation, e.g., the ASA preparation, e.g., the rhASA preparation, based, at least in part, upon the determination, and optionally, carrying out the decision.
• the disclosure features a method of making a batch of protein, e.g., glycoprotein, e.g., ASA, e.g., rhASA, having a preselected property, e.g., meeting a release specification, label requirement, or compendial requirement, e.g., a property described herein.
• protein e.g., glycoprotein, e.g., ASA, e.g., rhASA
• a preselected property e.g., meeting a release specification, label requirement, or compendial requirement, e.g., a property described herein.
• the method includes: providing a test protein preparation, e.g., glycoprotein preparation, e.g., ASA preparation, e.g., rhASA preparation; analyzing the test preparation according to a method described herein; determining if the test preparation includes the presence, distribution, or amount of one or more of the structures provided in Table 1, or Table 2; and selecting the test preparation to make the protein, thereby making a batch of protein.
• a test protein preparation e.g., glycoprotein preparation, e.g., ASA preparation, e.g., rhASA preparation
• the disclosure features a method of predicting or ensuring that a batch of protein, e.g., glycoprotein, e.g., ASA, e.g., rhASA, will have a preselected property, e.g., that it will meet a release specification, label requirement, or compendial requirement, e.g., a property described herein.
• the method includes: providing a test preparation, e.g., glycoprotein preparation, e.g., ASA preparation, e.g., rhASA
• test protein preparation analyzing the test protein preparation according to a method described herein, wherein satisfaction of the preselected reference, e.g., one or more references disclosed herein, by the test protein preparation, is predictive of or ensures that a batch of protein made from the test protein preparation will have a preselected property, e.g., it will meet a release specification, label requirement, or compendial requirement, e.g., a property described herein.
• the preselected reference e.g., one or more references disclosed herein
• the disclosure features a method of making one or more batches of a protein preparation, e.g., a glycoprotein preparation, e.g., an ASA preparation, e.g., an rhASA preparation, having one or more disulfide linkages, wherein one or more subject entities of the batches varies less than a preselected range or has some preselected relationship with a reference standard. For example, it is present at a lower, higher, or equivalent level as a standard or is within (or outside) a range of values.
• the method includes determining one or more subject entity (e.g., one or more structures or fractions) of one or more batches of a product, and selecting a batch as a result of the determination.
• the method can also include comparing the results of the determination to preselected values, e.g., a reference standard.
• evaluation of the value e.g., the presence of a subject entity, is made by a method described herein.
• the method further includes classifying or selecting one or more batches having a structural property that varies less than the preselected range, e.g., a range described herein.
• the method can further include adjusting the dose of the batch to be administered, e.g., based on the result of the determination of the subject entity.
• the disclosure features a method of determining a reference value for a protein composition, e.g., a glycoprotein preparation, e.g., an ASA
• evaluation of the value is made by a method described herein.
• the disclosure features a method for determining
• the method includes some or all of the following: providing or determining a value for the presence, amount, or distribution of one or more subject entities, e.g., one or more of the subject entities described herein, in a first protein preparation, e.g.
• a glycoprotein preparation e.g., an ASA preparation, e.g., an rhASA preparation; providing or determining the bioavailability of the preparation; providing a reference value, e.g., by providing or determining presence, amount, or distribution of one or more subject entities, e.g., one or more of the subject entities described herein, in a second protein preparation, e.g., a glycoprotein preparation, e.g., an ASA preparation, e.g., an rhASA preparation; and comparing the presence, amount, or distribution of one or more of the subject entities of the first preparation with the reference value, e.g., from the second protein preparation.
• evaluation of the one or more structures or fractions is made by a method described herein.
• the method further comprises monitoring for presence, tissue distribution, spatial distribution, temporal distribution or retention time, in a cell or a subject, e.g., an experimental animal.
• the method includes determining the presence, amount, or distribution of one or more subject entities described herein of one or more batches of a product.
• the method further includes selecting a batch as a result of the determination.
• the method further includes comparing the results of the determination to preselected values, e.g., a reference standard.
• the invention provides a method for determining the safety or suitability of a protein preparation, e.g., a glycoprotein preparation, e.g., an ASA preparation, e.g., an rhASA preparation, for use in a particular indication.
• the method includes one or more, typically all, of the following: determining the presence, amount, or distribution of one or more subject entities, e.g., one or more of the subject entities described herein, in the protein; providing a reference value or sample; determining if the protein is acceptable, e.g., by comparing a value for the presence, amount, or distribution of one or more subject entities of the protein with the reference value or with a value determined from the sample.
• the protein when the protein is rhASA, one or more of the structures, species, or fractions described herein can be used as a reference. When a preselected index of similarity is met, the protein can be determined to be safe or suitable.
• the reference sample is associated with one or more undesired effects.
• the reference sample is associated with one or more desired effects.
• evaluation of the presence, amount, or size distribution of the one or more structures or fractions, e.g., one or more structures or fractions described herein, in the protein is made by a method described herein.
• the indication is MLD.
• the disclosure features a method of one or more of: providing a report to a report receiving entity; evaluating a sample of protein, e.g., glycoprotein, e.g., ASA, e.g., rhASA, for compliance with a reference standard, e.g., an FDA requirement; seeking indication from another party that a sample of protein, e.g., glycoprotein, e.g., ASA, e.g., rhASA, meets some predefined requirement; and submitting information about a sample of protein, e.g., glycoprotein, e.g., ASA, e.g., rhASA, to another party.
