Production and purification of recombinant arylsulfatase A

Abstract

The present invention pertains to a process for production of recombinant arylsulfatase A in a cell culture system, the process comprising culturing a mammalian cell capable of producing rASA in liquid medium in a system comprising one or more bio-reactors; and concentrating, purifying and formulating the rASA by a purification process comprising one or more steps of chromatography. Other aspects of the invention provides a pharmaceutical composition comprising rASA, which is efficiently endocytosed via the mannose-6-phosphate receptor pathway in vivo as well as a rhASA a medicament and use of a rhASA for the manufacture of a medicament for reducing the galactosyl sulphatide levels within target cells in the peripheral nervous system and/or within the central nervous system in a subject. A final aspect of the invention provides a method of treating a subject in need thereof, said method comprising administering to said subject a pharmaceutical composition comprising a rhASA and thereby obtaining a reduction in the galactosyl sulphatide levels in target cells within said subject.

Claims

1. A method of treating metachromatic leukodystrophy (MLD) comprising a step of administering to a subject suffering from and/or diagnosed with metachromatic leukodystrophy a composition comprising recombinant arylsulfatase A in an amount and an administration interval for a treatment period effective to reduce the levels of galactosyl sulfatide by at least 10% within cells in the central nervous system of the subject, wherein the recombinant arylsulfatase A comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4, wherein the composition is administered systemically.

2. The method according to claim 1, wherein the composition comprising recombinant arylsulfatase A is administered intravenously.

3. The method according to claim 1, wherein the recombinant arylsulfatase A comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 3 or SEQ ID NO: 4.

4. The method according to claim 1, wherein the recombinant arylsulfatase A has a specific activity of at least 20 U/mg, wherein one unit (1 U) of enzyme activity is defined as the hydrolysis of 1 mol para-Nitrocatechol sulfate (pNCS) per minute at 37 C., pH 5.0.

5. The method according to claim 1, wherein the recombinant arylsulfatase A has a specific activity of at least 50 U/mg, wherein one unit (1 U) of enzyme activity is defined as the hydrolysis of 1 mol para-Nitrocatechol sulfate (pNCS) per minute at 37 C., pH 5.0.

6. The method according to claim 1, wherein the composition comprising recombinant arylsulfatase A is administered at a dose of between 0.1 and 100 mg arylsulfatase A per kg of subject body weight.

7. The method according to claim 1, wherein the composition comprising recombinant arylsulfatase A is administered daily, weekly, every other week, or monthly.

8. The method according to claim 7, wherein the composition comprising recombinant arylsulfatase A is administered weekly.

9. The method according to claim 7, wherein the composition comprising recombinant arylsulfatase A is administered every other week.

10. The method according to claim 1, wherein levels of galactosyl sulfatide are reduced by at least 13% within cells in the central nervous system of the subject.

11. The method according to claim 10, wherein levels of excess galactosyl sulfatide are reduced by at least 30% within cells in the central nervous system of the subject.

Description

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

(1) FIG. 1: (A) Schematic representation of the system for continuous cell propagation. (B) Outline of the purification process.

(2) FIG. 2: Ion exchange chromatography batch test of rhASA.

(3) FIG. 3: (A) HPLC-chromatogram of rhASA after completion of step (V) of the purification procedure according to the present invention. (B) Enlargement of the HPLC chromatogram of (A).

(4) FIG. 4: Sulfatide clearance in MLD-fibroblasts loaded with radiolabelled sulphatide after incubation with rhASA for 24 hours.

(5) FIG. 5: Sulfatide clearance in MLD-fibroblasts loaded with radiolabelled sulphatide after incubation with rhASA for 6, 24 and 48 hours.

(6) FIG. 6: rhASA serum levels 10 min after intravenous injection of rhASA

(7) FIG. 7: In vitro analyses of CHO-rhASA. (A) SDS-PAGE of native () and deglycosylated (+) enzyme. Gels were stained with Coomassie blue. The masses of protein standards (std) are indicated. Treatment with PNGase F causes a shift of the ASA band due to the loss of N-linked carbohydrates. (B) M6P-dependent endocytosis of CHO-rhASA by BHK cells analysed by ELISA. BHK cells were incubated with 1 g CHO-rhASA per ml medium for 20 h in the presence or absence of 10 mM M6P. Other dishes were supplemented with 10 mM glucose 6-phosphate (G6P) as a control. The complete block of endocytosis by M6P indicates that BHK cells internalize CHO-rhASA via M6P receptors. This indicates the presence of M6P residues on CHO-rhASA. The data are expressed as meansSD, n=3.

(8) FIG. 8: (A) MALDI-TOF analysis. rhASA isolated from secretions of CHO cells exhibits a correct size of 57 kDa and the enzyme preparation lacks major contaminants. The minor peak at 29 kDa (ASA/2) represents the doubly charged molecule. (B) MALDI-TOF analysis of CHO-rhASA treated with a low concentration of PNGase F. The limited deglycosylation yields four products. They presumably represent rhASA which bears three, two, one or no N-glycan(s). The mass pattern therefore suggests that all three potential N-glycosylation sites of the CHO-rhASA are glycosylated.

(9) FIG. 9. Pharmacokinetics of CHO-rhASA after a single injection into the tail vein of ASA-deficient mice. All data are expressed as meansSD. (A) rhASA levels in plasma in the first hour after injection of 20 mg/kg (closed circles, n=9) or 40 mg/kg (open circles, n=11). The concentration was determined by ELISA. (B) rhASA levels in liver at various times after administration of 40 mg/kg. The concentration was determined by ELISA (n=3). (C) Tissue kinetics of rhASA after administration of 40 mg/kg in kidney (open bars), sciatic nerve (hatched bars) and brain (closed bars) as determined by ELISA (n=3). ndnot determined.

(10) FIG. 10: Pharmacokinetics of CHO-rhASA after a single injection into the tail vein of ASA-deficient mice. All data are expressed as meansSD. (A) Tissue levels of rhASA 8 days after injection of different enzyme doses (n=5). The concentration was determined by ELISA for kidney (open bars), brachial plexus (closed bars) and sciatic nerve (hatched bars). Tissues from untreated wildtype mice and mock-treated ASA knockout mice were analysed as negative controls. Control homogenates from the sciatic nerve show some unspecific background signal since the incubation times had to be prolonged to quantify the specific immunoreactivity in the very small nerve samples from treated mice. (B) Relative tissue distribution of rhASA 8 days after injection of 20 mg/kg. The rhASA concentration was measured by ELISA and normalized on the level in liver (n=3).

(11) FIG. 11: Sulfatide clearance from tissues after single treatment of ASA-deficient mice with CHO-rhASA. The levels of sulfatide (closed bars), cholesterol (open bars) and sphingomyelin (hatched bars) were determined by TLC and are expressed as means of arbitrary unitsSD. Asterisks indicate a statistically significant difference to mock-treated controls (student's t-test, p<0.05). (A) Analysis of kidney lipids by TLC. Lipids were extracted from kidneys of different experimental groups and incubated under alkaline conditions to hydrolyze phosphoglycerolipids and cholesterylester. The reaction products were separated by TLC, visualized and analysed by densitometry. The loading volumes were normalized on the protein concentration of the crude tissue homogenate used for lipid extraction. Increasing amounts of cholesterol (chol), sulfatide (sulf) and sphingomyelin (sm) were loaded as standards. (B) Lipid levels in kidney at different times after injection of 40 mg/kg (n=3). (C) Lipid levels in kidney 8 days after injection of different enzyme doses (n=5).

