MODIFIED IDURONATE 2-SULFATASE AND PRODUCTION THEREOF

Abstract

Disclosed herein are a modified iduronate 2-sulfatase, a composition comprising a modified iduronate 2-sulfatase, as well as methods for preparing a modified iduronate 2-sulfatase and therapeutic use of such a iduronate 2-sulfatase. In particular, the present disclosure relates to a modified iduronate 2-sulfatase sulfatase comprising substantially no epitopes for glycan recognition receptors, wherein said iduronate 2-sulfatase has a catalytic activity of at least 50% of that of unmodified iduronate 2-sulfatase in vitro.

Claims

1. A modified iduronate 2-sulfatase comprising substantially no epitopes for glycan recognition receptors, wherein said iduronate 2-sulfatase has a catalytic activity of at least 50% of that of unmodified iduronate 2-sulfatase in vitro.

2. A modified iduronate 2-sulfatase according to claim 1, wherein said iduronate 2-sulfatase has catalytic activity in the brain of said mammal.

3. A modified iduronate 2-sulfatase according to claim 1, wherein said iduronate 2-sulfatase has catalytic activity in peripheral tissue.

4. A modified iduronate 2-sulfatase according to claim 3, wherein said peripheral tissue is joints, bone, connective tissue and/or cartilage.

5. The modified iduronate 2-sulfatase according to claim 1, having a relative content of natural glycan moieties being around 38% or less of the content of natural glycan moieties in unmodified recombinant iduronate 2-sulfatase.

6. The modified iduronate 2-sulfatase according to claim 1, wherein natural glycan moieties of said iduronate 2-sulfatase are disrupted by single bond breaks and double bond breaks, the extent of single bond breaks being at least 60% in oligomannose glycans.

7. The modified iduronate 2-sulfatase according to claim 1, comprising a polypeptide consisting of an amino acid sequence as defined in SEQ ID NO:1, or a polypeptide having at least 90% sequence identity with an amino acid sequence as defined in SEQ ID NO:1, wherein said epitopes are absent at at least five of the eight N-glycosylation sites: asparagine (N) in position 6 (N(6)), asparagine (N) in position 90 (N(90)), N in position 119 (N(119)), N in position 221 (N(221)), N in position 255 (N(255)), N in position 300 (N(300)), N in position 488 (N(488)) and N in position 512 (N(512)) of SEQ ID NO:1.

8. The modified iduronate 2-sulfatase according to claim 7, wherein the epitope at the glycosylation site asparagine (N) in position 90 (N(90)) is absent.

9. The modified iduronate 2-sulfatase according to claim 7, wherein said epitopes are absent at all of said eight N-glycosylation sites.

10. The modified iduronate 2-sulfatase according to claim 7, comprising a C-formylglycine residue in position 59 of SEQ ID NO:1 (FGly59) providing said catalytic activity.

11. An iduronate 2-sulfatase composition, comprising modified iduronate 2-sulfatase according to claim 1, said composition having a Ca-formylglycine (FGly) to serine (Ser) ratio at the active site that is greater than 1.

12. A method of preparing a modified iduronate 2-sulfatase, said method comprising: a) reacting a glycosylated iduronate 2-sulfatase with an alkali metal periodate, and b) reacting said iduronate 2-sulfatase with an alkali metal borohydride for a time period of no more than 2 h; thereby modifying glycan moieties of the iduronate 2-sulfatase and reducing the activity of the iduronate 2-sulfatase with respect to glycan recognition receptors, while retaining at least 50% catalytic activity of said iduronate 2-sulfatase in vitro.

13. The method according to 12, wherein step a) and step b) are performed in sequence without performing dialysis, ultrafiltration, precipitation or buffer exchange.

14. The method according to claim 12, wherein step a) is further characterized by at least one of: i) said alkali metal periodate is sodium meta-periodate; ii) said periodate is used at a concentration of no more than 20 mM; iii) said reaction is performed at a temperature of between 0 and 22 C.; iv) said reaction is performed for a time period of no more than 4 h; and v) said reaction of step a) is performed at a pH of 3-7.

15. The method according to claim 12, wherein step b) is further characterized by at least one of: i) said alkali metal borohydride is sodium borohydride; ii) said borohydride is used at a concentration of no more than 4 times the concentration of said periodate; iii) said reaction is performed for a time period of no more than 1.5 h; and iv) said reaction is performed at a temperature of between 0 and 8 C.

16. The method according to claim 12, wherein step a) is performed for a time period of no more than 3 h and step b) is performed for no more than 1 h, and said borohydride optionally is used at a concentration of no more than 4 times the concentration of said periodate.