• Exemplary receiving entities or other parties include a government, e.g., the U.S. federal government, e.g., a government agency, e.g., the FDA.
• the method includes one or more (and typically all) of the following: performing one or more steps in making and/or testing a batch of protein, e.g., glycoprotein, e.g., ASA, e.g., rhASA, in a first country, typically the United States; sending at least an aliquot of the sample outside the first country, e.g., sending it outside the United States, to a second country; preparing, or receiving, a report which includes data about the structure of the protein sample, e.g., data related to a structure, species, or fraction described herein, e.g., data generated by one or more of the methods described herein; and providing said report to a report recipient entity.
• protein e.g., glycoprotein, e.g., ASA, e.g., rhASA
• the report receiving entity can determine if a
• a response from the report receiving entity is received, e.g., by a manufacturer, distributor, or seller of the protein preparation, e.g., the rhASA preparation.
• protein e.g., rhASA
• the disclosure features a method of evaluating a sample of protein, e.g., glycoprotein, e.g., ASA, e.g., rhASA, that includes receiving data with regard to the presence or level of a structure or fraction described herein in a sample of protein, e.g., glycoprotein, e.g., ASA, e.g., rhASA, e.g., wherein the data was prepared by one or more methods described herein; providing a record which includes said data and optionally includes an identifier for a batch of protein, e.g., glycoprotein, e.g., ASA, e.g., rhASA; submitting said record to a decision-maker, e.g., a government agency, e.g., the FDA; optionally, receiving a communication from said decision maker; optionally, deciding whether to release market the batch of protein, e.g., glycoprotein, e.g., ASA, e.
• any of the methods described herein can further include determining and/or providing an analysis regarding one or more biological activities or properties of the preparation or sample.
• the biological activity can be one or more of arylsulfatase activity, molecular weight distribution, and average molecular weight.
• the methods can further include comparing any of arylsulfatase activity, molecular weight distribution, and average molecular weight to a reference standard, e.g., a reference standard described herein, for the protein, e.g., the glycoprotein, e.g., ASA, e.g., rhASA.
• the reference standard for arylsulfatase activity is about 20 to about 250 U/mg, e.g., about 50 to 140 about U/mg.
• the disclosure features an enriched, isolated, or purified preparation of a compound from Table 1, or Table 2.
• the disclosure features a set of standard preparations.
• the set includes a plurality of standards each having a different concentration of a compound of Table 1, or Table 2.
• the standard preparation is free of other subject entities, e.g., other structures, species, or fractions described herein.
• the standard preparations can be used to determine the identity of the subject entity.
• the standard preparations can also be used to evaluate the concentration of the subject entity. For example, concentrations can be evaluated in terms of weight/weight, weight/volume, or molarity.
• the compound is provided in a solvent.
• the set of standards can be used in the evaluation of one or more samples, e.g., one can assay for a subject entity and compare the assay result with a value obtained from one or more of the standards. For example, one can determine the absorbance or other parameters and compare that with a standard curve for the relevant parameter derived from the set of standard preparations and determine the concentration of the subject entity.
• the standard in a set is individually enriched, isolated, or purified.
• the set includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 standards.
• FIG. 1 depicts the primary structure of rhASA with disulfide linkages and unpaired cysteines. The sites of disulfide linkages and unpaired cysteines are indicated and the N- glycosylation motifs are underlined.
• FIG. 2A depicts the mass and charge of the pepsin-digested peptide with an unmodified (free) Cys20.
• FIG. 2B depicts the CID-MS2 spectrum of the precursor from FIG. 2 A.
• the sequence and theoretical mass of the peptide are indicated in the insert of FIG. 2 A.
• FIG. 3A depicts the mass and charge of the pepsin-digested peptide with a modified (fgly) Cys51.
• FIG. 3B depicts the CID-MS2 spectrum of the precursor from FIG. 3A.
• the sequence and theoretical mass of the peptide are indicated in the insert of FIG. 3A.
• FIG. 4A depicts the mass and charge of the pepsin-digested peptide with an unmodified (free) Cys51.
• FIG. 4B depicts the CID-MS2 spectrum of the precursor from FIG. 4A.
• FIG. 5A depicts the mass and charge of the pepsin-digested peptide with an unmodified
• FIG. 5B depicts the CID-MS2 spectrum of the precursor from FIG. 5A.
• FIG. 6A depicts the mass and charge of the Lys-C plus trypsin-digested peptide with a single disulfide (Cys282 with Cys396).
• FIG. 6B depicts the CID-MS2 spectrum of the precursor from FIG. 6A.
• FIG. 6C depicts the ETD-MS2 spectrum of the precursor from FIG. 6A.
• FIG. 7 depicts the nested disulfides in a tryptic peptide.
• FIG. 8 depicts the digestion strategy for nested disulfides.
• FIG. 9 depicts the digestion strategy for nested disulfides using Lys-C+Trypsin+PNGase F+Asp-N.
• FIG. 10A depicts the mass and charge of the Lys-C plus trypsin plus Asp-N plus PNGaseF-digested peptide with two disulfides (Cysl38 with Cysl54 and Cysl43 with Cysl50). The sequence and theoretical mass of the peptide are indicated in the insert.
• FIG. 10B depicts the CID-MS2 spectrum of the 2+ and 3+ charged precursor from FIG. 10A.