(12) FIG. 12: Sulfatide clearance from tissues after single treatment of ASA-deficient mice with CHO-rhASA. The levels of sulfatide (closed bars), cholesterol (open bars) and sphingomyelin (hatched bars) were determined by TLC and are expressed as means of arbitrary unitsSD. Asterisks indicate a statistically significant difference to mock-treated controls (student's t-test, p<0.05). (A) Lipid levels in brain at different times after injection of 40 mg/kg (n=3). (B) Lipid levels in the sciatic nerve 8 days after injection of different enzyme doses (n=5). (C) Lipid levels in the brachial plexus 8 days after injection of different enzyme doses (n=5).

(13) FIG. 13. Lipid levels in (A) kidney (B) brain, (C) sciatic nerve and (D) brachial plexus after repeated dosing of 20 mg CHO-rhASA/kg once weekly. The knockout mice were treated with up to four infusions of CHO-rhASA. Controls were mock-treated with four injections of buffer. The levels of sulfatide (closed bars), cholesterol (open bars) and sphingomyelin (hatched bars) were analysed 8 days after the last dosing and are expressed as meansSD (n=3). Asterisks indicate a significant difference in the sulfatide level compared to mock-treated controls (student's t-test, p<0.05).

(14) FIG. 14. Sulfatide storage in the CNS as histochemically demonstrated by incubation with alcian blue (see Methods). Coronal thick sections (100 m) through the brain stem of a mock-treated ASA knockout mouse (A, C, E, G) and an enzyme-treated ASA knockout mouse (B, D, F, H). (A, B) overview to outline the regions shown at higher magnification in the photomicrographs below. 7nroot of facial nerve, CbNcerebellar nucleus, icpinferior cerebellar pedunculus, PnCpontine reticular nucleus, VCventral cochlear nucleus, Vevestibular nucleus. (C, D) Abducens nucleus (6N) and adjacent regions. 7 ggenu of facial nerve. In the mock-treated mouse alcianophilic material (sulfatide) is seen in many cells, of which phagocytes and neurons can be identified (marked by triangles and circles, respectively, in the inset). In the rhASA-treated mouse alcianophilic material is seen mainly in neurons. (E, G) and (F, H) Inferior cerebellar pedunculus (icp) as an example of a white matter tract. In the mock-treated mouse numerous large alcianophilic phagocytes are seen only a few of which are in the optic focus in G (some are marked by circles). The small alcianophilic granules may be associated to oligodendrocytes which can, however, not be identified at this magnification. In the rhASA-treated mouse the icp shows only small alcianophilic structures suggesting that the sulfatide-storing phagocytes are decreased in size and/or number. The overall staining of the cerebellar and vestibular nuclei in F is reduced since alcianophilia is restricted mainly to neurons and has largely disappeared from phagocytes. Bars: 500 m in A and B; 100 m in C and D; 200 m in E and F; 10 m in G, H and insets of C and D.

(15) FIG. 15. Sulfatide storage in the kidney as histochemically demonstrated by incubation of 100 m slices with alcian blue (see Methods). (A) wild type mouse; weak staining is seen in the inner stripe of the outer medulla (iS-oM), whereas the outer stripe of outer medulla (oS-oM) and the cortex (C) are unstained. (B, C) Mock-treated ASA knockout mouse. Severe sulfatide storage (alcianophilic material) is seen in the tubules of the inner stripe of outer medulla; in the outer stripe and cortex several profiles show sulfatide storage. (D, E) ASA knockout mouse treated with four doses of 20 mg CHO-rhASA/kg. The cortex is devoid of alcianophilic material. In the outer stripe staining is reduced. In the inner stripe staining appears unchanged as compared with the mock-treated animal. Bars represent 500 m in A, B, D and 200 m in C, E. (F-H and J) Semithin sections stained with toluidine blue. (F, H) Mock-treated ASA knockout mouse, thick ascending limb (TAL) in the outer stripe and distal convoluted tubules (DCT) in cortex. The intensely stained cytoplasmic inclusions correspond to lysosomes filled with sulfatide as shown previously (Lullmann-Rauch, R. et al., Histochem. Cell Biol., 116, 161-169). (G, J) ASA knockout mouse treated with four doses of 20 mg CHO-rhASA/kg, representative segments of the nephron corresponding to those in F and H. The storage material is reduced in the TAL and absent from the DCT profiles. Gglomerulus, PTproximal tubule. Bar represents 20 m in F-H and J.

(16) FIG. 16. Functional effects of repeated dosing with 20 mg CHO-rhASA/kg once weekly. (A) Neurophysiological parameters determined in wildtype mice (closed bars), mock-treated ASA knockout mice (open bars) and ASA knockout mice 6 days after the fourth injection of CHO-rhASA (hatched bars). The indicated parameters were measured in the intrinsic foot musculature after distal stimulation of the sciatic nerve. Data are expressed as meansSD (n=7-8). Asteriscs indicate a significant difference (student's t-test, p<0.05). (B) Rotarod performance of mice at a mean age around 9 mo (9.21.1 mo). ASA knockout mice (closed triangles, n=7) were analysed at day 2 and 3 after the third treatment with 20 mg CHO-rhASA/kg. Age-matched wildtype mice (circles, n=10) and mock-treated ASA knockout mice (open triangles, n=10) were analysed as controls in parallel. The percentages of mice, which were able to balance on a slowly rotating rod for at least 4 min were determined in four consecutive trials. (C) Rotarod performance of mice at a mean age of 12 mo (11.81.1 mo). Also in this experiment the treated mice were tested at day 2 and 3 after the third injection of 20 mg CHO-rhASA/kg. Legend and group sizes as in B, except n=14 for rhASA-treated knockout mice.

(17) FIG. 17: Presentation of electrophysological parameters from studies on nerve motor conductivity. (Corresponds in part to panel (A) of FIG. 16).

EXAMPLES

Example 1

Continuous Cell Propagation

(18) A continuous cell propagation system and a small to medium size purification process for rhASA in 200-400 ml column scale intended for scale-up to large-scale production (column scale >2 L) is developed. A schematic representation of the system is given in FIG. 1. The quality and purity of the final product (rhASA) is very high and suitable for toxicology testing (including steps 1-4+7) and finally also suitable for clinical trials (including all steps described). As described above, the process will include a capture step, 1-2 intermediate purification steps, 1 polishing step, 1-2 virus removal steps and 1 formulation step. 1 or more buffer exchange steps will also be included.

(19) Experimental Design:

(20) Several different chromatography gels are tested and performance of the different steps (with respect to removal of contaminants, yield and purity) are analysed with a battery of analytical methods described briefly below.

