Modified sulfamidase and production thereof
09982243 · 2018-05-29
Assignee
Inventors
- Charlotta Berghard (Stockholm, SE)
- Erik Nordling (Danderyd, SE)
- Stefan Svensson Gelius (Älvsjö, SE)
- Agneta TJERNBERG (Stockholm, SE)
Cpc classification
A61P43/00
HUMAN NECESSITIES
C12Y310/01001
CHEMISTRY; METALLURGY
C12N9/96
CHEMISTRY; METALLURGY
International classification
C12N9/96
CHEMISTRY; METALLURGY
Abstract
Disclosed herein are a modified sulfamidase, a composition comprising a modified sulfamidase, as well as methods for preparing a modified sulfamidase and therapeutic use of such a sulfamidase. In particular, the present disclosure relates to a modified sulfamidase comprising substantially no epitopes for glycan recognition receptors, thereby enabling transportation of said sulfamidase across the blood brain barrier of a mammal, wherein said sulfamidase has catalytic activity in the brain of said mammal.
Claims
1. A method of preparing a modified sulfamidase, said method comprising: a) reacting a glycosylated sulfamidase with an alkali metal periodate, a2) optionally quenching the reaction resulting from step a), and b) reacting said sulfamidase with an alkali metal borohydride for a time period of no more than 2 h, wherein the steps are performed in sequence without performing an intermediate step.
2. The method according to claim 1, and wherein the concentration of said alkali metal borohydride is between 10 and 80 mM.
3. The method according to claim 1, wherein step a) is performed for a time period of no more than 4 h.
4. The method according to claim 1, wherein step a) is further characterized by at least one of i)-iii): i) said alkali metal periodate is sodium meta-periodate; ii) the concentration of said alkali metal periodate is no more than 20 mM, and iii) said reaction is performed at a temperature of between 0 and 22 C.
5. The method according to claim 1, wherein said reacting of step a) is performed at a pH of 3-7.
6. The method according to claim 1, wherein step b) is further characterized by at least one of i)-iv): i) said alkali metal borohydride is sodium borohydride; ii) the concentration of said alkali metal borohydride is no more than 4 times the concentration of said alkali metal periodate; iii) said reaction is performed for a time period of no more than 1.5 h, and iv) said reacting is performed at a temperature of between 0 and 8 C.
7. The method according to claim 1, wherein each of step a) and step b) is individually performed for a time period of no more than 2 h, and said alkali metal borohydride is optionally used at a concentration of 0.5-4 times the concentration of said alkali metal periodate.
8. The method according to claim 1, wherein step a) is performed for a time period of no more than 3 h, and step b) is performed for a time period of no more than 1 h, and said alkali metal borohydride optionally is used at a concentration of no more than 4 times the concentration of said alkali metal periodate.
9. The method according to claim 1, wherein the method comprises step a2) after step a), wherein the reaction resulting from step a) is quenched.
10. The method according to claim 1, further comprising a step b2) after step b), wherein the reaction resulting from step b) is quenched.
11. The method according to claim 1, wherein said glycosylated sulfamidase comprises glycan moieties at at least four asparagine residues.
12. The method according to claim 11, wherein said alkali metal periodate oxidizes cis-glycol groups of the glycan moieties to aldehyde groups.
13. The method according to claim 12, wherein said alkali metal borohydride reduces said aldehydes to alcohols.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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EXAMPLES
(19) The examples which follow disclose the development of a modified sulfamidase polypeptide according to the present disclosure.
Example 1
Cultivation, Purification and Characterization of Sulfamidase
(20) Material and Methods
(21) Construction of Expression Vectors for Sulfamidase:
(22) Synthetic genes encoding human sulfamidase were synthesized by Geneart (Life Technologies), both in codon optimized versions for H. sapiens or C. griseus (CHO cells) and the original human sequence. The synthetic genes were cloned in different mammalian expression vectors, such as pcDNA3.1(+) (Invitrogen) or pQMCF1 (Icosagen).
(23) Production of Sulfamidase:
(24) Two transient expression systems were evaluated for sulfamidase production, transient expression in HEK293 cells using pcDNA3.1(+) vectors and the Quattromed Cell Factory (QMCF) episomal expression system (Icosagen AS) using the pQMCF1 vector. In both systems cells were grown in standard medium and secreted protein was harvested typically 6-8 days after transfection. In addition, a stable cell line established using a commercially available CHO expression system was evaluated for production of sulfamidase.
(25) Sulfamidase was captured from medium by anion exchange chromatography (AIEX) on a Q sepharose column (GE Healthcare) equilibrated with 20 mM Tris, 1 mM EDTA, pH 8.0 and eluted by a NaCl gradient. Captured sulfamidase was further purified by 4-Mercapto-Ethyl-Pyridine (MEP) chromatography; sulfamidase containing fractions were loaded on a MEP HyperCel chromatography column and subsequently eluted by isocratic elution in 50 mM NaAc, 0.1 M NaCl, 1 mM EDTA, 1 mM DTT, pH 4.6. Final polishing was achieved by cation exchange chromatography (CIEX) on a SP Sepharose FF (GE Healthcare) column equilibrated in 25 mM NaAc, 2 mM DTT, pH 4.5. A NaCl gradient was used for elution. Purity and identity of sulfamidase batches from the different expression systems were analyzed by SDS-PAGE and MALDI-TOF-MS, data not shown.