• FIG. IOC depicts the ETD-MS2 spectrum of the 2+ and 3+ charged precursor from FIG. 10A.
• FIG. 11 depicts the precursor ion mass (2+) and its corresponding CID-MS2 spectra of the scrambled disulfides.
• FIG. 12 depicts the precursor ion mass (4+) and its corresponding CID-MS2 spectra of the nested disulfide-linked peptide (derived from pepsin digestion).
• FIG. 13A depicts the mass and charge of the pepsin-digested peptide with three disulfides (Cys470 with Cys482, Cys471 with Cys484 and Cys475 with Cys481). The sequence and theoretical mass of the peptide are indicated in the insert.
• FIG. 13B depicts the CID-MS2 spectrum (using the LTQ) of the 3+ charged precursor from FIG. 13A.
• FIG. 13C depicts the ETD-MS2 spectrum (using the Orbitrap) of the 4+ charged precursor from FIG. 13A.
• FIG. 13D depicts the ETD-MS2 spectrum (using the Orbitrap) of the 5+ charged precursor from FIG. 13A.
• FIG. 13E depicts the CID-MS3 spectrum (using the Orbitrap) of m/z 1312.6 from FIG. 13C.
• FIG. 13F depicts the CID-MS3 spectrum (using the LTQ) of m/z 1312.6 from FIG. 13C.
• the disclosure relates, at least in part, to methods of analyzing and/or preparing samples (e.g., protein samples) that contain one or more disulfide linkages.
• the methods described herein can include one or more of enzymatic digestion, chromatography, and mass spectrometry, e.g., multi-enzyme digestion and/or liquid chromatography-mass spectrometry (LC-MS). Characterization of proteins such as rhASA during or after biopharmaceutical manufacturing can increase manufacturers' ability to maintain the drug function and to control the stability or batch-to-batch variability. Definitions
• protein preparation refers to both protein drug substance preparations and protein drug product preparations.
• rhASA preparation refers to both rhASA drug product preparations and rhASA drug substance preparations.
• drug product preparation refers to a preparation having the purity required for and being formulated for pharmaceutical use.
• drug substance preparation refers to a preparation for pharmaceutical use but is not necessarily in its final formulation and/or comprises one or more non-product contaminant (e.g., one or more inorganic product such as sulfate, chloride, acetate and phosphates, protein contaminant, process by-product such as benzyl alcohol and benethonium).
• non-product contaminant e.g., one or more inorganic product such as sulfate, chloride, acetate and phosphates, protein contaminant, process by-product such as benzyl alcohol and benethonium.
• enriched preparation or "enriched fraction” as used herein refers to a preparation or fraction which is significantly enriched for a subject entity, e.g., a subject entity described herein.
• Significant enrichment can, by way of example, be based on weight/weight, weight/volume, or molarity. Enrichment can be with respect to a naturally occurring material, e.g., protein, e.g., glycoprotein, e.g., ASA.
• the subject entity in the case of a subject entity which is present in the naturally occurring protein, the subject entity is present in the enriched preparation at least 2, 5, 10, 50 or 100 times the concentration (as determined, e.g., by weight/weight, weight/volume, or molarity) that is found in the protein that has not been enriched. In some embodiments, in the case of a subject entity which is present in rhASA, the subject entity is present in the enriched preparation at least 2, 5, 10, 50 or 100 times the concentration (as determined, e.g., by weight/weight, weight/volume, or molarity) that is found in rhASA that has not been enriched. In some embodiments, the subject entity can be accompanied by a solvent, diluent, or carrier.
• the subject entity is substantially free of a solvent, diluent, or carrier.
• the subject entity can be accompanied by a medium, e.g., a buffer, matrix, or other material used to effect separation and/or eluent, used in its enrichment.
• the preparation is substantially free of such elements.
• the preparation is provided in an enclosure which is substantially free of contaminants.
• isolated preparation or "isolated fraction” as used herein refers to a preparation or fraction which is significantly isolated for a subject entity, e.g., a subject entity described herein. Significant isolation can, by way of example, be based on weight/weight, weight/volume, or molarity.
• Isolation can be with respect to a naturally occurring material, e.g., protein, e.g., glycoprotein, e.g., ASA.
• a naturally occurring material e.g., protein, e.g., glycoprotein, e.g., ASA.
• the subject entity in the case of a subject entity which is present in the naturally occurring protein, the subject entity is present in the isolated preparation at least 2, 5, 10, 50 or 100 times the concentration (as determined, e.g., by weight/weight, weight/volume, or molarity) that is found in the protein that has not been isolated.
• the subject entity in the case of a subject entity which is present in rhASA, the subject entity is present in the isolated preparation at least 2, 5, 10, 50 or 100 times the concentration (as determined, e.g., by weight/weight, weight/volume, or molarity) that is found in rhASA that has not been isolated.
• the subject entity can be accompanied by a solvent, diluent, or carrier.
• the subject entity is substantially free of a solvent, diluent, or carrier.
• the subject entity can be accompanied by a medium, e.g., a buffer, matrix, or other material used to effect separation and/or eluent, used in its isolation.
• the preparation is substantially free of such elements.
• the preparation is provided in an enclosure which is substantially free of contaminants.
• purified preparation or “purified fraction” as used herein refers to a preparation or fraction which is significantly purified for a subject entity, e.g., a subject entity described herein.
• Significant purification can, by way of example, be based on weight/weight, weight/volume, or molarity. Purification can be with respect to a naturally occurring material, e.g., protein, e.g., glycoprotein, e.g., ASA.