17. The method according to claim 12, further comprising a2) quenching of the reaction resulting from step a); and/or b2) quenching of the reaction resulting from step b).

18. The method according to claim 12, wherein the active site of said iduronate 2-sulfatase is made inaccessible to oxidative and/or reductive reactions during at least one of steps a) and b).

19. The method according to claim 18, wherein at least one of steps a) and b) of the method is/are performed in the presence of a protective ligand.

20. The method according to claim 18, wherein steps a) and b) of the method are performed while iduronate 2-sulfatase is immobilized on a resin.

21. A modified iduronate 2-sulfatase obtainable by the method according to claim 12.

22. (canceled)

23. (canceled)

24. A method of treating a mammal afflicted with a lysosomal storage disease, comprising administering to the mammal a therapeutically effective amount of a modified iduronate 2-sulfatase according to claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0123] FIG. 1 is a picture outlining the differences between the methods for chemical modification developed by the inventors, disclosed in Example 3, and the known method, disclosed in WO 2008/109677.

[0124] FIG. 2A shows a SDS-PAGE gel of iduronate 2-sulfatase (lane 2) and iduronate 2-sulfatase modified according to the known method (lane 3). Two additional protein bands, denoted A and B, generated by the glycan modification procedure were identified in lane 3.

[0125] FIG. 2B shows a SDS-PAGE gel of iduronate 2-sulfatase (lane 2), iduronate 2-sulfatase modified according to the known method (lane 3) as well as iduronate 2-sulfatase modified according to new method 1, 2, 3, 4 as disclosed herein (lanes 4, 5, 6, 7, respectively).

[0126] FIG. 3 is a diagram showing the relative amounts of different naturally occurring glycans at asparagine in position 90 (N(90)) of a peptide fragment of iduronate 2-sulfatase (black bars), iduronate 2-sulfatase modified according to the known method (grey bars) as well as iduronate 2-sulfatase modified according to new method 1 (checkered bars). The glycans correspond to the following; GOF: Asialo-, agalacto-, fucosylated biantennary oligosaccharide (Oxford notation name: FA2); G1F: Monogalactosylated, fucosylated biantennary oligosaccharide (Oxford notation name: FA2[3]G1 or FA2[6]G1); G2F: Asialo-, fucosylated biantennary oligosaccharide (Oxford notation name: FA2G2); A1F: Monosialo-, fucosylated biantennary oligosaccharide (Oxford notation name: FA2G2S1); A2F: Disialo-, fucosylated biantennary oligosaccharide (Oxford notation name: FA2G2S2).

[0127] FIG. 4 is a diagram showing the activity of iduronate 2-sulfatase as well as iduronate 2-sulfatase modified according to new method 3 and 4.

[0128] FIG. 5 is a diagram visualizing the receptor mediated endocytosis in human primary fibroblast cells of unmodified recombinant iduronate 2-sulfatase (black squares) and iduronate 2-sulfatase modified according to new method 1 as described herein (black circles).

[0129] FIG. 6A shows the time dependence of serum concentrations of iduronate 2-sulfatase and iduronate 2-sulfatase chemically modified according to new method 2 in mice after i.v. administration at a dose of 1 mg/kg.

[0130] FIG. 6B shows the time dependence of serum concentrations of iduronate 2-sulfatase and iduronate 2-sulfatase chemically modified according to new method 3 in mice after i.v. administration at a dose of 3 mg/kg.

[0131] FIG. 7 is a schematic drawing of the three archetypal N-glycan structures generally present in proteins of mammalian origin and the typical N-glycan present in yeast proteins. The left glycan represents the oligomannose type, the second from the left the complex type, and the second from right, the hybrid type and the one on the far right is the polymannose type of yeast proteins. In the Figure the following compounds are depicted: black filled diamonds correspond to N-acetylneuraminic acid; black filled circles correspond to mannose; squares correspond to N-acetylglucosamine; black filled triangle corresponds to fucose; circle corresponds to galactose. Sugar moieties marked with an asterisk can be modified by the periodate/borohydride treatment disclosed herein.

[0132] FIG. 8A is a schematic drawing illustrating predicted bond breaks on mannose after chemical modification.

[0133] FIG. 8B is a schematic drawing illustrating a model of a Man-6 glycan. The sugar moieties susceptible to bond breaks upon oxidation with periodate are indicated. Grey circles correspond to mannose, black squares correspond to N-acetylglucosamine, T13 corresponds to the tryptic peptide NITR including the N-glycosylation site N(131) of SEQ ID NO:2 of the related enzyme sulfamidase.