(26) Glycosylation Analysis:
(27) The glycosylation pattern was determined for the different sulfamidase batches produced. Prior to glycopeptide analysis, sulfamidase (ca 10 g) was reduced, alkylated and digested with trypsin. Reduction of the protein was done by incubation in 5 l DTT 10 mM in 50 mM NH.sub.4HCO.sub.3 at 70 C. for 1 h. Subsequent alkylation with 5 l iodoacetamide 55 mM in 50 mM NH.sub.4HCO.sub.3 was performed at room temperature (RT) and in darkness for 45 min. Lastly, the tryptic digestion was performed by addition of 30 l of 50 mM NH.sub.4HCO.sub.3, 5 mM CaCl.sub.2, pH 8, and 0.2 g/l trypsin in 50 mM acetic acid (protease: protein ratio 1:20 (w/w)). Digestion was allowed to take place over night at 37 C.
(28) Five peptide fragments of the trypsin digested sulfamidase contained potential N-glycosylation sites. These peptide fragments containing potential glycosylation sites N(x), where x refers the position of the asparagine in the sulfamidase amino acid sequence as defined in SEQ ID NO:1, were:
(29) N(21) containing fragment (residue 4-35 of SEQ ID NO:1, 3269.63 Da)
(30) N(122) containing fragment (residue 105-130 of SEQ ID NO:1, 2910.38 Da)
(31) N(131) containing fragment (residue 131-134 of SEQ ID NO:1, 502.29 Da)
(32) N(244) containing fragment (residue 239-262 of SEQ ID NO:1, 2504.25 Da)
(33) N(393) containing fragment (residue 374-394 of SEQ ID NO:1), 2542.22 Da
(34) The asparagine of each potential glycosylation site is indicated in bold and the molecular mass of each peptide fragment is given.
(35) Possible glycosylation variants of the five tryptic peptide fragments were investigated by glycopeptide analysis. This was performed by liquid chromatography followed by mass spectrometry (LC-MS) on an Agilent 1200 HPLC system coupled to an Agilent 6510 Quadrupole time-of-flight mass spectrometer (Q-TOF-MS). Both systems were controlled by MassHunter Workstation. LC separation was performed by the use of a Waters XSELECT CSH 130 C18 column (1502.1 mm), the column temperature was set to 40 C. Mobile phase A consisted of 5% acetonitrile, 0.1% propionic acid, and 0.02% TFA, and mobile phase B consisted of 95% acetonitrile, 0.1% propionic acid, and 0.02% TFA. A gradient of from 0% to 10% B for 10 minutes, then from 10% to 70% B for another 25 min was used at a flow rate of 0.2 mL/min. The injection volume was 10 l. The Q-TOF was operated in positive-electrospray ion mode. During the course of data acquisition, the fragmentor voltage, skimmer voltage, and octopole RF were set to 90, 65, and 650 V, respectively. Mass range was between 300 and 2800 m/z.
(36) Results
(37) Transient expression in HEK293 cells resulted in low levels of secreted sulfamidase (less than 0.3 mg/L medium, SEQ ID NO:3). The QMCF stabile episomal expression system (Icosagen AS), resulted in sulfamidase (SEQ ID NO:2) production in titers of above 10 mg/L in CHO cells. The stable cell line established from a CHO expression system resulted in sulfamidase (SEQ ID NO:1) titers in excess of 40 mg/L.
(38) Sulfamidase was purified to apparent homogeneity with a molecular mass in the 61-63 kDa range. Based on the theoretical peptide chain mass of 55 kDa this indicates the presence of glycans with a total molecular mass of 6-8 kDa. Purity and identity of sulfamidase batches were analyzed by SDS-PAGE and MALDI-TOF-MS (results not shown).
(39) Glycosylation analysis for the tryptic digested peptides was performed by LC-MS. Manual search for 30 different kinds of glycosylation on each glycopeptide was performed. Relative quantitation was performed by measuring the peak areas from reconstructed ion chromatograms (without correction for ionization efficiency). Four of the five putative N-glycosylation sites (N(21), N(131), N(244) and N(393)) were glycosylated consistently through all sulfamidase batches. N(21) and N(393) were predominantly occupied by complex glycans, with a low degree of full sialylation. N(131) were completely occupied by oligomannose type of glycans. The degree of glycan phosphorylation was roughly 50% for all batches. The N(131) site was resistant to dephosphorylation by alkaline phosphatase. The fourth site, N(244), differed in composition between CHO cell (SEQ ID NO:2) and HEK293 cell (SEQ ID NO:3) produced sulfamidase by being oligomannose in CHO batches and a oligomannose/complex mixture in HEK293 batches. The tryptic peptide containing N(122) was found without any glycans attached.
Example 2
Chemical Modification of Sulfamidase According to Previously Known Method
(40) Material and Methods
(41) Chemical Modification According to the Known Method (as Disclosed in WO 2008/109677):
(42) In order to modify glycan moieties of sulfamidase, sulfamidase (SEQ ID NO:2), produced as described in Example 1 in Quattromed Cell Factory (QMCF) episomal expression system (Icosagen AS), was initially incubated with 20 mM sodium meta-periodate at 0 C. for 6.5 h in 20 mM sodium phosphate, 100 mM NaCl (pH 6.0). Glycan oxidation was quenched by addition of ethylene glycol to a final concentration of 192 mM. Quenching was allowed to proceed for 15 min at 0 C. before performing dialysis against 20 mM sodium phosphate, 100 mM NaCl (pH 6.0) over night at 4 C. Following dialysis, reduction was performed by addition of sodium borohydride the reaction mixture to a final concentration of 100 mM. The reduction reaction was allowed to proceed over night. Finally, the enzyme preparation was dialyzed against 20 mM sodium phosphate, 100 mM NaCl (pH 7.5). All incubations were performed in the dark.
(43) Results
(44) Modified sulfamidase was produced in triplicates in accordance with the sequence of steps depicted in
Example 3
Analyses of Sulfamidase Modified According to Known Method
(45) Material and Methods
(46) The sulfamidase modified according to Example 2, corresponding to the known method, was subjected to the following analyses.