• the subject entity in the case of a subject entity which is present in the naturally occurring protein, the subject entity is present in the purified preparation at least 2, 5, 10, 50 or 100 times the concentration (as determined, e.g., by weight/weight, weight/volume, or molarity) that is found in the protein that has not been purified. In some embodiments, in the case of a subject entity which is present in rhASA, the subject entity is present in the purified preparation at least 2, 5, 10, 50 or 100 times the concentration (as determined, e.g., by weight/weight, weight/volume, or molarity) that is found in rhASA that has not been purified. In some embodiments, the subject entity can be accompanied by a solvent, diluent, or carrier.
• the subject entity is substantially free of a solvent, diluent, or carrier.
• the subject entity can be accompanied by a medium, e.g., a buffer, matrix, or other material used to effect separation and/or eluent, used in its purification.
• the preparation is substantially free of such elements.
• the preparation is provided in an enclosure which is substantially free of contaminants.
• Arylsulfatase A (or cerebroside-sulfatase) is an enzyme that breaks down cerebroside 3-sulfate (or sulfatide) into cerebroside and sulfate.
• galactosyl sulfatide is normally metabolized by the hydrolysis of 3-O-sulphate linkage to form galactocerebroside through the combined action of the lysosomal enzyme arylsulfatase A (EC 3.1.6.8) (Austin et al. Biochem J. 1964, 93, 15C-17C) and a sphingolipid activator protein called saposin B.
• MLD leukodystrophy
• ASA is an acidic glucoprotein with a low isoelectric point. Above pH 6.5, the enzyme exists as a monomer with a molecular weight of approximately 100 kDa. ASA undergoes a pH-dependent polymerisation forming a dimer at pH 4.5. In human urine, the enzyme consists of two nonidentical subunits of 63 and 54 kDa (Laidler PM et al. Biochim Biophys Acta. 1985, 827, 73-83). ASA purified from human liver, placenta, and fibroblasts also consist of two subunits of slightly different sizes varying between 55 and 64 kDa (Draper RK et al. Arch Biochemica Biophys.
• ASA is synthesized on membrane-bound ribosomes as a glycosylated precursor. It then passes through the endoplasmic reticulum and Golgi, where its N-linked oligosaccharides are processed with the formation of phosphorylated and sulfated oligosaccharide of the complex type (Waheed A et al. Biochim Biophys Acta.
• the methods described herein can be used to purify ASA from any source, e.g., from tissues, or cultured cells (e.g., human cells (e.g., fibroblasts) that recombinantly produce ASA).
• any source e.g., from tissues, or cultured cells (e.g., human cells (e.g., fibroblasts) that recombinantly produce ASA).
• the length (18 amino acids) of the human ASA signal peptide is based on the consensus sequence and a specific processing site for a signal sequence. Hence, from the deduced human ASA cDNA (EMBL GenBank accession numbers J04593 and X521151) the cleavage of the signal peptide occurs in all cells after residue number 18 (Ala), resulting in the mature form of the human ASA.
• ASA The active site of ASA contains an essential histidine residue (Lee GD and Van Etten RL, Arch Biochem Biophys. 1975, 171, 424-434) and two or more arginine residues (James GT, Arch Biochem Biophys. 1979, 97, 57-62). Many anions are inhibitors of the enzyme at concentrations in the millimolar range or lower.
• a protein modification has been identified in two eukaryotic sulfatases (ASA and arylsulfatase B (ASB)) and for one from the green alga Volvox carteri (Schmidt B et al. Cell. 1995, 82, 271-278, Selmer T et al. Eur J Biochem. 1996, 238, 341-345).
• This modification leads to the conversion of a cysteine residue, which is conserved among the known sulfatases, into a 2-amino-3-oxopropionic acid residue (Schmidt B et al. Cell. 1995, 82, 271-278).
• the novel amino acid derivative is also recognized as C- formylglycin (FGly).
• Cys-69 is referred to the precursor ASA which has an 18 residue signal peptide.
• cysteine residue is Cys-51.
• the human ASA gene structure has been described.
• the ASA gene is located near the end of the long arm of chromosome 22 (22ql3.31-qter), it spans 3.2 kb (Kreysing et al. Eur J Biochem. 1990, 191, 627-631) and consists of eight exons specifying the 507 amino acid enzyme unit (Stein et al. J Biol Chem. 1989, 264, 1252-1259).
• Messenger RNAs of 2.1, 3.7, and 4.8 kb have been detected in fibroblast cells, with the 2.1-kb message apparently responsible for the bulk of the active ASA generated by the cell (Kreysing et al. Eur J Biochem. 1990, 191, 627-631).
• the ASA sequence has been deposited at the EMBL GenBank with the accession number X521150. Differences between the published cDNA and the coding part of the ASA were described by Kreysing et al. (Eur J Biochem. 1990, 191, 627-631).
• the cDNA sequence originally described by Stein et al. (J Biol Chem. 1989, 264, 1252-1259) and the cDNA sequence described by Kreysing et al. (Eur J Biochem. 1990, 191, 627-631) have been deposited at the EMBL GenBank with the following accession numbers J04593 and X521151, respectively.
• ASA gene Several polymorphisms and more than 40 disease-related mutations have been identified in the ASA gene (Gieselmann et al. Hum Mutat. 1994, 4, 233-242, Barth et al. Hum Mutat. 1995, 6, 170-176, Draghia et al. Hum Mutat. 1997, 9, 234-242).