(21) Analytical Methods

(22) Enzyme activity: Arylsulfatase assay Total protein concentration: BCA analysis rhASA concentration: rhASA ELISA Purity: rpHPLC, SDS-PAGE Identity: rpHPLC, Western Blot rhASA HCP proteins: HCP-ELISA, Western blot HCP proteins Endotoxin level: According to European Pharmacopoeia (Ph. Eur.) method 2.6.14. For i.v.-administration the acceptable value is 5 IU/kg/h. With a maximal dose of 1 mg/kg/h and a concentration of the product of 5 mg/ml, the limit is 25 IU/ml. Osmolality: According to Ph. Eur. method 2.2.35. Since no acceptable value is stated in the European Pharmacopoeia for this exact product the value (250-350 mOsmol/kg) is defined because it compares to an isotonic solution of (0.9%) NaCl, which is well-tolerated in-vivo. DNA content: DNA threshold pH: According to Ph. Eur. method 2.2.3. Since no acceptable value is stated in the European Pharmacopoeia for this exact product. The value (7.0-8.0) is defined because it is neutral pH and well-tolerated in-vivo. Bacterial count: Ph. Eur. method 2.6.12 (membrane filtration) will be used to test the API and Bulk Substance. There is no acceptable value stated in the European Pharmacopoeia for this exact product. The value (10 cfu/ml) is defined to ensure an adequate minimal bioburden prior to sterilisation. The final product for i.v.-administration will be sterile and tested according to Ph.Eur. method 2.6.1
Description of Analytical Methods
Aryl Sulfhatase Assay

(23) In addition to its natural substrates ASA is also able to catalyze the hydrolysis of the synthetic, chromogenic substrate, para-Nitrocatechol sulfate (pNCS), see Fig. The product, para-Nitrocatechol (pNC), absorbs light at 515 nm. The method is described by Fluharty et al. 1978, Meth. Enzymol. 50:537-47

(24) Materials and Equipment

(25) Spectrophotometer Spectra MAX Plus from Molecular Devices or equivalent.

(26) Cuvette 1 ml (glass or plastic) with 1 cm path-length suitable for 515 nm.

(27) Flat bottomed 96 well micro-titer plate.

(28) Chemicals and Reagents

(29) pNCSp-NitroCatechol Sulfate (no. N-7251, Sigma)

(30) BSABovine Serum Albumin Frac. V

(31) NaAcSodium Acetate trihydrate

(32) Triton X-100

(33) Tris-HCl molecular biology grade

(34) PBS, pH 7.4 w/o Ca.sup.2+, Mg.sup.2+: 0.20 g/l KCl, 0.20 g/l KH.sub.2PO.sub.4, 8 g/l NaCl, 1.15 g/l Na.sub.2HPO.sub.4. Adjust pH.

(35) All other solvents and chemicals were of p.a. quality (Merck) a. 2 ASA substrate solution: 30 mM pNCS, 10% (w/v) NaCl and 1 mg/ml BSA in 0.5 M NaAc pH 5.0. b. TBS, pH 7.5: 10 mM Tris-HCl and 150 mM NaCl in H.sub.2O. c. Stop solution: 1 M NaOH

(36) Since many anions and kations, such as SO.sub.4.sup.2, PO.sub.4.sup.3, SO.sub.3.sup.2, F.sup., Ag.sup.+, Cu.sup.2+ and Hg.sup.2+, are inhibitors of the enzyme at concentrations in the millimolar range or lower the sample is transferred to a suitable buffer (e.g. TBS) before activity is measured. This is done by dialysis or buffer exchange on a gel filtration column (e.g. PD10 from Amersham Pharmacia Biotech).

(37) a. Measurement of ASA Activity in Cell Supernatants

(38) The used medium is centrifuged (110g, 5 minutes) and the supernatant is transferred to a clean tube. The buffer is changed to TBS by dialysis or by using a gel filtration column.

(39) b. Measurement of Intracellular ASA Activity

(40) Cells in suspension are washed once with PBS and then once with TBS before they are lysed in 0.5 ml TBS+0.5% TritonX-100 for 10 minutes, RT. After vortexing the lysates are centrifuged (13.200 rpm, 10 minutes) and supernatants collected in clean tubes. Alternatively, the cells are resuspended in TBS and then lysed by repeated freeze-thawing cycles.

(41) c. Measurement of ASA Activity in In-Process Samples and Final Product

(42) The buffer is changed to TBS before activity is measured and protein concentration in the samples is determined using the BCA Protein Assay Reagent kit (see below). In order to assure linearity a final absorbance between 0.1 and 2 (see reference 2) is aimed at. Samples are diluted in TBS if necessary. a. 50 l of sample diluent (TBS or TBS+TritonX-100) is added in at least duplicates to a micro-titer plate and use as blanks. b. 50 l of samples or diluted samples is added in duplicates to the micro-titer plate. c. 50 l of 2ASA substrate solution is added into each well. The plate is sealed and incubated at 37+/0.5 C. for exactly 30 minutes. d. The reaction is stopped by adding 50 l of stop solution (1 M NaOH) into all wells. e. Pre-read is done using a micro-titer plate filled with 0.15 ml MilliQ water/well to correct for scattering effects. Subsequently the absorbance at 515 nm is measured within 30 minutes using a plate reader. The absorbance measured from the micro-titer plate to a 1 cm path length by the use of an application named Path Check. f. The delta absorbance (A) is calculated by subtracting the absorbance value of the blank from the measured absorbance of each of the samples. The molar extinction coefficient (M) for the product pNC is 12 400 M-1 cm-1.
Calculations
Definition:

(43) One Unit (1 U) of enzyme activity is defined as the hydrolysis of 1 mol pNCS per minute at 37 C., pH 5.0.

(44) The following equation is used in order to calculate the enzyme activity in mol pNCS hydrolysed/minml (=Units/ml):

(45) $\begin{array}{cc}\frac{\mathrm{Vtot}\left(\mathrm{ml}\right)}{{\mathrm{.Math.}}_M/1000\mathrm{Vsample}\left(\mathrm{ml}\right)\mathrm{Incubation}\mathrm{time}\left(\min \right)}A=\mathrm{Units}\text{/}\mathrm{ml}& \left(1\right)\end{array}$
where:
AA=bsorbance of sampleabsorbance of blank
Vtot (ml)=total reaction volume in ml (in this case 0.15 ml)
Vsample (ml)=added sample volume in ml (in this case 0.05 ml)
.sub.M=the molar extinction coefficient for the product pNC, which in this case is 12 400 M.sup.1 cm.sup.1

(46) Equation 1 could more simplified be written as:
A(0.15/(12 400/10000.0530))=X mol/(minuteml)(=Units/ml)(1)

(47) To calculate the specific activity in mol pNC consumed/(minutemg) (=Units/mg) divide equation 1 with the protein concentration of the sample:
Eq. 1/Protein conc. (mg/ml)=Y mol/(minutemg)=Units/mg(2)
BCA Analysis

(48) A commercially available assay kit (Pierce BCA Protein assay kit, no. 23225) is used according to the manufacturers instructions.

(49) rhASA ELISA for Determination of rhASA Concentrations

(50) The procedure is an enzyme-linked immunosorbent assay (ELISA) for quantitative determination of recombinant human Arylsulfatase (rhASA) in solutions, such as buffers, cell culture medium and serum.