(47) SDS-PAGE Analysis:
(48) 5 g of modified sulfamidase was loaded into each well on a NuPAGE 4-12% Bis-Tris gel. Seeblue 2 plus marker was used and the gel was colored with Instant Blue (C.B.S Scientific).
(49) Analysis by Size Exclusion Chromatography (SEC):
(50) The modified enzyme was analyzed by analytical size exclusion chromatography, performed on a KTAmicro system (GE Healthcare). A Superdex 200 PC 3.2/30 column with a flow rate of 40 L/min of formulation buffer was used. The sample volume was 10 L and contained 10 g enzyme.
(51) Dynamic Light Scattering (DLS) Analysis:
(52) The modified sulfamidase was degassed by centrifugation at 12000 rpm for 3 min at room temperature (RT). DLS experiments were performed on a DynaPro Titan instrument (Wyatt Technology Corp) using 25% laser power with 3 replicates of 75 L each.
(53) In-Gel Digestion and MALDI-TOF MS Analysis:
(54) The SDS-PAGE analysis revealed some extra bands, which were excised, destained and processed by in-gel digestion with trypsin. The procedure was the following: i. Coomassie destaining: the excised gel bands were placed in eppendorf tubes. The tubes were agitated twice in 100 mM NH.sub.3HCO.sub.3 in 50% acetonitrile at 30 C. for 1 h. The supernatants were discarded. ii. Reduction and alkylation: the gel pieces were dehydrated in acetonitrile, dried in a Speed Vac, and subsequently covered with 10 mM DTT in 100 mM NH.sub.3HCO.sub.3. Reduction was allowed to proceed for 1 h at 57 C. The supernatant was discarded and replaced by 55 mM iodoacetamide in 100 mM NH.sub.3HCO.sub.3. Alkylation was performed for 45 min at RT, in darkness and under slight agitation. The supernatant was once again discarded. The gel was washed with 100 mM NH.sub.3HCO.sub.3 in 50% acetonitrile for 20-30 min at 30 C., whereafter the supernatant was discarded. The gel pieces were dried completely in a Speed Vac. iii. In-gel digestion with trypsin: 2-5 L 50 mM NH.sub.3HCO.sub.3 was added to the dried gel pieces, whereafter 5 L trypsin solution (0.1 g/L in 1% acetic acid) was added. More 50 mM NH.sub.3HCO.sub.3 was added to cause swelling of the gel. Digestion was performed over night at 37 C. (with agitation). The supernatant was transferred to a new tube and extracted with 60% acetonitrile, 0.1% TFA (320 min) at RT. The resulting supernatants were evaporated in a Speed Vac to near dryness. The concentrated solution was mixed 1:1 with alpha-cyano-4-hydroxycinnamic acid solution (10 mg/mL) and 0.6 L was applied on a MALDI plate.
(55) Molecular masses of the tryptic peptide fragments were determined using a Sciex 5800 matrix-assisted laser desorption/ionization-time-of-flight mass spectrometer (MALDI-TOF/TOF MS). The analyses were performed in positive ion reflectron mode with a laser energy of 3550 and 400 shots.
(56) Preservation of Active Site:
(57) Any effect of the chemical modification on the active site of sulfamidase was investigated by the use of LC-MS and LC-MS/MS analyses. The samples were prepared according to the LC-MS method described under section Glycosylation analysis in Example 1. The resulting tryptic peptides containing cysteine 50 variants (cysteine50 (alkylated), oxidized cysteine 50, FGly50 and Ser50) were all semiquantified using peak area calculations from reconstructed ion chromatograms. The identity of the peptides were performed by MSMS sequencing. The MSMS parameters were as follows: the collision energies were set to 10, 15, and 20V, scan range 100-1800 m/z, and scan speed 1 scan/sec.
(58) Results
(59) SDS-PAGE Analysis:
(60) As apparent by SDS-PAGE analysis, several peptides of sizes distinct from that of full length sulfamidase were formed as a result of the chemical modification (
(61) Analysis by SEC and DLS:
(62) The chemical modification procedure was found to promote aggregation of sulfamidase, as demonstrated as a pre-peak in the chromatogram of
(63) The DLS analysis moreover revealed that the same material contained 15-20% of protein of the total protein content in high molecular weight forms (i.e. above 10.sup.10 kDa) (
(64) Analysis of SDS-PAGE Bands:
(65) By MALDI-TOF MS analysis, the four gel bands #1-4 observed on SDS-PAGE (
(66) The gel bands #1 and #2 of
(67) Thus, it was found that chemical modification of sulfamidase in accordance with the known method not only modifies glycans but also generates strand breaks at specific positions in the sulfamidase polypeptide chain.
(68) It was also found that the chemical modification according to the known method introduces oxidation on several methionine residues on sulfamidase, in particular on methionine 184 and methionine 443, which were almost completely oxidized. Methionine 226 (found in tryptic peptide T23, which corresponds to amino acid residues 226-238) was oxidized to a much lower degree, but this oxidation appeared to give rise to a more unstable protein than sulfamidase as such, generating the 41 kDa N-terminal truncation (gel band#3 of
(69) Consequently, the known procedure for modification of glycans catalyses oxidation of amino acid residues crucial for structural integrity of the enzyme.
(70) Preservation of Active Site:
(71) Moreover, by the use of LC-MSMS the reduction step (
(72) TABLE-US-00001 TABLE 1 Conversion of FGly to Ser at active site Chemical modification of sulfamidase Ser formation (%) FGly/Ser ratio None 0 Known method 56.0 0.3 (n = 3) 0.8
(73) Thus, the known chemical modification procedure, in addition to the modifications mentioned above, causes reduction of amino acid residues crucial for catalytic activity of the enzyme.