• the disease-related mutations in the ASA gene can be categorised in two broad groups that correlate fairly well with the clinical phenotype of MLD.
• One group (I) produces no active enzyme, no immunoreactive protein, and expresses no ASA activity when introduced into cultured animal cell lines.
• the other group (A) generates small amounts of cross-reactive material and low levels of functional enzyme in cultured cells.
• the crystal structure of human ASA shows that there are a total of 15 cysteines, six disulfide linkages, and three free cysteines including one that is posttranslationally modified to formylglycine (Lukatela et al., Biochem. 1998, 37, 3654-3664).
• a cystiene knot is formed at the C-terminal end of the molecule that consists of three disulfide linkages: Cys470 with Cys482, Cys471 with Cys484, and Cys475 with Cys481.
• cysteine knot refers to a protein structural motif where three disulfides (6 cysteine residues in close proximity in a protein backbone), with one of the disulfide passing through a ring, formed by the other two disulfide bonds (Le Nguyen Biochimie. 1990, 72(6-7): 431-5). Cystine knots are known to enhance protein structural stability, and they can be found in many proteins with a wide range of biological functions, such as inhibition, growth stimulation, and cyclization (Alvarez Reprod. Biol. Endocrinol. 2009, 7: 90; Daly et al., Curr. Opin. Chem. Biol. 2011, 15: 362-8). However, the cysteine-knot family shows low sequence homology, and it is therefore hard to predict cysteine-knot signatures by sequence alignment.
• ASA conformation of ASA is also pH-dependent. ASA forms a homo-dimeric protein at neutral pH but becomes a homo-octamer at acidic pH, such as in the lysosome.
• the stability of the enzyme seems to relate to the dimer-to-octamer transition in the lysosomal milieu, in which the octamerization process has been shown to be disrupted by a phenylalanine replacement mutation at Cys282 (Marcao et al., Biochem. Biophy. Res. Commun., 2003, 306, 293-297).
• a sample described herein can be lyophilized and/or dried in a vacuum oven, e.g., at about 40°C, 43°C, 46°C, 49°C, 52°C or 55°C, for about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 hours.
• the sample can be lyophilized and/or dried under one of the following conditions: 40°C for 12 hours; 46°C for 8 hours; 49°C for 6 hours; 52°C for 4 hours.
• a lyophilized o dried sample can be reconstituted in water or a suitable buffer at a concentration of about 1, 2, 5, 10, 15, 20, 50 mg/mL.
• the sample e.g., an ASA preparation
• Enzyme can be added to the protein solution at 1: 5, 1: 10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1: 90, 1: 100 (w/w), or less.
• the enzyme digestion can be performed at room temperature or at 37°C.
• the time of incubation can be, e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more hours.
• the protein can be digested by two or more enzymes at the same time.
• the protein can be digested sequentially by two or more enzymes.
• Exemplary enzymes include, but not limited to endoproteinases and endoglycosidases.
• one or more of the pepsin, trypsin, Lys-C, and PNGase F can be selected.
• Pepsin hydrolyzes only peptide bonds. It does not hydrolyze non-peptide amide or ester linkages. Pepsin exhibits preferential cleavage for hydrophobic, preferably aromatic, residues in PI and ⁇ positions. Increased susceptibility to hydrolysis occurs if there is a sulfur-containing amino acid close to the peptide bond, which has an aromatic amino acid. Pepsin will also preferentially cleave at the carboxyl side of phenylalanine and leucine and to a lesser extent at the carboxyl side of glutamic acid residues. Pepsin will not cleave at valine, alanine, or glycine linkages. Amidation of the C-terminal carboxyl group prevents hydrolysis by pepsin.
• Trypsin specifically hydrolyzes peptide bonds at the carboxyl side of lysine and arginine residues.
• Lys-C is a serine protease that specifically hydrolyzes amide, ester, and peptide bonds at the carboxylic side of Lys.
• Asp-N is an endoproteinase which selectively cleaves peptide bonds N-terminal to Asp, Glu, and Cys residues.
• PNGaseF cleaves an entire glycan from a glycoprotein provided the glycosylated asparagine moiety is substituted on its amino and carboxyl terminus with a polypeptide chain.
• Liquid chromatography-mass spectrometry is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities of mass
• LC-MS is a technique that has high sensitivity and selectivity and that can be used for various applications.
• LC-MS can be used for detection and potential identification of chemicals in the presence of other chemicals (in a complex mixture).
• the scale of chromatography in LC-MS is generally smaller than that in traditional HPLC, e.g., with respect to the internal diameter of the column and the flow rate.
• 1 mm columns with an internal diameter equal to or less than 1 mm e.g., 300 ⁇ or 75 ⁇
• the flow rates approach 100 nL/min and can be used with nanospray sources.
• standard bore (4.6 mm) columns are used the flow is often split -10: 1. This can be beneficial by allowing the use of other techniques in tandem such as MS and UV. However splitting the flow to UV will decrease the sensitivity of spectrophotometric detectors.
• the mass spectrometry on the other hand will give improved sensitivity at flow rates of 200 ⁇ / ⁇ or less.
• mass analyzers can be used in LC-MS.
• Exemplary mass analyzers include, but not limited to, single quadrupole, triple quadrupole, ion trap, TOF (time of flight), and quadrupole-time of flight (Q-TOF).