(51) rhASA is captured on maxisorp 96-well plates coated with the IgG fraction of rabbit antiserum to affinity-purified rhASA. The captured rhASA is detected with a monoclonal antibody to rhASA, followed by horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulins. HRP will convert the substrate tetramethylbenzidine (TMB) to a blue product, which turns yellow upon acidification. The absorbance is measured at 450 nm and a standard curve from known rhASA concentrations is used to calculate rhASA concentrations of the samples.

(52) Equipment

(53) Spectrophotometer for plates, i.e. Spectramax Plus, Molecular Devices with SOFTmax PRO software for calculations

(54) Plate washer

(55) Plate shaker

(56) Pipettes; single and multi-channel

(57) Materials

(58) Maxisorp 96-well plates

(59) Sealing tape

(60) Reagents

(61) Coating buffer

(62) Tris-buffered saline (TBS): 10 mM Tris-HCl, 0.15 M NaCl, pH 7.4.

(63) Washing Buffer

(64) TBS (coating buffer) is supplemented with 0.1% tween-20.

(65) 1 ml tween-20 is added to 1 liter of TBS.

(66) Blocking Buffer

(67) SuperBlock blocking buffer in TBS (Pierce).

(68) Dilution Buffer

(69) 10 ml blocking buffer is added to 90 ml TBS (coating buffer).

(70) Polyclonal Immunoglobulins to rhASA

(71) Medium from rhASA-CHO cells is affinity-purified on a column with monoclonal antibody to rhASA (5.7) cross-linked to Protein A. Rabbits are immunized with affinity-purified rhASA (DAKO) and the antisera are verified to react to rhASA with western blotting. Antiserum from rabbit is purified on HiTrap protein G column(s).

(72) The IgG fraction is stored in 50% glycerol, 10 mM Na-Pi, 75 mM NaCl, pH 7.2 at 4 C. The protein concentration is 1.25 mg/ml, determined with BCA protein assay kit.

(73) rhASA Standard

(74) Purified rhASA, batch M0208, is used as a standard. The standard is purified from rhASA-CHO cell supernatant with three consecutive purification steps, DEAE sepharose, HIC octyl sepharose and Mustang Q.

(75) The stock is stored in 50% glycerol, 10 mM Tri-HCl, pH 7.5 at 4 C. The concentration, determined with BCA protein assay kit, is estimated to 100 g/ml.

(76) Monoclonal Antibody to rhASA

(77) Supernatant from a rhASA monoclonal antibody (mab) producing hybridoma (19-16-3 from Prof. Gieselmann, Bonn) is purified on a HiTrap protein A column.

(78) The mab is stored at 20 C. in 20 mM Na-Pi, 0.145 M NaCl, pH 7.2 (PBS) supplemented with 0.02% sodium azid. A working portion is kept at 4 C. for 6 months.

(79) HRP-Anti-Mouse Immunoglobulins

(80) Horseradish peroxidase-conjugated, affinity-isolated, goat anti-mouse immunoglobulins are purchased from DAKO (P 0447) and stored at 4 C.

(81) TMB Substrate.

(82) The One-Step Substrate system containing 3,3,5,5-tetramethylbenzidine (TMB) is purchased from DAKO (S 1600) and stored at 4 C.

(83) Stop Solution

(84) 1 M H.sub.2SO.sub.4

(85) Method

(86) Coating

(87) The stock of anti-rhASA polyclonal IgG is diluted 1:1000 in TBS to 1.25 g/ml and 100 l/well is added to a maxisorp the 96-well plate. The plate is incubated over night at room temperature and washed twice with 250 l washing buffer.

(88) Blocking

(89) 200 l of blocking buffer is added per well prior to incubation at room temperature under agitation for at least 15-60 minutes.

(90) Capturing of rhASA

(91) 100 l diluting buffer is added to all wells.

(92) rhASA Standard:

(93) The rhASA standard stock solution is diluted 2000 times in dilution buffer in triplicates 50 ng/ml. Triplicates of 100 l standard are transferred to the 96-well plate and serial two-fold dilutions are prepared.

(94) Samples:

(95) The samples are diluted in triplicates in dilution buffer to estimated rhASA concentration around 25 ng/ml. 100 l of each sample are transferred to the 96-well plate. 2-8 two-fold dilutions are prepared in the plate.

(96) The plate is incubated for 100-140 minutes at room temperature and under agitation and is subsequently washed four times with 250 l washing buffer.

(97) Detection with Monoclonal Antibody

(98) The monoclonal antibody (mab) to rhASA is diluted 1:2000 to 185 ng/ml in dilution buffer and 100 l is added to each well. The plate is incubated for 70-120 minutes at room temperature under agitation and is subsequently washed four times with 250 l washing buffer.

(99) Detection of Complexed Mab with Anti-Mouse IgG-HRP

(100) Anti-mouse IgG-HRP is diluted 1:2000 to 500 ng/ml in TBS (coating buffer) and 100 l is added to each well. The plates are incubated for at 70-120 minutes at room temperature and under agitation and subsequently washed four times as above.

(101) Colour Development

(102) TMB substrate (100 l) is added to each well and the plates are incubated for 15 minutes at room temperature without agitation. The reaction by adding 100 l/well 1 M H.sub.2SO.sub.4 (stop solution) and the absorbance is measured at 450 nm with endpoint reading in the plate spectrophotometer.

(103) Evaluation

(104) rhASA concentrations are calculated using the SOFTmax PRO software ( . . . ) according to the manufacturers instructions.

(105) The linear part of the standard curve is plotted using linear regression and the concentration of unknown samples is read from the standard curve.

(106) Reversed Phase HPLC for Analysis of rhASA

(107) The purity of Arylsulfatase A (rhASA) is determined by reversed phase HPLC, monitoring the UV absorption at 220 nm. The elution is obtained with an increasing concentration of organic modifier (acetonitrile) in the mobile phase. The retention times for rhASA and other components in the sample are dependent on their ability to adsorb and desorb to the non-polar stationary phase, which in turn depends on factors such as protein conformation, hydrophobicity and sequence.

(108) Materials and Equipment

(109) Hewlett Packard model 1090 HPLC system equipped with a tertiary pump system, auto injector, diode array detector, controlled by HP Chemstation version A.06.03. Equivalent HPLC systems may be used provided that the system suitability test verifies an adequate performance.

(110) Filter for sample concentration: Centriplus YM-30, Millipore corp.

(111) Analytical column: Zorbax 300SB-C18, 2.1*150 mm 5-micron, Rockland Technologies Scientific, Inc.

(112) Inline filter: Inline filter A-102X and inline filter cartridge 1*1 mm, Upchurch Scientific, Inc.

(113) Filter for sample preparation: Whatman Anatope 10 LC

(114) Chemicals and Reagents

(115) Milli-Q water, HPLC grade water or equivalent

(116) Acetonitrile, far UV, HPLC grade (VWR, LiChrosolve or equivalent)

(117) Trifluoroacetic acid (TFA), ampoules 101 g (Pierce)

(118) Tris base p.a. quality (Angus or equivalent)

(119) Guanidinium chloride p.a. quality (VWR biochemistry grade or equivalent)

(120) Mobile phase A: Dissolve 1 ampoule TFA (1 g) in 1 liter of Milli-Q water

(121) Mobile phase B: Dissolve I ampoule TFA (1 g) in 1 liter of acetonitrile

(122) Sample diluent: 20 mM Tris-HCl, pH 7.5

(123) Column cleaning solution 1: 50% Isopropanol p.a. quality in water

(124) Column cleaning solution 2: 6 M Guanidinium choloride

(125) rhASA standard (purified on a mabASA/protein A Sepharose column. Reported in experimental summary Exp. No: M-6). If affinity purified rhASA not is available, samples of lower purity from the rhASA purification scheme may be used as a standard.