[0134] FIG. 9A is a diagram visualizing the extent of bond breaking of the tryptic peptide T13+Man-6 glycan after chemical modification of sulfamidase, a related lysosomal enzyme, according to the previously known method (black bar), new method 1 (black dots), new method 2 (white), and new method 3 (cross-checkered).

[0135] FIG. 9B is a diagram visualizing the relative abundance of single bond breaks in the tryptic peptide T13+Man-6 glycan after chemical modification of sulfamidase according to the previously known method (black bar), new method 1 (black dots), new method 2 (white), and new method 3 (cross-checkered).

[0136] FIG. 10 is a table listing amino acid sequences of human iduronate 2-sulfatase, (SEQ ID NO:1) and human sulfamidase (SEQ ID NO:2; related to Examples 8 and 9).

EXAMPLES

[0137] The Examples which follow disclose the development of a modified iduronate 2-sulfatase polypeptide according to the present disclosure.

[0138] Unless stated otherwise, the recombinant iduronate 2-sulfatase used in the Examples below was the medicinal product Elaprase. Elaprase was purchased from a pharmacy (Apoteket farmaci, Sweden), stored according to the manufacturer's specifications and treated under sterile conditions.

Example 1

Chemical Modification of Iduronate 2-Sulfatase According to Previously Known Method

Material and Methods

[0139] Prior to chemical modification iduronate 2-sulfatase was diluted to 0.58 mg/ml in Elaprase drug product buffer.

Chemical Modification According to WO 2008/109677:

[0140] In order to modify glycan moieties, iduronate 2-sulfatase (SEQ ID NO:1), was initially incubated with 20 mM sodium meta-periodate at 0 C. for 6.5 h in 20 mM sodium phosphate, 137 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, 137 mM NaCl (pH 6.0) over night at 4 C. Following dialysis, reduction was performed by addition of sodium borohydride to the reaction mixture at 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, 137 mM NaCl (pH 6.0). All incubations were performed in the dark.

Example 2

Analyses of Iduronate 2-Sulfatase Modified According to Known Method

Material and Methods

[0141] The iduronate 2-sulfatase modified according to known method as described in Example 1 was subjected to the following analyses.

SDS-PAGE Analysis:

[0142] 5 g of iduronate 2-sulfatase and modified iduronate 2-sulfatase 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).

Enzymatic Activity:

[0143] Catalytic activity of iduronate 2-sulfatase was assessed by incubating preparations of iduronate 2-sulfatase with the substrate 4-Methylumbeliferone iduronide-sulfate. The concentration of substrate in the reaction mixture was 50 M and the assay buffer was 50 mM sodium acetate, 0.005% Tween 20, 0.1% BSA, 0.025% Anapoe X-100, 1.5 mM sodium azide, pH 5. After the incubation, further desulphation was inhibited by addition of a stop buffer containing 0.4 M sodium phosphate, 0.2 M citrate pH 4.5. A second 24 hour incubation with iduronate 2-sulfatase (assay concentration 0.83 g/mL) was performed to hydrolyze the product (4-methylumbeliferone iduronide) and release 4-Methylumbeliferone, which was monitored by fluorescence at 460 nm after quenching the reaction with 0.5 M sodium carbonate, 0.025% Triton X-100, pH 10.7.

Glycan Analysis by LC/MS of Tryptic Fragments:

[0144] The glycosylation pattern was determined for the different iduronate-2-sulfatase batches produced. Prior to glycopeptide analysis, iduronate-2-sulfatase (ca 20 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 NH4HCO3 at 60 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.

[0145] Seven peptide fragments of the trypsin digested iduronate-2-sulfatase contained potential N-glycosylation sites, N(x), where x refers the position of the asparagine in the iduronate-2-sulfatase amino acid (aa) sequence as defined in SEQ ID NO:1, were:

N(6) peptide, aa 1-23, 2500.30 Da
N(90) peptide, aa 86-99, 1607.81 Da
N(119) peptide, aa 111-139, 3301.47 Da
N(221) peptide, aa 216-246, 3504.76 Da
N(255) peptide, aa 249-269, 2356.21 Da
N(300) peptide, aa 289-322, 3678.83 Da
N(488) and N(512) peptide, aa 474-525, 5980.70 Da
The molecular mass of each peptide fragment is given.

[0146] For the investigation of possible glycosylation variants, the N(90) tryptic peptide fragment was selected for further glycopeptide analysis. The analysis 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, Agilent Technologies). 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 MS 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.