• the quadrupole mass analyzer consists of 4 circular rods, set parallel to each other.
• the quadrupole is the component of the instrument responsible for filtering sample ions, based on their mass-to-charge ratio (m/z). Ions are separated in a quadrupole based on the stability of their trajectories in the oscillating electric fields that are applied to the rods.
• a triple quadrupole mass spectrometer is a tandem mass spectrometer consisting of two quadrupole mass spectrometers in series, with a (non mass-resolving) radio frequency (RF) only quadrupole between them to act as a collision cell for collision- induced dissociation.
• the first (Q and third (Q 3 ) quadrupoles serve as mass filters, whereas the middle (q 2 ) quadrupole serves as a collision cell.
• This collision cell is an RF only quadrupole (non-mass filtering) using an inert gas such as Ar, He, or N 2 gas to provide collision-induced dissociation of a selected precursor ion that is selected in Subsequent fragments are passed through to Q where they may be filtered or scanned.
• This configuration is often abbreviated QqQ, here Qiq 2 Q 3 .
• An ion trap is a combination of electric or magnetic fields that captures ions in a region of a vacuum system or tube. Ion traps can be used in mass spectrometery while the ion's quantum state is manipulated.
• Time-of-flight mass spectrometry is a method of mass spectrometry in which an ion's mass-to-charge ratio is determined via a time measurement. Ions are accelerated by an electric field of known strength. This acceleration results in an ion having the same kinetic energy as any other ion that has the same charge. The velocity of the ion depends on the mass-to-charge ratio. The time that it subsequently takes for the particle to reach a detector at a known distance is measured. This time will depend on the mass-to-charge ratio of the particle (heavier particles reach lower speeds). From this time and the known experimental parameters one can find the mass-to-charge ratio of the ion.
• a quadrupole-time of flight is a triple quadrupole with the last quadrupole section replaced by a TOF analyzer.
• the interface can be an electrospray ion source or variant such as a nanospray source. Atmospheric pressure chemical ionization interface can also be used. Various deposition and drying techniques have also been used such as using moving belts, e.g., off-line MALDI deposition. In addition, Direct-EI LC-MS interface, couples a nano HPLC system and an electron ionization equipped mass spectrometer, can also be used.
• collision-induced dissociation also known as collisionally activated dissociation (CAD)
• CID collision-induced dissociation
• CAD collisionally activated dissociation
• CID and the fragment ions produced by CID are used for several purposes.
• Partial or complete structural determination can be achieved. In some cases, identity can be established based on previous knowledge without determining structure. Another use is in simply achieving more sensitive and specific detection. By looking for a unique fragment ion you can detect a given molecule in the presence of other molecules of the same nominal molecular weight, essentially reducing the background and increasing the limit of detection.
• the first quadrupole termed “Ql” can act as a mass filter and transmits a selected ion and accelerates it towards "Q2" which is termed a collision cell.
• Q2 which is termed a collision cell.
• the pressure in Q2 is higher and the ions collide with neutral gas in the collision cell and fragments by CID.
• the fragments are then accelerated out of the collision cell and enter Q3 which scans through the mass range, analyzing the resulting fragments (as they hit a detector). This produces a mass spectrum of the CID fragments from which structural information or identity can be gained.
• Many other experiments using CID on a triple quadrupole exist such as precursor ion scans that determine where a specific fragment came from rather than what fragments are produced by a given molecule.
• SORI-CID sustained off-resonance irradiation collision-induced dissociation
• Fourier Transform Ion Cyclotron Resonance mass spectrometry which involves accelerating the ions in cyclotron motion (in a circle inside of an ion trap) and then increasing the pressure resulting collisions that produce CID fragments.
• CID can be performed on charge-reduced species, e.g., isolated from ETD fragment ions. This type of CID is termed CRCID.
• Electron-transfer dissociation is a method of fragmenting ions in a mass spectrometer. ETD induces fragmentation of cations (e.g. peptides or proteins) by transferring electrons to them. ETD does not use free electrons but employs radical anions (e.g. anthracene or azobenzene) for this purpose. ETD cleaves randomly along the peptide backbone (so called c and z ions) while side chains and modifications such as phosphorylation are left intact. The technique is suitable for higher charge state ions (z>2). ETD is also suitable for the fragmentation of longer peptides or even entire proteins. ETD is also effective for peptides with modifications such as phosphorylation.
• cations e.g. peptides or proteins
• ETD does not use free electrons but employs radical anions (e.g. anthracene or azobenzene) for this purpose.
• ETD cleaves randomly along the peptide backbone (s
• a reference value can be a value determined from a reference sample ⁇ e.g., a commercially available sample or a sample from previous production).
• a reference value can be a value for the presence of a subject entity in a sample, e.g., a reference sample.
• the reference value can be numerical or non-numerical, e.g., it can be a qualitative value, e.g., yes or no, or present or not present at a preselected level of detection, or graphic or pictorial.
• the reference value can also be values for the presence of more than one subject entity in a sample.
• the reference value can be a map of structures present in a protein, e.g., ASA, e.g., rhASA, when analyzed by LC-MS, e.g., an LC-MS method described herein.
• the reference value can also be a release standard (a release standard is a standard which should be met to allow
• ASA e.g., rhASA
• the reference standard can be derived from any of a number of sources.