(126) All other chemicals and reagents were of p.a. quality if not otherwise stated and purchased through common commercial sources.

(127) Method

(128) Instrumental conditions

(129) Mobile phase composition: A: Water, 0.1% TFA B: Acetonitrile (AcN), 0.1% TFA

(130) Flow rate 0.2 ml/min

(131) Temperature: +40 C.

(132) Sample Injection Volume:

(133) Crude extract 20 l (if concentrated to 0.3 mg/ml)

(134) In-process samples: 20 l (if concentrated to 0.3 mg/ml)

(135) Affinity purified samples: 5 l (if 1.0 mg/ml)

(136) Gradient:

(137) TABLE-US-00001 Time (min) % A % B 1.00 70 30 10.00 40 60 15.00 5 95 20.00 5 95 25.00 70 30 30.00 (post time) 70 30

(138) Column wash (performed every 5.sup.th injection):

(139) Injection of 25 l of 50% isopropanol (p.a. grade) as a sample and a run of the gradient stated above in order to clean the column.

(140) Sample and standard preparation

(141) rhASA samples with a protein concentration less than 100 g/ml are concentrated in a Centriplus centrifugal filter device (model YM-30, Millipore Corp.).

(142) The obtained retentate is adjusted to a protein concentration of 1.0-0.3 mg/ml with 20 mM Tris-HCl pH 7.5 and filtrated through a 0.22 m filter in order to remove any particles and precipitated proteins. In case of small sample volumes the filtration can be replaced by centrifugation at 10.000 g for 10 minutes.

(143) Chromatography

(144) The samples are loaded and run on the chromatograph while the temperature is kept low (+8 C.) if possible.

(145) Integration and Calculation of Purity

(146) Area under the curve measured at 220 nm for the rhASA peak is calculated and related to total integrated area. Purity is reported as percentage rhASA of total protein. Use the integration parameters in the appendix (designed for Hewlett Packard/Agilent Chemstation 06.03 software) as base for integration. Since integration of the rhASA main peak is not always optimal with the preset integration parameters, manual integration might be necessary. Different HPLC software might also require different integration parameters, which has to be tested individually for each system.

(147) Evaluation

(148) Identity: The retention of the main peak of the sample should be within 0.5 minutes as compared to the rhASA standard.

(149) Purity: The purity of the sample is determined by comparing the integrated area of the main peak compared to total integrated area. Purity is reported as % main peak (rhASA).

(150) Raw data

(151) Raw data files are stored on a server or CD-ROM discs.

(152) Appendix

(153) Integration Parameters

(154) Integration parameters are highly instrument and system dependent and have to be evaluated for different systems used. The integration parameters below are optimized for Agilent/Hewlett Packard ChemStation HPLC software version 06.03.

(155) TABLE-US-00002 Event Value Time Slope sensitivity 10.0 initial Peak width 0.2 initial Area reject 5.0 initial Height reject 1.0 initial Detect shoulders drop initial Integration OFF 0.000 Integration ON 5.000
Outline of Continuous Cell Propagation

(156) The continuous mammalian cell propagation has been developed in B. Braun 5 L bioreactors equipped with Bio-Sep cell retention devices from AppliSens. The principle of the process presented schematically in FIG. 2. The mammalian cell are cells capable of amplification and production of foreign proteins as a suspension culture in bioreactors or large-scale fermenters.

(157) During the process development the cell line is maintained and propagated in Excell 302 medium (catalog number 81045 from JRH Biosciences). This is a serum-free medium, which is devoid of proteins of animal or human origin. Furthermore, the medium, which does not contain phenol red, has been supplemented with insulin-like growth factor-1 (IGF-1) and with glucose. The glucose concentration is monitored and adjusted to optimal levels during the process.

(158) The recombinant human ASA produced by the continuous culture process in B. Braun 5 L bioreactor is presently expressed in CHO DG44 cells. The amplification of the CHO cells after thawing is initiated in T-flasks and the cells are later transferred to spinner flasks. Before splitting and inoculation of the bioreactor culture, the spinnner culture has a cell density of 1.3 10.sup.6 cells/ml with a viability of 96%.

(159) In preparation for the culture process the cells are transferred from the spinner flasks to the bioreactors. Data on cell densities in the bioreactor before and after inoculation can be deducted from table 1 below. Also typical initial values for viability, glucose, agitation, pH, pO.sub.2 and temperature are reported.

(160) When propagated and maintained as described above the cells do not clump, and propagate and produce as suspension cultures.

(161) Part of the propagation, maintenance and production from the CHO DG44 culture is the harvest of 1-4 reactor volumes of media per day. To compensate for the harvest, the culture is supplemented with the same amount of fresh medium per day.

(162) The continuous culture process can be maintained over a period of more than 500 hours, and a production phase of 2 weeks or more is preferable. In order to increase the yield it is desirable to lower the temperature from 37 C. to 32-35 C. once the plateau of the production phase is reached. Cell density values at and above the 1.210.sup.7 cells/ml are obtained and productivities above 3.0 pg/cell/day resulting in >20 mg rhASA/L is demonstrated in this system. During the process the parameters; glucose, lactate, glutamine, ammonium and osmolarity are measured and controlled.

(163) TABLE-US-00003 TABLE 1 Main parameters for the cell culture system._Cell Retention Efficiency (CRE) is a measure, reported as a percentage, of the efficacy with which the cell retention device separates the cells from the medium and bring back the cells to the culture vessel. Bleeding is a deliberate harvest of cell containing medium. Proportional Integral Differential (PID) parameter is relevant when controlling the way a process reaches and maintains defined set-points. Steady production-state is a set of process parameters, chosen because the are believed to support an optimal production. The aim is to maintain the process at these parameters for a longer period, the steady production-state, and harvest product during this period. Cell culture is performed in medium without serum and with the addition of less than 1 mg/L recombinant human proteins with a molecular weight of less than 10 kDa. Parameters Values Volume 1000 ml, 5000 ml, 15 L 100 L, 400 L, 700 L Agitation 100-165 rpm Temperature 37 (reduce to 32 C.-35 C.) Re-circulation rate 3 to 4 times the perfusion rate Separation parameters CRE above 95% Bleeding 0-10% of bioreactor volume per day Glucose 2-4 g/l Lactate 0.13-5.0 g/l Oxygenation Pure oxygen sparging + PID parameter adjustments pO.sub.2 30-40% Perfusion rate Up to 4 vol/day Cell viability at inoculation >93% Cell viability in production >90% phase Cell density at inoculation 3.6 10.sup.5 cell/ml Cell density 10-14 10.sup.6 cells/ml during production Specific ASA production 1.5-3.0 pg/cell/day Protein output per bioreactor >170 mg/day (5 L reactor volume) per day >1 g/day (100 L reactor volume) pH 6.8-7.3 Steady production state To be defined
Outline of Purification Process
Clarification and Virus Reduction

(164) 20 L of medium (ASA activity in the range of 0.3-1.5 U/ml) is clarified through a sequence of depth filters from Millipore (Polygard D5 5 m+Opticap FF and Opticap 0.45 m). For virus reduction Tween 80 is added to a final concentration of 1% and left at least 30 min (over night also possible) at +4 C.