Example 4
New Methods for Chemical Modification of Sulfamidase
(74) Material and Methods
(75) New Method 1:
(76) Sulfamidase produced in Quattromed Cell Factory (QMCF) episomal expression system (Icosagen AS) according to Example 1, was oxidized by incubation with 20 mM sodium meta-periodate at 0 C. in the dark for 120 min in phosphate buffers having a pH of 6.0. Glycan oxidation was quenched by addition of ethylene glycol to a final concentration of 192 mM. Quenching was allowed to proceed for 15 min at 6 C. before sodium borohydride was added to the reaction mixture to a final concentration of 50 mM. After incubation at 0 C. for 120 min in the dark, the resulting sulfamidase preparation was ultrafiltrated against 20 mM sodium phosphate, 100 mM NaCl, pH 6.0. The new method 1 for chemical modification is depicted in
(77) New Method 2:
(78) Performed as New method 1 with the exception that the concentration of sodium borohydride in the reduction step was 10 mM. The new method 2 for chemical modification is depicted in
(79) New Method 3:
(80) Sulfamidase produced in a stable cell line according to Example 1 was oxidized by incubation with 10 mM sodium meta-periodate at 0 C. in the dark for 180 min in acetate buffer having an initial pH of between 4.5 to 5.7. Glycan oxidation was quenched by addition of ethylene glycol to a final concentration of 192 mM. Quenching was allowed to proceed for 15 min at 6 C. before sodium borohydride was added to the reaction mixture to a final concentration of 25 mM. After incubation at 0 C. for 60 min in the dark, the resulting sulfamidase preparation was ultrafiltrated against 10 mM sodium phosphate, 100 mM NaCl, pH 7.4. The new method 3 for chemical modification is depicted in
(81) New Method 4:
(82) Sulfamidase produced in a stable cell line according to Example 1 was oxidized by incubation with 10 mM sodium meta-periodate at 8 C. in the dark for 60 min in acetate buffer having an initial pH of 4.5. Glycan oxidation was quenched by addition of ethylene glycol to a final concentration of 192 mM. Quenching was allowed to proceed for 15 min at 6 C. before sodium borohydride was added to the reaction mixture to a final concentration of 25 mM. After incubation at 0 C. for 60 min in the dark, the resulting sulfamidase preparation was ultrafiltrated against 10 mM sodium phosphate, 100 mM NaCl, pH 7.4.
(83) Investigation of a Second Quenching Step:
(84) The effect of quenching the second step was investigated by the addition of 0.1 M Acetone after the sodium borohydride incubation step in new method 1. Material was produced in parallel according to new method 1 up to and including the sodium borohydride addition and incubation, after that reaction in one sample was quenched by the addition of acetone. Both samples were then treated according to the ultrafiltration step in new method 1.
(85) New Method 5:
(86) Sulfamidase produced in a stable cell line according to Example 1 was oxidized by incubation with 10 mM sodium meta-periodate at 8 C. in the dark for 60 min in acetate buffer having an initial pH of 4.5. Glycan oxidation was quenched by addition of ethylene glycol to a final concentration of 192 mM. Quenching was allowed to proceed for 15 min at 6 C. before sodium borohydride was added to the reaction mixture to a final concentration of 25 mM. After incubation at 0 C. for 45 min in the dark and quenching the reaction with 0.1 M acetone, the resulting sulfamidase preparation was ultrafiltrated against 10 mM sodium phosphate, 100 mM NaCl, pH 7.4.
(87) Results
(88) As already accounted for elsewhere herein, sodium meta-periodate is an oxidant that converts cis-glycol groups of carbohydrates to aldehyde groups, whereas borohydride is a reducing agent that reduces the aldehydes to more inert alcohols. The carbohydrate structure is thus irreversibly destroyed.
(89) In order to provide an improved method for chemical modification of glycans, in particular a procedure that provides a modified sulfamidase with improved properties, different reaction conditions were evaluated. It could be concluded that both oxidation by sodium meta-periodate and reduction by sodium borohydride introduced polypeptide modifications and aggregation; properties that negatively impact on catalytic activity and immunogenic propensity.
(90) Conditions were discovered for an improved chemical modification procedure. Surprisingly, these conditions facilitated that the reduction step could be performed immediately after the ethylene glycol quenching step, omitting buffer change and long exposure of sulfamidase to reactive aldehyde intermediates. The new chemical modification procedures are depicted in
Example 5
Analyses of Sulfamidase Modified According to New Methods
(91) Material and Methods
(92) The sulfamidase modified according to the new methods of Example 4 were subjected to the following analyses.
(93) SDS-PAGE Analysis:
(94) 5 g of sulfamidase modified in accordance with the known method (Example 2) as well as with the new method 1 and 2 (Example 4) were loaded into separate individual wells in accordance with the description in Example 3. Similarly, 5 g of sulfamidase modified in accordance with the new method 1 (Example 4) as well as with the new method 3, 4 and 5 were loaded into separate individual wells in accordance with the description in Example 3.
(95) Analysis by Size Exclusion Chromatography (SEC):
(96) Sulfamidase modified according to new method 1-4 was analyzed by analytical size exclusion chromatography in accordance with Example 3.
(97) Dynamic Light Scattering (DLS) Analysis:
(98) Sulfamidase modified according to new method 1 was analyzed by DLS in accordance with Example 3.
(99) Preservation of Active Site:
(100) Any effect of the chemical modification on the active site of sulfamidase, produced according to new method 1-5, as well as the second investigated quenching step was investigated in accordance with the description in Example 3.