Results

[0147] As apparent by SDS-PAGE analysis, two major peptides of sizes distinct from that of monomeric iduronate 2-sulfatase were formed as a result of the chemical modification (FIG. 2A, lane 3). The first peptide denoted A is roughly twice the size of monomeric iduronate 2-sulfatase, and most probably represents a covalently linked dimer, whereas the second peptide denoted B is roughly 5 kDa smaller than iduronate 2-sulfatase and represents a peptide cleavage product. The main peptide band, representing the monomer, is smaller for iduronate 2-sulfatase modified using the known method as compared to the unmodified iduronate 2-sulfatase, indicative of loss of molecular weight by the chemical modification procedure (FIG. 2A, lane 3 versus lane 2). The molecular weight of iduronate 2-sulfatase is somewhat reduced after modification due to the bond breaking within the glycan moieties.

[0148] Glycan analysis was performed on the selected N(90) tryptic peptide fragment both prior and after chemical modification. Prior to chemical modification sialylated and fucosylated complex oligosaccharides were found on this asparagine. After the chemical modification no naturally occurring glycan structures were present at this position (FIG. 3).

[0149] The activity of iduronate 2-sulfatase modified according to the known method was below 50% of that of unmodified iduronate 2-sulfatase (results not shown).

Example 3

New Methods for Chemical Modification of Iduronate 2-Sulfatase

Material and Methods

[0150] Prior to chemical modification iduronate 2-sulfatase was diluted to 0.58 mg/ml in Elaprase drug product buffer.

Chemical Modification According to New Method 1:

[0151] Iduronate 2-sulfatase was initially incubated at 15 mM sodium meta-periodate at 0 C. for 1 h in 20 mM sodium phosphate, 137 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. Thereafter sodium borohydride was added to the reaction mixture to a final concentration of 35 mM and was allowed to proceed for 1.5 h at 4 C. Finally, the enzyme preparation was ultrafiltrated against 20 mM sodium phosphate, 137 mM NaCl (pH 6.0). All incubations were performed in the dark. The new method 1 for chemical modification is depicted in FIG. 1B.

Chemical Modification According to New Method 2:

[0152] Iduronate 2-sulfatase was initially incubated at 15 mM sodium meta-periodate at 0 C. for 0.5 h in 20 mM sodium phosphate, 137 mM NaCl (pH 6.0). Glycan oxidation was quenched by addition of ethylene glycol to a final concentration of 96 mM. Quenching was allowed to proceed for 15 min at 0 C. Thereafter sodium borohydride was added to the reaction mixture to a final concentration of 38 mM and the resulting mixture was held at 0 C. for 0.5 h. Finally, the enzyme preparation was ultrafiltrated against 20 mM sodium phosphate, 137 mM NaCl (pH 6.0). All incubations were performed in the dark. The new method 2 for chemical modification is depicted in FIG. 1C.

Chemical Modification According to New Method 3:

[0153] Iduronate 2-sulfatase was initially incubated at 10 mM sodium meta-periodate at 0 C. for 0.5 h in 20 mM sodium phosphate, 137 mM NaCl (pH 6.0). Glycan oxidation was quenched by addition of ethylene glycol to a final concentration of 96 mM. Quenching was allowed to proceed for 15 min at 0 C. Thereafter sodium borohydride was added to the reaction mixture to a final concentration of 15 mM and the resulting mixture was held at 0 C. for 0.5 h. Finally, the enzyme preparation was ultrafiltrated against 20 mM sodium phosphate, 137 mM NaCl (pH 6.0). All incubations were performed in the dark. The new method 3 for chemical modification is depicted in FIG. 1D.

Chemical Modification According to New Method 4:

[0154] Reaction conditions were as described for new method 2, with the single exception that periodate oxidation was performed in the presence of 0.5 mg/mL heparin.

Results

[0155] 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.

[0156] In order to provide an improved method for chemical modification of glycans, in particular a procedure that provides a modified iduronate 2-sulfatase 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.

[0157] 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 iduronate 2-sulfatase to reactive aldehyde intermediates. The new chemical modification procedures are depicted in FIG. 1B, FIG. 1C and FIG. 1D.

Example 4

Analyses of Iduronate 2-Sulfatase Modified According to New Methods

Material and Methods

[0158] The iduronate 2-sulfatase modified according to the new methods of Example 3 were subjected to the following analyses.

SDS-PAGE Analysis:

[0159] 2 g of iduronate 2-sulfatase modified in accordance with the known method (Example 1) as well as with the new method 1, 2, 3 and 4 (Example 3) were loaded into separate individual wells in accordance with the description in Example 2.

Glycan Analysis by LC/MS of Tryptic Fragments:

[0160] The glycan analysis was performed as described in Example 2.

Enzymatic Activity:

[0161] Activity was determined according to the procedure described in Example 2.