(165) Application to production in 15 L Bioreactor: 300 L (1-2 U/ml) harvest/cultivation (36 days). How many filters are needed? Suggestion: Clarify every third day during perfusion, 45 L/filtration. Application to production in 100 L Bioreactor: 2000 L harvest/cultivation. 300 L/filtration.

(166) Concentration/Diafiltration with Tangential Flow Filtration (TFF)

(167) The filtrate is concentrated 10-20 times in volume using TFF at transmembrane pressure (TMP) 15 psi on a Sartoflow system with a Sartorius frame (Sartorius). A Millipore Biomax 30 kDa screen type A with 0.1 m.sup.2 area is used. After concentration diafiltration is performed against 20 mM Tris-HCl pH 7.5 or against 10 mM sodium phosphate buffer (standard buffer), pH 7.5, approximately 2 volumes until the conductivity is approx. 4 mS/cm. The medium was finally filtered through a Opticap 0.45 m filter

(168) Example: To 20 L filtrate (clarified harvest) a 0.1 m.sup.2 membrane is used. The expected yield is 90-100%.

(169) Application to production in 15 L Bioreactor: Concentrate totally 300 L filtrate to 15 L (20-40 U/ml) and change 2 volumes of buffer. Suggestion: TFF every 6.sup.th day during perfusion. Concentrate 90 L to 4.5 L, 3-4 times per cultivation.

(170) Application to production in 100 L Bioreactor: 2000 L to 100 L and change 2 volumes of buffer. Concentrate 300 L to 15 L, 3-4 times/cultivation.

(171) Step 1: Capture StepDEAE Sepharose FF (Amhersam Biotech)

(172) Sample from step 1 (corresponding to 50.000 U of total activity) is applied on a 800 ml DEAE sepharose packed in a 70 mm diameter column (Pharmacia Index 70/500) equilibrated with standard buffer. Flow rate is 80-120 cm/hr. Protein bound to the DEAE gel is then washed with 2-3 column volumes (CV) of standard buffer followed by 2-3 CV's of 0.1 M NaCl in standard buffer.

(173) rhASA is eluted with 3-4 CV's of 0.3 M NaCl in standard buffer. Fractions containing rhASA activity are pooled and used for further purification. Normal yield is 90% and purity approximately 30-40%.

(174) For large scale production the capture step is preferably performed using Expanded Bed Adsorption technologySTREAMLINE DEAE

(175) A STREAMLINE DEAE is equilibrated in a Direct STREAMLINE column with sodium phosphate buffer pH 7.1+200 mM mannitol (final concentration). The resin expands to 3 times the sedimented bed volume (SBV). The arylsulfatase A containing sample is mixed, preferably online, with 300 mM mannitol, 1:1, and applied on the column. Alternatively, the sample is stirred with a top spinner continuously after mixing. Conductivity is 7 mS/cm. The resin is washed with 2 SBV of equilibration buffer followed by 8 SBV of sodium phosphate buffer pH 7.1+0.06 M NaCl. and the rhASA is eluted with 8 SBV sodium phosphate buffer pH 7.1+0.35 M NaCl and 4-6 SBV are collected.

(176) Flow is upward and 300 cm/hr.

(177) Estimated yield is 95% and estimated purity is 30-40%.

(178) Capacity is 80 U ASA (1 mg)/ml adsorbent.

(179) CIP immediately.

(180) Application to Production in 5 L and 15 L Bioreactor:

(181) 1.4 L STREAMLINE Direct 95/1.0 column=20 cm sedimented bedheight (60 cm expanded). Harvest is loaded twice/week. For a 15 L Bioreactor the load corresponds to 135 and 180 L after dilution. 5.5-8 L rhASA pool is eluted at each run.

(182) Capacity limit of column: 80 U/ml adsorbent. Maximum rhASA load on 1.4 L adsorbent=112 000 Units (1.4 g rhASA), which corresponds to maximum 1.2 U/ml harvest (15 mg/L if specific activity is 80 U/mg) in harvest from a 4 days-pool and max 1.7 U/ml (21 mg/L) from a 3 days-pool.

(183) Application to Production in 100 L Bioreactor:

(184) 12.3 L STREAMLINE Direct 280 column=20 cm sedimented bedheight (60 cm expanded).

(185) Harvest is loaded twice/week, corresponding to 900 and 1200 L load after dilution. 50-70 L rhASA pool is eluted at each run.

(186) Capacity: 984 000 U corresponds to 1.6 U/ml (20 mg/ml) in harvest from a 4-days pool and 2.2 U/ml (27 mg/ml) in a 3 days-pool.

(187) When using a 30-50 cm bedheight and 15.4-30.8 L adsorbent capacity is 1.2-2.5 10.sup.6 U, corresponding to 2.0-4.1 U/ml (4-days pool) and 2.7-5.5 U/ml (3-days-pool).

(188) Replacing the conventional anion chromatography (DEAE sepharose FF) with Expanded Bed Adsorption technology is favoured for large scale production since it renders prior concentration/Diafiltration with Tangential Flow Filtration (TFF) (step 1) redundant.

(189) Step 2: Intermediate Step 1Butyl Sepharose FF (Amhersam Biotech)

(190) Sample pool from step 2 is mixed 1:1 with 1.0 M Na.sub.2SO.sub.4 in standard buffer and applied on a 800 ml octyl sepharose FF packed in a 70 mm diameter column (Pharmacia Index 70/50) equilibrated with standard buffer+0.5 M Na.sub.2SO.sub.4. Flow rate is 60-120 cm/hr. Column is washed with 1-2 CV of equilibration buffer followed by 1-2 CV's of 1.8 M Na-Acetate in standard buffer pH 7.5. rhASA is eluted with 1.5-3 CV's of 0.9 M Na-Acetate in standard buffer pH 7.5 and fractions containing activity are pooled and used for further purification. Normal yield is 90% and purity 70-87%.

(191) As an example, the sample from step 1 corresponding to maximum tested 53 000 U of arylsulfatase activity is applied on a 600 ml Butyl Sepharose 4FF column (packed in a Pharmacia Index 70/50 column). The capacity is 100-300 U/ml gel.

(192) Application to Production in 15 L Bioreactor:

(193) Volume of HIC column is from 1.1-3.5 L. Three eluates from step 1 are mixed with 33-50 L 1 M Na.sub.2SO.sub.4, and loaded twice per Bioreactor cultivation. 11 L (or 3.5 L) rhASA pool is eluted/run. On condition that the eluted rhASA can be stored without risk for bacteria contamination, the two runs on HIC could be exchanged to a single run on a larger column followed by a single steps

(194) Application to Production in 100 L Bioreactor:

(195) 25 (or 8) L column.