(101) Results
(102) SDS-PAGE Analysis:
(103) Several peptides of sizes distinct from that of full length sulfamidase were formed as a result of the new chemical modification method 1 (
(104) Strand-breaks in the sulfamidase polypeptide prepared by the new methods is thus limited compared to strand-break occurrence in the sulfamidase prepared according to Example 2.
(105) Analysis by Size Exclusion Chromatography (SEC):
(106) It was found that the sulfamidase modified according to the new method 1 contained less aggregates compared to the sulfamidase modified by the known method. This is demonstrated in the chromatograms of
(107) Dynamic Light Scattering (DLS) Analysis:
(108) The DLS analysis (
(109) Preservation of Active Site:
(110) The reduction of FGly to Ser in position 50 at the active site of sulfamidase was determined by LC-MS/MS and the tryptic peptides containing FGly and Ser were positively identified. The relative amount of the peptide fragments was analyzed with LC-MS by measuring the peak areas from reconstructed ion chromatograms of the doubly charged ions (without correction for ionization efficiency). The samples generated from the methods used in Example 4 for the chemical modification were prepared and analyzed. (Table 2).
(111) TABLE-US-00002 TABLE 2 Conversion of FGly to Ser at active site Chemical modification of sulfamidase Ser formation (%) FGly/Ser ratio None 0 New method 1 45.4 0.9 (n = 3) 1.2 New method 2 11.5 1.3 (n = 3) 7.7 New method 3 44.1 2.0 (n = 2) 1.2 New method 4 36.1 (n = 1) 1.6 New method 5 41.7 1.1 (n = 2) 1.4
(112) Loss of active site FGly is limited considerably by the new methods. The new methods of modifying glycans on sulfamidase significantly decreased the amount of Ser formation, from 56% using the known method (see Table 1, Example 3), to 45%, 44%, 36% and 42% (new method 1, 3, 4 and 5, respectively, Table 2). The Ser formation of the new method 2 was about 11%, thus indicating that the conversion of FGly to Ser was highly dependent on sodium borohydride concentration.
(113) The second quenching step of the reaction provided modified sulfamidase comparable to the sulfamidase produced without quenching the reaction (modified sulfamidase produced according to new method 1 has 45% Ser formation compared to 43% Ser formation after quenching the second reaction with acetone). This was further confirmed by new method 5, which also encompasses a quenching step.
Example 6
Receptor Mediated Endocytosis In Vitro
(114) Material and Methods
(115) Sulfamidase was prepared as described in Example 1, 2 and 4, produced in Quattromed Cell Factory (QMCF) episomal expression system (Icosagen AS) and modified according to the known method and new methods 1 and 2 Endocytosis was evaluated in MEF-1 fibroblasts expressing M6P receptors. The MEF-1 cells were incubated for 24 h in DMEM medium supplemented with 75 nM of sulfamidase. The cells were washed twice in DMEM and once in 0.9% NaCl prior to cell lysis using 1% Triton X100. Lysate sulfamidase activity and total protein content were determined and lysate specific activity was calculated. Activity was monitored by fluorescence intensity at 460 nm using 0.25 mM 4-methylumbelliferyl-alpha-D-N-sulphoglucosaminide as substrate in 14.5 mM diethylbarbituric acid, 14.5 mM sodium acetate, 0.34% (w/v) NaCl, and 0.1% BSA. Total protein concentration was determined using the BCA kit (Pierce) with BSA as standard. Data are presented as mean+SD (n=4).
(116) Results
(117) Sulfamidase activity could be detected in cell homogenate for all preparations evaluated in the endocytosis assay. Modified sulfamidase prepared by the known method as well as the new methods 1 and 2 showed specific activities in cell homogenate below 10% of that obtained with unmodified recombinant sulfamidase (
(118) It can therefore be concluded that chemical modification render sulfamidase less prone to cellular uptake which is a consequence of removal of epitopes for glycan recognition receptors as M6PR. On a macroscopic level, this loss of molecular interactions translates into a reduced clearance from plasma when administrated intravenously. The reduced clearance of the protein could allow for less frequent dosing for the patients.
Example 7
In Vivo Plasma Clearance of Modified Sulfamidase Produced by New Method 1
(119) Material and Methods
(120) Plasma clearance (CL) of unmodified and modified recombinant sulfamidase produced as described in Example 1, in Quattromed Cell Factory (QMCF) episomal expression system (Icosagen AS) and modified according to the new method 1 of Example 4 was investigated in mice (C57BL/6J). The mice were given an intravenous single dose administration in the tail vein of 10 mg/kg sulfamidase and 10 mg/kg modified sulfamidase. Sulfamidase and modified sulfamidase were formulated at 2 mg/mL and administered at 5 mL/kg. Blood samples were taken from vena saphena or vena cava at different time points up to 24 h post dose (3 mice per time point). The blood was collected in EDTA tubes stored on ice and plasma was prepared by centrifugation. The plasma levels of sulfamidase and modified sulfamidase were analyzed by ECL. Plasma clearance was calculated using WinNonlin software version 6.3 (Non-compartmental analysis, Phoenix, Pharsight Corp., USA).
(121) Quantification of Sulfamidase and Modified Sulfamidase by Electrochemiluminescence (ECL) Immunoassay:
(122) Sulfamidase and modified sulfamidase in plasma PK samples were determined by ECL immunoassay using the Meso Scale Discovery (MSD) platform. A Streptavidin coated MSD plate was blocked with 5% Blocker-A in PBS. The plate was washed and different dilutions of standard and PK samples were distributed in the plate. A mixture of a biotinylated anti-sulfamidase mouse monoclonal antibody and Sulfo-Ru-tagged rabbit anti-sulfamidase antibodies was added and the plate was incubated at RT. Complexes of sulfamidase and labelled antibodies bind to the Streptavidin coated plate via the biotinylated mAb. After washing, the amount of bound complexes was determined by adding a read buffer to the wells and the plate was read in a MSD SI2400 instrument. The recorded ECL counts were proportional to the amount of sulfamidase in the sample and evaluated against a relevant sulfamidase standard.