Results

SDS-PAGE Analysis:

[0162] The new chemical modification methods 1, 3-4 (FIG. 2B, lane 4, 6 and 7) resulted in single peptides of sizes identical to that of unmodified full length iduronate 2-sulfatase. New method 2 (FIG. 2B, lane 5) however also resulted in a peptide corresponding to the band denoted A in FIG. 2B. However, compared to the iduronate 2-sulfatase modified according to the known method, new method 2 gave rise to less unwanted covalent dimerization. Thus, the decrease in monomer size, which was apparent for the iduronate 2-sulfatase modified with the known method, was not observed for any of the new methods 1-4. In addition, strand-breaks in the iduronate 2-sulfatase polypeptide prepared by the new methods could not be observed or were very limited compared to strand-break occurrence in the iduronate 2-sulfatase prepared according to Example 1. Importantly, the use of a ligand protecting the active site (heparin in new method 4) was compatible with the procedure and resulted in modified iduronate 2-sulfatase that by SDS-PAGE analysis was indistinguishable from that where the ligand was omitted (new method 3).

[0163] In conclusion, process related impurities, limiting the quality and safety of a medicament produced by the modification methods, are significantly reduced by the new methods as compared to the previously known methods.

Glycan Analysis by LC/MS of Tryptic Fragments:

[0164] Glycan analysis of the selected N(90) tryptic peptide fragment showed that no naturally occurring glycan structures were present at this position after chemical modification (FIG. 3).

Enzymatic Activity:

[0165] Iduronate 2-sulfatase prepared according to new method 1, 2, 3 and 4 showed an activity that was comparable to that of unmodified iduronate 2-sulfatase (FIG. 4).

Example 5

Receptor Mediated Endocytosis In Vitro

Material and Methods

[0166] Endocytosis of Iduronate 2-sulfatase and Iduronate 2-sulfatase modified according to the new method 1 was evaluated in human primary fibroblasts expressing M6P receptors. The fibroblast cells were incubated for 24 h in DMEM medium supplemented with iduronate 2-sulfatase (2, 0.5 and 0.12 g/mL), Iduronate 2-sulfatase modified according to the new method 1 (4, 1 and 0.25 g/mL) or PBS. The cells were washed twice in DMEM and once in 0.9% NaCl prior to cell lysis using 100 L 1% Triton X100. Lysate iduronate 2-sulfatase protein content was determined using the electrochemiluminescence immunoassay described in Example 6.

Results

[0167] Iduronate 2-sulfatase could be detected in cell homogenate for both preparations evaluated in the endocytosis assay. Modified iduronate 2-sulfatase prepared by new method 1 had a protein concentration in cell homogenate below 25% of that obtained with unmodified recombinant iduronate 2-sulfatase (FIG. 5). The protein concentration retained in cells first loaded with and then grown in the absence of iduronate 2-sulfatase for 2 days were comparable for all preparations showing that chemical modification do not negatively impact on lysosomal stability.

[0168] It can therefore be concluded that chemical modification render iduronate 2-sulfatase less prone to cellular uptake which is a consequence of removal of epitopes for glycan recognition receptors such 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 6

In Vivo Serum Clearance of Modified Iduronate 2-Sulfatase Produced by New Method 2 & 3

Material and Methods

[0169] Serum clearance (CL) of unmodified and modified recombinant iduronate 2-sulfatase modified according to the new method 2 and 3 of Example 3 was investigated in mice (C57BL/6J). The mice were given an intravenous single dose administration in the tail vein. Iduronate 2-sulfatase modified according to the new method 2 was studied together with unmodified iduronate 2-sulfatase at a dose of 1 mg/kg. Both enzymes were formulated at 0.2 mg/mL and administered at 5 mL/kg. Iduronate 2-sulfatase modified according to the new method 3 was studied together with unmodified iduronate 2-sulfatase at a dose of 3 mg/kg. Both enzymes were formulated at 0.6 mg/mL and administered at 5 mL/kg. Blood samples were taken at different time points up to 24 h post dose (3 mice per time point). The serum levels of iduronate 2-sulfatase and modified iduronate 2-sulfatase were analyzed by ECL. Serum clearance was calculated using WinNonlin software version 6.3 (Non-compartmental analysis, Phoenix, Pharsight Corp., USA).