• the reference standard can be one which was set or provided by (either solely or in conjunction with another party, e.g., a regulatory agency, e.g., the FDA) the manufacturer of the drug or practitioner of a process to make the drug.
• the reference standard can be one which was set or provided by (either solely or in conjunction with another party, e.g., a regulatory agency, e.g., the FDA) a party other than the party manufacturing a drug and practicing a method disclosed herein, e.g., another party which manufactures the drug or practices a process to make the drug.
• the reference standard can be one which was set or provided by (either solely or in conjunction with another party) a regulatory agency, e.g., the FDA, to the manufacturer of the drug or practitioner of the process to make the drug, or to another party licensed to market the drug.
• a regulatory agency e.g., the FDA
• the reference standard can be a production, release, or product standard required by the FDA.
• a reference standard is a standard required of a pioneer drug (e.g., a drug marketed under an approved NDA) or a generic drug (e.g., a drug marketed or submitted for approval under an AND A).
• the reference standard can be one which was set or provided by Shire, its fully owned subsidiaries, its successors and assigns or agents, either solely or in conjunction with another party, e.g., a regulatory agency, e.g., the FDA, for production or release of a protein, e.g., ASA, e.g., rhASA.
• a regulatory agency e.g., the FDA
• ASA e.g., rhASA
• the reference value can be a statistical function, e.g., an average, of a number of values.
• the reference value can be a function of another value, e.g., of the presence or distribution of a second entity present in the sample, e.g., an internal standard.
• Evaluation against a reference value can be used to determine if a particular subject entity is present in an ASA sample, e.g., an rhASA sample.
• the following examples describe the characterization of unpaired cysteines, disulfide linkages (e.g., nested pairs of cysteines and cystine knot), and posttranslational modification of a cysteine to formylglycine for rhASA preparations.
• the statuses of these cysteines are critical structure attributes for rhASA function and stability that requires precise examination.
• the disulfide linkages including the cystine knot and a pair of nested cysteines, unpaired cysteines, and the posttranslational modification of a cysteine to formylglycine, were all determined.
• the primary structure of rhASA is shown in FIG. 1.
• the disulfide linkages observed were Cysl38 with Cysl54, Cysl43 with Cysl50, Cys282 with Cys396, Cys470 with Cys482, Cys471 with Cys484, and Cys475 with Cys481.
• Cys20 and Cys276 were free cysteines, and Cys51 was largely converted to formylglycine (> 70%).
• Samples rhASA, GMP lot JPT11001, manufactured by Shire Human Genetic Therapies (Lexington, MA, USA) was provided at 39.1 mg/mL. The sample was aliquoted (10 ⁇ ⁇ or 391 ⁇ g per vial) and stored at -80° C before analysis.
• Enzymatic Digestion The protein solution (10 ⁇ ⁇ of 4.9 mg/mL) was buffer exchanged with 100 mM ammonium bicarbonate (pH 8) or 50 mM Tris-HCl buffer (pH 6.8) using a 10 kDa molecular weight cutoff filter and concentrated to 1 mg/mL (49 ⁇ ). In addition to pH 8, a slightly less than alkaline pH (pH 6.8) was used to examine the effect of pH on the formation of alternative disulfide linkages during the digestion procedure. If a difference was observed, pepsin digestion at pH 2 was used to eliminate the scrambling that can occur at high pH conditions. For a pepsin digestion, the protein solution was buffer exchanged with 10 mM HC1 (pH 2).
• Pepsin (1: 10, w/w) was added to the protein solution and incubated at 37° C for 30 min. The reaction was quenched by adjusting the pH to 6 with sodium hydroxide.
• the protein solution pH 6.8 or 8
• endoproteinase Lys-C (1:50 w/w)
• trypsin (1:50 w/w)
• 8 hr room temperature
• second time (1:50 w/w for each enzyme
• the protein solution (pH 6.8 or 8) was treated with endoproteinase Lys-C (1:50 w/w), trypsin (1:50 w/w), Asp-N (1:50 w/w), and PNGase F (10 units/mg) for 8 hr at room temperature and then added a second time (the same ratio for each enzyme) and allowed to incubate for an additional 12 hr at room temperature.
• digestion was terminated by the addition of 1% formic acid. An aliquot of 2 ⁇ g of the enzyme digest was analyzed per LC-MS run.
• LC-MS An Ultimate 3000 nano-LC pump (Dionex, Mountain View, CA) and a o
• the LTQ-Orbitrap-ETD XL mass spectrometer was operated in the data-dependent mode to switch automatically between MS (scan 1 in the Orbitrap), CJD-MS2 (scan 2 in the LTQ), and ETD-MS2 (scan 3 in the LTQ). Briefly, after a survey MS spectrum from m/z 300 to 2000, subsequent CID-MS2, and ETD-MS2 steps were performed on the same precursor ion with a +2.5 m/z isolation width (Yu et ah, FEBS Lett. 2007, 581: 5561-5565). CID-MS2 and ETD-MS2 spectra were repeated by targeting specific ions, in order to gain additional linkage information not obtained in the initial run.
• Disulfide assignment The expected disulfide-linked tryptic or multi-enzyme digested peptide masses with different charges were first calculated, and then matched to the observed masses in the LC-MS chromatogram. The matched masses (with ⁇ 5 ppm mass accuracy for highly abundant ions and ⁇ 20 ppm for low abundant ions) were further verified by analysis of the corresponding CID-MS2 and ETD-MS2 fragmentation spectra, as well as the CID-MS3 fragmentation spectra, as needed. Digestion strategy
• the digestion strategy for analyzing the disulfide linkages in a protein with a complicated disulfide structure ⁇ e.g., rhASA) is, at least in part, based on the following considerations.