(123) Results
(124) The plasma clearance in mice of modified sulfamidase was roughly 10-fold lower as compared to unmodified sulfamidase, see Table 3 below. This is probably at least partly due to the inhibition of receptor mediated uptake in peripheral tissue following chemical modification of sulfamidase (as demonstrated in the cellular uptake studies of Example 6).
(125) The data on clearance in mice obtained for modified sulfamidase produced in the stable cell line according to Example 1 and modified according to new method 3 of Example 4, was in agreement with the data presented in Table 3 for modified sulfamidase produced in the QMCF system and modified by new method 1. The reduced clearance of the protein could allow for less frequent dosing for the patients.
(126) TABLE-US-00003 TABLE 3 Plasma clearance of sulfamidase and modified sulfamidase Dose Plasma CL Test article (mg/kg) (L/(h .Math. kg)) sulfamidase (SEQ ID NO: 2) 10 0.17 modified sulfamidase 10 0.014 (New method 1, SEQ ID NO: 2)
Example 8
In Vivo Effect of Modified Sulfamidase on Brain Heparan Sulfate Storage
(127) Materials and Methods
(128) The effect of intravenously (i.v.) administrated modified sulfamidase produced as described in Example 1, in Quattromed Cell Factory (QMCF) episomal expression system (Icosagen AS) and modified according to new method 1 of Example 4 on brain heparan sulfate storage in vivo was investigated.
(129) Test Article Preparation:
(130) Modified sulfamidase was formulated at 6 mg/mL, sterile filtrated and frozen at 70 C. until used. Frozen modified sulfamidase and corresponding vehicle solution were thawed on the day of injection at RT for minimum one hour up to two hours before use. Chlorpheniramine was dissolved in isotonic saline to a concentration of 0.5 mg/mL, and stored at 20 C.
(131) Animals:
(132) Male mice having a spontaneous homozygous mutation at the mps3a gene, B6.Cg-Sgsh.sup.mps3a/PstJ (MPS IIIA)(Jackson Laboratories, ME, USA), were used. The animals were housed singly in cages at 231 C. and 40-60% humidity, and had free access to water and standard laboratory chow. The 12/12 h light/dark cycle was set to lights on at 7 pm. The animals were conditioned for at least two weeks before initiating the study. Wild-type siblings from the same breeding unit were also included as controls. In study A, mice were 23-24 weeks old whereas mice were 9-10 weeks old in study B.
(133) Experimental Procedure Study A:
(134) Modified sulfamidase at 30 mg/kg (n=8) and vehicle (n=7) were administered intravenously to MPS IIIA mice every other day for twenty-five days (13 injections). Chlorpheniramine was dosed (2.5 mg/kg) subcutaneously 30-45 min before administration of modified sulfamidase or vehicle. Dosing started approximately at 07.00 in the morning. The test article and vehicle were administered at 5 mL/kg. The final administration volume was corrected for the actual body weight at each dosing occasion. This scheme was repeated for vehicle. The study was finished 2 h after the last injection. Untreated age-matched wild-type mice (n=5) were included in conjunction with the test article-treated groups. The mice were anaesthetized by isoflurane. Blood was withdrawn from retro-orbital plexus bleeding. Perfusion followed by flushing 20 mL saline through the left ventricle of the heart. Tissues were dissected (brain, liver, spleen, lung, and heart), weighed and frozen rapidly in liquid nitrogen. The tissues and blood were prepared to measure hexosamine N-sulfate [-1,4] uronic acid (HNS-UA) levels using LC-MS/MS. HNS-UA is a disaccharide marker of heparan sulfate storage, and thus a decrease in HNS-UA levels reflects degradation of heparan sulfate. The HNS-UA data were calculated in relative units vs. internal standard, expressed per mg tissue and normalized to the average of the control group. The data were analyzed by one-way ANOVA test and if overall significance was demonstrated also by Bonferroni's multiple comparison post-hoc test for test of significance between groups (*P<0.05, **P<0.01, ***P<0.001).
(135) Experimental Procedure Study B:
(136) Modified sulfamidase at 30 mg/kg (n=6), 10 mg/kg (n=6) and vehicle (n=6) were administered intravenously to MPS IIIA mice once weekly for 10 weeks (10 injections). Chlorpheniramine was dosed (2.5 mg/kg) subcutaneously 30-45 min before administration of modified sulfamidase or vehicle. The final administration volume was corrected for the actual body weight at each dosing occasion. This scheme was repeated for vehicle. The study was finished 24 h after the last injection. Untreated age-matched wild-type mice (n=6) were included in conjunction with the test article-treated groups. The mice were anaesthetized by isoflurane. Blood was withdrawn from retro-orbital plexus bleeding. Perfusion followed by flushing 20 mL saline through the left ventricle of the heart. Tissues were dissected (brain, liver, spleen), weighed and frozen rapidly in liquid nitrogen. The tissues and blood were prepared to measure HNS-UA levels using LC-MS/MS. The HNS-UA data were calculated in relative units vs. internal standard, expressed per mg tissue and normalized to the average of the control group. The data were analyzed by one-way ANOVA test and if overall significance was demonstrated also by Bonferroni's multiple comparison post-hoc test for test of significance between groups (*P<0.05, **P<0.01, ***P<0.001).