Quantification of Iduronate 2-Sulfatase and Modified Iduronate 2-Sulfatase by Electrochemiluminescence (ECL) Immunoassay:

[0170] Iduronate 2-sulfatase and modified iduronate 2-sulfatase in serum PK samples were determined by ECL immunoassay using the Meso Scale Discovery (MSD) platform. The wells of a 96 well streptavidin gold plate (#L15SA-1, MesoScaleDiscovery (MSD)) were blocked with 1% Fish Gelatin in Phosphate buffer saline (PBS), washed with wash buffer (PBS+0.05% Tween-20) and incubated with a biotinylated, affinity purified goat-a-human Iduronate 2-sulfatase polyclonal antibody (BAF2449, R&D) after washing different dilutions of standard and PK samples in sample diluent (1% Fish Gelatin in PBS+0.05% Tween 20+1% C57BL6 serum pool) were incubated in the plate at 700 rpm shake and RT for 2 h. The plate was washed and a iduronate 2-sulfatase specific Rutenium (SULFO-TAG, MSD) tagged goat polyclonal antibody (AF2449, R&D) was added and allowed to bind to the captured iduronate 2-sulfatase or chemically modified iduronate 2-sulfatase. The plate was washed and 2 Read Buffer (MSD) was added. The plate content was analyzed using a MSD Sector 2400 Imager Instrument. The instrument applies a voltage to the plate electrodes, and the SULFO-TAG label, bound to the electrode surface via the formed immune complex, will emit light. The instrument measures the intensity of the emitted light which is proportional to the amount of iduronate 2-sulfatase or chemically modified iduronate 2-sulfatase in the sample. The amount of iduronate 2-sulfatase or chemically modified iduronate 2-sulfatase was determined against a relevant iduronate 2-sulfatase or chemically modified iduronate 2-sulfatase standard.

Results

[0171] The serum clearance in mice of modified iduronate 2-sulfatase by method 2 was reduced 4-fold as compared to unmodified iduronate 2-sulfatase, see Table 1 below and FIG. 6. Whereas for iduronate 2-sulfatase modified according to method 3 it was reduced by 1.7 fold. Thus, both methods give a robust prolongation of serum half live of iduronate 2-sulfatase. This is probably at least partly due to the inhibition of receptor mediated uptake in peripheral tissue following chemical modification of iduronate 2-sulfatase.

TABLE-US-00001 TABLE 1 Serum clearance of iduronate 2-sulfatase and modified iduronate 2-sulfatase Dose Serum CL Test article (mg/kg) (mL/(h .Math. kg)) iduronate 2-sulfatase (SEQ ID NO: 1) 1 60 modified iduronate 2-sulfatase (New 1 14 method 2, SEQ ID NO: 1) iduronate 2-sulfatase (SEQ ID NO: 1) 3 50 modified iduronate 2-sulfatase (New 3 30 method 3, SEQ ID NO: 1)

Example 7

Potency of Modified Iduronate 2-Sulfatase on Glucosaminoglycan Storage in the Brain of a Living Animal

[0172] The usefulness of iduronate 2-sulfatase, produced according to the new methods described in Example 4, to treat neurological complications associated with MPS-II is evaluated in a mouse model of the disease in a manner similar to that described in Assunta-Polito et al, Hum Mol Genet. 19:4871-4885 (2010). This mouse is deficient in iduronate 2-sulfatase and shows cellular and pathological phenotypes similar to the human patients.

[0173] Modified iduronate 2-sulfatase is administered i.v., e.g. every other day for one month. A primary measure of the efficacy of the modified 2-sulfatase is the glucosaminoglycan levels in the brain of the mouse.

Example 8

Analysis of Glycan Structure after Chemical Modification of Sulfamidase According to Previously Known Method

[0174] In order to characterize the end product of chemical modification according to the previously known method, another sulfatase, namely sulfamidase (SEQ ID NO:2) was chemically modified according to the known method and characterized. Sulfamidase is due to its glycopeptide characteristics a suitable model protein for precise product identification after chemical modification.

Material and Methods

Chemical Modification According to the Known Method:

[0175] The chemical modification of sulfamidase according to the known method was performed as described in Example 1.

Glycosylation Analysis:

[0176] The glycosylation pattern was determined for unmodified and different modified sulfamidase batches. 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.

[0177] 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:2, were:

N(21) containing fragment (residue 4-35 of SEQ ID NO:2, 3269.63 Da)
N(122) containing fragment (residue 105-130 of SEQ ID NO:2, 2910.38 Da)
N(131) containing fragment (residue 131-134 of SEQ ID NO:2, 502.29 Da)
N(244) containing fragment (residue 239-262 of SEQ ID NO:2, 2504.25 Da)
N(393) containing fragment (residue 374-394 of SEQ ID NO:2), 2542.22 Da
The molecular mass of each peptide fragment is given.