(196) Step 3: Concentration and Diafiltration with TFF

(197) Sample pool from step 3 is concentrated to approximately 1 mg/ml with TFF against a Biomax A-screen, 30 kDa. Diafiltration is performed against 3-5 volumes of 20 mM Na-Acetate, pH 5.4-5.7. Normal yield is 90-100% and purity the same as the previous step. Alternatively, the mixture is concentrated to 4 mg (total protein)/ml and the buffer is changed to 2 mM sodium phosphate, pH 7.5 by 6 volumes of diafiltration. Diafiltration is performed at transmembrane pressure (TMP) 15 psi with Biomax 30 kDa, screen A, polyethersulfone membrane (Millipore). Yield is 90-100% and purity is the same as step 4.

(198) Application to Production in 15 L Bioreactor

(199) Concentrate 11 L to 2 L and change buffer with 6 volumes of 2 mM Na-Pi, pH 7.5. Twice/cultivation.

(200) Application to Production in 100 L Bioreactor:

(201) Concentrate 80 L to 16 L.

(202) Optionally, concentration and diafiltration is preceeded by virus-inactivation by Tween-80: The eluate from step 2 is mixed with Tween-80 (C.sub.18H.sub.124O.sub.26) to a final concentration of 1% and left for at least 1 hour.

(203) Step 4: Polishing Step

(204) Mustang-S Membrane or Blue Sepharose (Passive Step)+Anion Exchanger or Membrane (Active Step)

(205) Brief Description:

(206) A Mustang-S membrane or Blue Sepharose is coupled in series with a high resolving anion exchanger (e.g. Source-Q from Amhersam Biotech or Mustang Q membrane). The columns are equilibrated with >10 CV's of 20-100 mM Sodium Acetate pH 5.4-6.0. Sample pool from step 4 is loaded on the columns after adjustment of the pH by mixing 1:1 with 0.1 M NaAc, pH 5.6 (rhASA will pass through the Mustang-S membrane/Blue Sepharose and be captured on the high resolving anion exchanger). The Mustang-S membrane/Blue Sepharose is uncoupled and the high resolving anion exchanger is washed with 2-10 CV's of 20-75 mM Sodium Acetate pH 4.8.

(207) The anion exchanger is re-equilibrated with >10 CV's of 20 mM Tris-HCl pH 7.5 (standard buffer) or, alternatively with 10 column volumes of 10 mM Na-Pi buffer pH 7.5. The column is washed with 0.1 M NaCl in standard buffer or, alternatively with 0.06 M NaCl in 10 mM Na-Pi, pH 7.5 and rhASA is eluted with a linear gradient of 0.1-0.3 M NaCl in standard buffer or, alternatively with a gradient of from 60-500 mM NaCl in Na-Pi, pH 7.5. The active rhASA fractions are collected.

(208) Flow rate is 100-120 cm/hr., estimated yield is 90% and purity 98-100%. Capacity >40 mg/ml for Blue Sepharose and 30 mg/ml for Source 30Q.

(209) Application to Production in 15 L Bioreactor:

(210) 200 ml Blue Sepharose and a 300 ml Source 30Q column run twice/cultivation. Load the pool from 4 after lowering pH by dilution 1:1 with 0.1 M NaAc pH 5.6=4 L.

(211) Application to Production in 100 L Bioreactor:

(212) 1.3 L Blue Sepharose and 2 L Source 30Q, twice/cultivation.

(213) Step 5: Virus Filtration Step

(214) Virus filtration will be performed on the product pool from step 5 using a 0.1 micron sterile filter followed by a DV 20 nano filter from Pall with an applied constant pressure of 20-50 psi. Estimated flow through in process scale is 25 L/hr.

(215) As an alternative, 1% of Tween 20 or 80 could be applied to the supernatant (contact time 30-60 minutes) before the first concentration and diafiltration step (step 1).

(216) Step 6: Diafiltration/Formulation Step

(217) Tangential flow filtration (TFF) against a Millipore Biomax 30 kDa screen type A against 5-10 volumes of formulation buffer is performed. The most likely formulation buffers are presented below

(218) Formulation Buffer 1.

(219) TABLE-US-00004 Na.sub.2HPO.sub.4 3.50-3.90 mM NaH.sub.2PO.sub.4 0-0.5 mM Glycine 25-30 mM Mannitol 230-270 mM Water for injection (WFI)
Formulation Buffer 2.

(220) TABLE-US-00005 Tris-HCl 10 mM Glycine 25-30 mM Mannitol 230-270 mM Water for injection (WFI)
Formulation Buffer 3.

(221) TABLE-US-00006 Na.sub.2HPO.sub.4 3.50-3.90 mM NaH.sub.2PO.sub.4 0-0.5 mM Glycine 25-30 mM Mannitol 230-270 mM Water for injection (WFI)

(222) The pH and osmolality in both formulation buffers will be balanced to 7.50.2 and 30050 mOsm/kg respectively. Final protein concentration should be according to the specification (>5 mg/ml).

(223) Step 7: Formulation, Filling

(224) Formulation and Dosage Form

(225) In the development of the dosage form, the stability of rhASA is an important factor to consider. At present, all stability data points towards an aqueous stabile solution. Freeze-dried powder is currently our back-up strategy.

(226) The options at present are the two different formulation buffers described in step 7: Formulation buffer 1 and 2.

(227) Both these formulations are known to stabilize proteins in aqueous solutions as well as in freeze-dried powders. The pH and osmolality in both Formulation buffers will be balanced to 7.50.2 and 30050 mOsm/kg respectively. Final protein concentration should be according to the specification and in the range 5-20 mg/ml.

(228) The filling of rhASA will be performed in a production unit according to EU GMP practice and in a room classified as Class A. During production the filling zone is monitored with particle count and settle plates. The personnel are regularly trained according to EU GMP and monitored after each production with glove prints. The sterility of equipment and materials are secured by validated sterilization procedures.

(229) Conclusion

(230) The described purification process consists of 7 steps and two sub-batches is produced per Bioreactor cultivation. The overall yield is 60-70%. The purity is at least 95%. The Host cell proteins content should be <200 ng/mg with a target value<100 ng/ml. To reduce HCP's further it might be necessary to reduce the yield for either the intermediate or the polishing step.

(231) TABLE-US-00007 TABLE 2 Flow chart of the purification process. Standing time at +5 C. of product Step In process analysis from the step Harvest 42 ~4 days 1. Capture: EBA 42 30 days 2. Intermediate: Butyl 42, CMC-A280 nm At least over Sepharose night 3. TFF 42, CMC-A280, 38 At least 15 days 4. Polish: Blue + 42, CMC-A280, 38 30 days Source Q 5. Virus filtration 42, CMC- A280 30 days 6. TFF, bulk drug 42, 34 or/and CMC-A280, Stable substance 38, LAL, bacterial count, pH, osmolality, CMC-HCP ELISA 7. Filling To be decided: 42, 34 Stable or/and CMC-A280, 38, LAL, bacterial count osmolality, HCP ELISA CMC

(232) TABLE-US-00008 TABLE 3 Analytical methods. Dora No Methods for analysis 34 Protein determination of rhASA by BCA Protein assay Kit Microtiter Plate Protocol or OD CMC-A280 Protein determination OD at A280 nm 35 SDS-PAGE analysis of recombinant human Arylsulfatase A (rhASA) 38 Reversed Phase HPLC analysis of recombinant Human Arylsulfatase A (rhASA) 9213 Carbohydrate composition quantification of glycoproteins by reversed phase HPLC with fluorescence detection 40 Western blotting from SDS-PAGE for analysis of CHO host cell proteins 43 ELISA method for determination of CHO host cell proteins CMC-HCP ELISA method for determination of CHO host ELISA cell proteins/CMC 42 Enzyme assay for analyzing activity of Arylsulfatase A, ASA. Microtiter Plate Protocol 28 ELISA method for determination of recombinant human Arylsulfatase A (rhASA) concentrations

(233) TABLE-US-00009 TABLE 4 Analysis performed at Zymenex Dora No 1.1.1.1.1 When 9213 After step 6 or later, occasionally 43 After step 6 or later, occasionally 40 After step 6 or later occasionally 35, 28 After any step, occasionally
Clean in Place (CIP) Procedures:

(234) Step 1: STREAMLINE DEAE: Upward flow, 100 cm/hr immediately after each run. 1 M NaCl 8-10 SBV, 1 M NaOH 5 SBV to waste, then recirculation >6 hrs, 1120, citric acid/HAc if needed. Store 20% EtOH.