(137) LC-MS/MS Analysis of HNS-UA in Tissue Samples:
(138) Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of hexosamine N-sulfate [-1,4] uronic acid (HNS-UA) in tissue samples was conducted partly according to methods described by Fuller et al (Pediatr Res 56: 733-738 (2004)) and Ramsay et al (Mol Genet Metab 78:193-204 (2003)). The tissues (90-180 mg) were homogenized in substrate buffer (29 mM diethylbarbituric acid, 29 mM sodium acetate, 0.68% (w/v) NaCl, 100 mL water, pH 6.5) using a Lysing Matrix D device (MP Biomedicals, LLC, Ohio, US). Homogenization was performed for 25 s in a Savant FastPrep FP120/Bio101 homogenizer (LabWrench, ON, Canada) and the homogenate was subsequently centrifuged in an Eppendorf centrifuge 5417R at 10000 rcf. The supernatant was evaporated to near dryness. 150 L derivatizing solution (250 mM 3-methyl-1-phenyl-2-pyrazolin-5-one (PMP), 400 mM NH.sub.3, pH 9.1) and 5 L of the internal standard Chondroitin disaccharide di-4S sodium (UA-GalNAc4S, 0.1 mg/mL) stock solution was added. The derivatization was performed at 70 C. for 90 min under agitation and then the solutions were acidified with 200 L of 800 mM formic acid. Deionized water was added to the samples to a final volume of 500 L, and extraction was performed with chloroform (3500 L) to remove excess PMP. Centrifugation was performed at 13000g for 5 min and the upper phase was transferred to a new vial. To remove any excess of formic acid and NH.sub.4COOH, the aqueous phase was evaporated to dryness in a speed vac (Savant Instruments Inc., Farmingdale, N.Y.), The samples were reconstituted to a total of 100 L of 5% acetonitrile/0.1% acetic acid/0.02% TFA.
(139) LC-MS/MS analysis was performed on Waters Ultra Performance Liquid Chromatography (UPLC), coupled to Sciex API 4000 triple quadrupole mass spectrometer. Instrument control, data acquisition and evaluation were done with Analyst software.
(140) LC separation was performed by the use of an Acquity C18 CSH column (502.1 mm, 1.7 m). Mobile phase A consisted of 5% acetonitrile/0.5% formic acid, and mobile phase B consisted of 95% acetonitrile/0.5% formic acid. A gradient from 1% to 99% B in 7 min was used at a flow rate of 0.35 mL/min. The injection volume was 10 L. The API 4000 was operated in electrospray negative ion multiple reaction monitoring (MRM) mode. The ion spray voltage was operated at 4.5 kV, and the source temperature was 450 C. Argon was used as collision gas. Collision energy was 34 V. The MRM transitions were 764.4/331.2 (PMP-HNS-UA) and 788.3/534.3 (PMP-internal standard). The relative amount of the HNS-UA was calculated with respect to the level of the internal standard.
(141) Results
(142) The results from study A shown in
(143) In addition, treatment with the modified sulfamidase totally abolished HNS-UA levels in liver (
(144) The results from study B are shown in
(145) These results thus demonstrate that a sulfamidase protein modified according to the new method 1 described herein causes, after long-term treatment, a robust reduction of HNS-UA levels in brain as well as an essentially complete reduction of HNS-UA levels in peripheral organs.
Example 9
Optimization of Sulfamidase Modification
(146) The chemical modification process can generally be divided into two parts where the oxidation step is the first step, denoted R1 hereinafter, and the reduction is the second step, denoted R2. To optimize the two steps a full factor design of experiment (DoE) investigating the effect of temperature, concentration and time for the two steps was set up.
(147) Materials and Methods
(148) Sulfamidase produced as described in Example 1 in Quattromed Cell Factory (QMCF) episomal expression system (Icosagen AS) were modified essentially as described in Example 4 for new method 1, however parameters subjected to investigation were varied in accordance with Table 4 (below). The investigation of R1 was carried out with the same reduction and parameters work-up as described in Example 4 (method 1). The end-points for the analysis are degree of oxidation of glycans described in Example 1, and the level of cell uptake of the modified protein, described in Example 6.
(149) TABLE-US-00004 TABLE 4 Parameters varied in R1 and R2 Variable R1 R2 T ( C.) 0, 8, 22 0, 8, 22 t (min) 30, 60, 120 30, 60, 120 c (mol/L) 10, 20, 40 1.2x (c in R1), 2.5x (c in R1), 5x (c in R1)
(150) The number of parameters and the type of design selected yields ten experiments for each step, the results of which were evaluated using the MODDE 10 software (Umetrics AB).
(151) In addition the influence of the second quenching step was tested on sulfamidase produced with the R1 parameters 8 C., 60 min and 20 mM sodium meta-periodate. Two additional reactions were run in parallel to the DoE experiment and quenched using 0.1 M acetone or by addition of acetic acid until a pH of 6.0 or lower was obtained. The final work-up followed the scheme for the other reactions. The sulfamidase thus produced was evaluated using the SDS-PAGE method described in Example 5.
(152) The R2 experiments were conducted with sulfamidase modified according to the parameters found to be optimal after the analysis of the DoE of R1.
(153) Results
(154) The R1 results are summarized in table 5 below:
(155) TABLE-US-00005 TABLE 5 R1 experiments and results Cell uptake Remaining original (natural) % of Varied parameters N-Glycan (%) unmodified T t c N N N N sulfamidase ( C.) (min) (mmol/L) (21) (131) (244) (393) uptake 0 30 10 0 0.9 0 0 12 0 120 10 0 0.4 0 0 9.5 22 30 10 0 0.2 0 0 7.1 22 120 10 0 0.1 0 0 8.5 0 30 40 0 0.3 0 0 3.8 0 120 40 0 0.1 0 0 3.5 22 30 40 0 0.1 0 0 2.7 22 120 40 0 0.01 0 0 3.5 8 60 20 0 0.2 0 0 6.1 8 60 20 0 0.2 0 0 6.5
(156) In addition, a glycosylation analysis according to Example 1 was conducted for sulfamidase modified according to the known method. No remaining original N-glycans were detected at the N-glycosylation sites N(21), N(131), N(244), and N(393).