[0178] 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

[0179] 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. Resulting modifications on the glycan moieties on four tryptic peptide fragments containing the N glycosylation sites N(21), N(131), N(244) and N(393) were investigated by LC-MS analysis.

Results

Glycosylation Analysis:

[0180] 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).

[0181] 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.

[0182] 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. FIG. 8A illustrates an example of predicted bond breaks on mannose after chemical modification. FIG. 8B depicts a model of Man-6 glycan showing the theoretical bond breaks that may take place after oxidation with sodium periodate.

[0183] The tryptic peptide NITR with Man-6 glycan attached to N(131) (T13+Man-6 glycan) was analyzed by mass spectrometry, prior to and after chemical modification according to the previously known method (results not shown). Ions corresponding to the chemically modified glycopeptide with various degree of bond breaking were identified. For Man-6 glycan, there can theoretically be a maximum of 3 double bond breaks and one single bond break. When the modification was performed according to the known method, the most intense ion signal in the mass spectrum was found to be corresponding to 2 double bond breaks and 2 single bond breaks, while the second most intense ion signal corresponded to 3 double bond breaks and one single bond break, which is the most extensive bond breaks possible. A diagram visualizing the extent of bond breaking found on T13+Man-6 glycan after chemical modification according to the known method is shown in FIG. 9 (due to isotopic distribution from the ions observed, the results are approximate but comparable).

[0184] The reproducibility of the chemical modification was tested by using three different batches of chemically modified sulfamidase produced according to the previously known method. The ions corresponding to different degree of bond breaking showed very similar distribution in the MS spectra from the three different batches.

Example 9

Analysis of Glycan Structure after Chemical Modification of Sulfamidase According to New Methods 1, 2, and 3

Material and Methods

[0185] Sulfamidase, another sulfatase, was chemically modified according to the following new methods.

New Method 1:

[0186] Sulfamidase produced in Quattromed Cell Factory (QMCF) episomal expression system (Icosagen AS), 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.

New Method 2:

[0187] Sulfamidase 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.

New Method 3:

[0188] 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.

Glycosylation Analysis:

[0189] The glycosylation analysis was performed according to the LC-MS method described in Example 8. 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.

Results

Glycosylation Analysis:

[0190] Detailed characterization of the modified glycan profile on sulfamidase, chemically modified according to new methods 1, 2, and 3, was performed on the most abundant chemically modified glycopeptides. In this Example, the modification on Man-6 glycan after chemical modification according to new methods 1, 2, and 3, was investigated.

[0191] 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 FIG. 8B a model of Man-6 glycan showing the bond breaks possible to occur after oxidation with sodium periodate). When the modification was performed according to the new method 1, the most intense ion signal in the mass spectrum (not shown) was found to be corresponding to one double bond break and 3 single bond breaks, while the second most intense ion signal corresponded to 2 double bond breaks and 2 single bond breaks. When the modification was performed according to new methods 2 and 3, the bond breaks on Man-6 glycan were even further shifted to preferentially single bond breaks. In FIG. 9A is shown a diagram visualizing the extent of bond breaking of the tryptic peptide T13+Man-6 glycan after chemical modification.

[0192] The reproducibility of the chemical modification was tested by using triplicates (new method 1) or duplicates (new methods 2) of chemically modified sulfamidase.

[0193] 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, 2, and 3, there was a large difference in degree of bond breaking. This is illustrated in FIG. 9A, where the distribution of the different degrees of bond breaking is plotted for the four methods (due to isotopic distribution from the ions observed, the results are approximate, but comparable).

[0194] FIG. 9B shows the relative abundance of single bond breaks for the methods used. The previously known method provides a modified sulfamidase having 45% single bond breaks in the investigated Man-6-glycan, while the new methods 1, 2, and 3 have 70, 80, and 82% single bond breaks, respectively, after chemical modification.

[0195] Subsequently, the milder methods of chemical modification described herein provides a product with significantly less double bond breaks in modified glycans.

Example 10

Analysis of Glycan Structure after Chemical Modification of Iduronate 2-Sulfatase According to Previously Known Method

[0196] As evident from SDS-PAGE analysis (FIG. 2A), chemical modification according to the previously known method resulted in the lowering of the molecular weight of iduronate 2-sulfatase. In order to characterize iduronate 2-sulfatase modified according to the previously known method, glycosylation analysis is performed according to the methods described in Example 8.

Material and Methods

Chemical Modification According to the Known Method:

[0197] The chemical modification of sulfamidase according to the known method is performed as described in Example 1.

Glycosylation Analysis:

[0198] The glycosylation analysis is performed according to the LC-MS method described in Example 2 and 8.