• proteases which can cut proteins to peptide sizes containing only a single disulfide are desired for identification of a single disulfide linkage because there is usually only one possibility for connection.
• intertwined disulfides or a cysteine-rich region in a protein ⁇ e.g., rhASA may prevent enzyme digestion to the desired peptide size.
• Peptide sizes are preferred to be 1 to 5 kDa since recovery and electrospray ionization efficiency can be a problem for larger peptides while smaller peptides less than 1 kDa may not retain well on a typical reversed-phase column.
• disulfide assignment will require further adjustment of peptide sizes to generate peptide lengths with sufficiently high-charge states that result in a low mass-to- charge ratio ⁇ i.e., m/z ⁇ 900) for effective ETD fragmentation.
• selection of proper enzymes or multiple enzymes needs to be considered to achieve ideal peptide length.
• an additional PNGase F treatment step should be considered to reduce the complexity of the mass spectra.
• the digestion pH for the selected enzymes needs also to be optimized to maintain sufficient enzyme activity while avoiding scrambling.
• the remaining unpaired cysteines were identified in a similar manner, for example, as shown in FIGS. 3A-3B (Cys51 converted to formylglycine), FIGS. 4A-4B (Cys51 as a free cysteine), and FIGS. 5A-5B (Cys276 as a free cysteine).
• Table 1 (#1, #2, #3, and #4) summarizes the assignments for all the unpaired cysteines. At Cys51, it contains more than 70% of formylglycin form.
• the observed accurate mass matched the theoretical peptide mass with one disulfide (a loss of 2 H from the backbone sequence).
• the corresponding CID-MS2 spectrum, b and y ions in FIG. 6B verified the correct sequence.
• the disulfide bond was preferentially dissociated, which resulted in two dissociated peptides designated as PI and P2 (FIG. 6C), that confirms that the two peptides are linked together.
• cysteines for the nested disulfides are located in Cysl38- Cysl54, and Cysl43-Cysl50. Since there are four cysteines, other potential linkages could be either as two separate disulfides (Cysl38-Cysl43 and Cysl50-Cys 154) as well as two crossed disulfides (Cysl38-Cysl50 and Cysl43-Cysl54) (FIG. 7). Furthermore, the complexity is increased by two N-linked glycosylation sites, one within, and the other next to the two disulfides (N underlined in FIG. 1).
• the disulfide linkages could be conclusively assigned as long as cleavages can be observed in the backbone between the CDGGC amino acid residues.
• the yl, y3, bl 1, and bl2 fragments in the CID-MS2 spectrum provide strong evidence for the linkages Cysl38 with Cysl54, and Cysl43 with Cysl50.
• the corresponding ETD-MS2 spectrum confirms that the two linked peptides (PI and P2) are connected.
• ETD was also tested to fragment the pepsin- digested disulfide but was not successful, due to minimal fragmentation and mainly charge -reduced species in the ETD spectrum.
• CID-MS3 and even MS4 have been attempted to fragment the charge-reduced species, the fragmentation efficiency was still poor for the peptide of this size.
• an additional enzyme i.e., Asp-N
• Cystine knot Cys470-Cys482, Cys471-Cys484, and Cys475-Cys481
• the cystine knot could not be broken by all the enzymes or the combination of the enzymes.
• CID fragmentation could not produce backbone cleavages within the cystine knot.
• ETD was examined.
• pepsin digestion was selected in order to obtain the proper peptide length with less acidic residues for effective fragmentation by ETD (i.e., eliminated additional glutamic and aspartic acid residues as compared to the corresponding tryptic fragment).
• the corresponding mass and charge of the pepsin-digested peptide is shown in FIG. 13A.
• the monoisotopic mass matched the expected peptide mass with three disulfides (a loss of 6 H from the backbone sequence).
• FIG. 13B Limited sequence information was obtained by CID-MS2 (FIG. 13B). Nevertheless, and significantly, ETD-MS2 dissociated the disulfides, which allowed cleavage of the peptide backbone, as shown in FIGS. 13C- 13D. The fragmentation of this disulfide-linked peptide but for two different charge states is shown in FIG. 13C (m/z 656.30, 4+) and FIG. 13D (m/z 525.20, 5+). The fragmentation data from the two different charge states demonstrates consistency with respect to cleavage sites and verifies that the linkage assignments are correct.
• the MS3 spectra contain additional disulfide and backbone cleavages, such as yl7 and b8, confirming the connection between Cys470 and Cys482.
• the fragmentation pattern and assignments were also observed with the same CID-MS3 spectra generated in the LTQ ion trap (FIG. 13F), which makes this method applicable to makes the method applicable even with low resolution MS instruments.
• the non-dissociated (the third) disulfide was left with the only possible remaining connection, which was a linkage between Cys475 and Cys481.
• ETD-MS2 and CID-MS3 mass spectral analysis confirms the linkage sites as Cys470 with Cys482, Cys471 with Cys484, and Cys475 with Cys481.
• the combination of ETD-MS2 and CID-MS3 mass spectral analysis confirms the linkage sites as Cys470 with Cys482, Cys471 with Cys484, and Cys475 with Cys481.
• the theoretical and observed fragment ions are listed in Table 2.

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