(157) The MODDE evaluation of R1 (oxidation) showed that an optimum for R1 at a temperature of around 8 C., a reaction duration of around 1 h and a concentration of around 10 mmol/L of sodium meta-periodate. The overall protein health (e.g. structural integrity) seems to benefit from the lowest oxidant concentration as possible that still limits the cellular uptake via glycan recognition receptors to the level of new method 1 (see Example 6 for details).
(158) Among the various conditions disclosed for R1 reaction time was considered as an important parameter for degree of glycan modification. In addition, periodate concentration may influence degree of glycan modification.
(159) The R2 (reduction) design thus used the above identified preferred parameters for R1, i.e. used for oxidation of sulfamidase. The critical end-point for R2 is FGly content since it was found to influence the activity of the modified sulfamidase (cf Examples 3 and 5). See Table 6 below for results. The relative amount of the peptide fragments containing FGly50 and Ser50 was analyzed with LC-MS by measuring the peak areas from reconstructed ion chromatograms (without correction for ionization efficiency).
(160) TABLE-US-00006 TABLE 6 Summary of DoE for R2 and confimatory experiments Varied parameters Active site T c Ser formation FGly/Ser t (min) ( C.) (mmol/L) (%) Ratio 30 0 12 10 9.0 90 0 12 11 8.1 30 22 12 15 5.7 90 22 12 17 4.9 30 0 50 40 1.5 90 0 50 50 1.0 30 22 50 64 0.6 90 22 50 72 0.4 60 8 25 42 1.4 30 0 20 25 3.0 30 0 50 45 1.2 60 0 15 15 5.7 60 0 25 33 2.0 60 8 12 15 5.7 60 8 50 62 0.6
(161) The DoE for R2 showed that the Ser formation is related to concentration of sodium borohydride and temperature. Taking into account Ser formation and the presence of high molecular weight forms (data not shown, the results are analogous with the ones received for new method 2 in Example 4), the preferred conditions for R2 are a temperature of around 0 C., a reaction duration of around 1 h or less, and a sodium borohydride concentration of more than 12 mmol/L and up to and including 50 mmol/L.
(162) It was confirmed on SDS-PAGE (data not shown) that the sulfamidase produced in a reaction where the reduction step was quenched was comparable with the sulfamidase produced without quenching. This indicates that the introduction of the second quenching step do not negatively affect the quality of the material by either quenching with 0.1 M acetone or by lowering the pH to below 6 by addition of acetic acid.
Example 10
Analysis of Glycan Structure after Chemical Modification of Sulfamidase According to Previously Known Method
(163) Material and Methods
(164) Chemical Modification According to the Known Method:
(165) The chemical modification of sulfamidase according to the known method was performed as described in Example 2.
(166) Glycosylation Analysis:
(167) The analysis of glycan structure on sulfamidase after chemical modification was performed according to the LC-MS method described in Example 1.
(168) Resulting modifications on the glycan moieties on the four tryptic peptide fragments containing the N glycosylation sites N(21), N(131), N(244) and N(393) described in Example 1 were investigated by LC-MS analysis.
(169) Results
(170) Glycosylation Analysis:
(171) As described in Example 1, the type of glycosylation found on the four glycosylation sites prior to the chemical modification was predominantly complex glycans on N(21) and N(393), and oligomannose type of glycans on N(131) and N(244).
(172) After the chemical modification, detailed characterization of the modified glycan structure was performed on the most abundant chemically modified glycopeptides (less abundant glycans were not detectable due to significant decrease in sensitivity as a result of increased heterogeneity of the glycans after chemical modification). In this Example, the modification on Man-6 glycan after chemical modification according to the known method is investigated.
(173) Periodate treatment of glycans cleaves carbon bonds between two adjacent hydroxyl groups of the carbohydrate moieties and alter the molecular mass of the glycan chain.
(174) In
Example 11
Analysis of Glycan Structure after Chemical Modification of Sulfamidase According to New Methods 1, 3, and 4
(175) New Methods 1, 3, and 4:
(176) The chemical modifications of sulfamidase according to the new methods were performed as described in Example 4.
(177) Glycosylation Analysis:
(178) The glycosylation analysis was performed according to the LC-MS method described in Example 1. Resulting modifications on the glycan variants of the four tryptic peptide fragments containing the N glycosylation sites N(21), N(131), N(244) and N(393) were investigated by LC-MS analysis.
(179) Results
(180) Glycosylation Analysis:
(181) Detailed characterization of the modified glycan profile on sulfamidase, chemically modified according to new methods 1, 3, and 4, was performed on the most abundant chemically modified glycopeptides. In this Example 11, the modification on Man-6 glycan after chemical modification according to new methods 1, 3, and 4, was investigated.
(182) Ions corresponding to the chemically modified glycopeptide T13+Man-6 glycan with various degree of bond breaking were identified. Theoretically there can be a maximum of 3 double bond breaks and one single bond break (see
(183) The reproducibility of the chemical modification was tested by using triplicates (new method 1) or duplicates (new methods 3) of chemically modified sulfamidase.
(184) When comparing the Man-6 glycan modifications resulting from sulfamidase chemically modified according to the known method with the Man-6 glycan modifications resulting from sulfamidase chemically modified according to the new methods 1, 3, and 4, there was a large difference in degree of bond breaking. This is illustrated in
(185)