Results

[0199] Chemical modification of iduronate 2-sulfatase according to the known method is expected to give rise to modified glycopeptides(s) for which the extent of bond breaking is similar to the extent of bond breaking in glycopeptides of modified sulfamidase (see Example 8). Thus, the known method is expected to give rise to predominantly double bond breaks in the glycan moieties, which could explain the loss of molecular weight observed on SDS-PAGE (FIG. 2A, lane 2 vs. 3).

Example 11

Analysis of Glycan Structure after Chemical Modification of Iduronate 2 Sulfatase According to New Methods 1, 2, and 3

Material and Methods

[0200] Iduronate 2-sulfatase modified according to the methods described in Example 3 is subjected to glycosylation analysis.

Glycosylation Analysis:

[0201] The glycosylation analysis is performed according to the LC-MS method described in Example 10.

Results

[0202] The new methods of chemical modification described herein provides a product with significantly less double bond breaks in modified glycans, which is also reflected in the difference in molecular weight of iduronate 2-sulfatase produced by the different methods (FIG. 2B).

Example 12

Chemical Modification of Iduronate 2-Sulfatase in the Presence of an Active Site Protecting Ligand

[0203] As described in Example 3 new method 6, oxidation (step a)) can be performed in the presence of a ligand. The ligand can be a substrate as exemplified by 4-methylumbeliferone iduronide-sulfate. Alternatively, any other known ligand of iduronate 2-sulfatase, such as sulfate, can be used. Heparin or heparin sulfate of any origin could also be used as an additive throughout one or more of the reaction steps.

Example 13

Chemical Modification of Iduronate 2-Sulfatase Immobilized on a Gel Matrix

[0204] The modification method as described herein, and in particular, new method 1-6 of Example 3, is performed while iduronate 2-sulfatase is immobilized on a gel matrix. By using a POROS XQ Strong Anion Exchange column, iduronate 2-sulfatase is immobilized by loading the column using a sodium phosphate buffer with a pH of 7.5. Following loading of iduronate 2-sulfatase, the column is equilibrated with solutions for step a), quenching of step a), step b), and quenching of step b) in a consecutive fashion. Elution of chemically modified iduronate 2-sulfatase is performed by washing the column with a buffer containing 100 mM sodium phosphate and 150 mM sodium chloride with a pH of 5.6.

Example 14

Chemical Modification of Iduronate 2-Sulfatase in a Continuous Process

[0205] The modification method as described herein, and in particular, new methods 1-6 of Example 3, is performed in a continuous mode. By applying a continuous flow, e.g. by utilizing a HPLC pump or similar equipment, a solution of iduronate 2-sulfatase is transported through a tubing. The tubing can be of any inert material, e.g. ethylenetetrafluoroethylene or polytetrafluoroethylene. By adjusting the speed of flow and the inner tubing diameter, speed of transport within the system is precisely adjusted. By applying inlets at defined positions, stock solutions of the reagents of the chemical modification are added in a continuous mode. This can be achieved in a multi-pump HPLC system, e.g. an Akta avant (GE Healthcare). At each point of inlet a small-volume mixing chamber is added, similar to those present in most multi-pump HPLC systems.

[0206] In a specific continuous mode example of the new methods 1-6, reagents are added at an inlet (valve) at a flowrate that is approximately 10 of that for the iduronate 2-sulfatase solution. Stock solution of reagents are prepared at a concentration that is ten-fold higher the concentration accounted for in new method 1-6 in Example 3.

Example 15

Distribution of Modified Iduronate 2-Sulfatase to Brain of Iduronate 2-Sulfatase Deficient Mice

Materials and Methods

[0207] The distribution of intravenously (iv) administrated modified iduronate 2-sulfatase produced according to new method 2 of Example 3 to brain in vivo was investigated.

Test Article Preparation:

[0208] Modified iduronate 2-sulfatase was formulated at 2 mg/mL, sterile filtrated and frozen at 70 C. until used.

Animals:

[0209] Male mice, IDS-KO (B6N.Cg-Idstm1Muen/J)(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. The mice were given an intravenous administration in the tail vein of 10 mg/kg modified iduronate 2-sulfatase. The study was finished 24 h after the last injection. 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. Brain was dissected weighed and frozen rapidly in liquid nitrogen. Brain homogenates where prepared and activity was assessed using the method described in example 2 with addition of 10 mM lead acetate in the assay buffer as adjustment to the protocoll.

Results

[0210] Activity of modified iduronate 2-sulfatase in perfused brain homogenates of IDS-KO mice could be confirmed. An average activity of 1.80.4 M/min (n=4) was determined under the assay conditions used.