(235) Step 2 Butyl Sepharose: Upward flow, 30 cm/hr. After each run CIP at reversed flow with 1-2 CV H.sub.2O, 1-2 CV 1 M NaOH (40 min contact time), 1-2 CV H.sub.2O and 1-2 CV 20% EtOH. Store in 20% EtOH.

(236) Steps 3 and 6 TFF membrane, Biomax 30 kDa: Wash with distilled water followed by 0.5 M NaOH and then 0.1 M NaOH. Store in 0.1 M NaOH.

(237) Step 4 Blue Sepharose: After each run CIP at reversed flow with 2 CV 1 M NaCl, 2 CV H.sub.2O, 1-2 CV 0.1 M NaOH (40 min contact time), 1-2 CV H.sub.2O and 1-2 CV 20% EtOH. Store in 20% EtOH.

(238) Source Q: Upward flow After each run CIP at reversed flow with 2 CV 2 M NaCl, 2 CV H.sub.2O, 1-2 CV 1 M NaOH (40 min contact time), 1-2 CV H.sub.2O and 1-2 CV 20% EtOH, flow rate 30 cm/hr. Store in 20% EtOH.

(239) Results

(240) Data for preparations of rhASA obtained through a purification procedure as outlined above are presented in tables 5 and 6. In brief, the results show that the overall yield of the purification process correspond to 79% of the rhASA present in the starting material. The purity of rhASA in the resulting preparation corresponds to 98.0% as determined by reverse phase HPLC. Results are shown in FIG. 3. Specific conditions for the procedure for which data are shown are as follows:

(241) Step 1-3: As Described Above.

(242) Step 4: A 10 ml Mustang-S membrane is coupled in series with a high resolving anion exchanger (Resource-Q from Amhersam Biotech, 6 ml). The columns are equilibrated with >10 CV's of 20 mM Sodium Acetate pH 5.5. rhASA from Tox03HC20 is buffer exchanged to the equilibration buffer and loaded on the columns. After passing the Mustang-S membrane, rhASA will be captured on the Resource-Q column. The Mustang-S membrane is uncoupled and the Resource-Q column is washed with 3 CV's of 75 mM Sodium Acetate pH 4.8.

(243) The Resource-Q column is washed with >10 CV's of 20 mM Tris-HCl pH 7.5 (standard buffer) until the correct pH is reached. The column is washed with 0.1 M NaCl in standard buffer and rhASA is eluted with a linear gradient of 0.1-0.3 M NaCl in standard buffer. Fractions containing active rhASA are collected.

(244) TABLE-US-00010 TABLE 5 Purification scheme Tox03HC20, which have been used for evaluation of the polishing step. Enzyme activity in the scheme may vary due to changes of the method during development Total Purity Volume Activity Yiel(% (% by rp- Step (ml) (U) activity) HPLC) TFF 7990 54358 n.d. n.d. Capture: 2250 61537 n.d n.d. DEAE (high?) Intermediate: 720 42768 n.d n.d Butyl TFF 655 49125 90% 92% (based on (slightly on average the high activity 75 side) U/ml)* n.d. = not determined

(245) TABLE-US-00011 TABLE 6 Result from polishing test development in small scale using Tox03HC20 as start material. Test Mustang-S (passive) + Resource-Q (active) as polishing step. Purity Total Total Specific Yield (% by Volume Activity protein activity (% rp- Step (ml) (U) (mg) (U/mg) activity) HPLC) Start: 3.2 293 11 26.6 100. 91.2% Tox03HC20 Polishing 19.0 249 8.7 28.5 85% 98.0% pool (purifi- (based on cation activity) factor 79% of 1.07) (based on protein)
Product Specification

(246) Specification Bulk substance for i.v. Toxicology testing of recombinant human Arylsulfatase A (rhASA). The analytical tests are performed before sterile filtration and filling in vials at the end of the purification process.

(247) Description:

(248) Recombinant human Arylsulfatase A (rhASA) in solution for i.v. administration

(249) Shelf life is 6 month from production if stored at 20 C. In-use time is 1 week from thawing if stored at +5 C.

(250) TABLE-US-00012 TEST METHOD NO. LIMIT Content rhASA specific activity Enzyme activity 50 U/mg (Units/mg) of rhASA Actual: 60-120 U/mg rhASA protein concentration 2500-P- 5 mg/ml (BCA) (mg/ml) 1034 Identity Retention time main peak on 2500-P- Approved HPLC 1038 (relative to standard) Purity HPLC (% main peak) 2500-P- >95% 1038 Actual: >97% Host cell proteins (HCP) 2500-P- <200 ng/mg (ng/mg protein) 1041 Actual: 50-100 ng/mg Other Tests Bacterial count, membrane Ph. Eur. 10 cfu/ml filtration (cfu/ml) LAL (IU/mg) (max 2.5 mg Ph. Eur. 2 IU/mg rhASA rhASA/kg) Osmolality (mOs/kg) Ph. Eur. 250-350 pH Ph. Eur. 7.2-8.2

Example 2

Test of rhASA for Binding to Cation Exchange Resin and Anion Exchange Resin

(251) Experimental Description:

(252) rhASA (Tox03HC20) 5 mg/ml was mixed 1:10 with buffers at pH 4.8-7.2 Kation exchanger (Unosphere-S, BioRad)+Anionexchanger (DEAE FF, Amhersam Biotech) was portioned in test tubes and equilibrated with 20 mM Na-Acetate pH 4.8, 5.2, 5.6 and 6.05 or 20 mM Tris-HCl pH 7.2. (approx. 100 ul IEX media/tube). 170 ul rhASA 1:10 in resp. buffer was added to the IEX media with the same pH + to empty reference tubes. Mix several times and let sit for approx. 30 minutes. Spin down and measure activity in Supernatant.

(253) Conclusion:

(254) rhASA binds as expected to the cation exchanger, but not to the anionexchanger. Even at pH 4.8 rhASA binds strongly and unexpected to the resin. This binding may be explained by strong polarity or alternatively by a change from dimer to octamer below pH 5.8, which induces changes in exposed charged groups. Results are shown in FIG. 2.

Example 3

Degradation of Natural Sulfatides in Fibroblasts by rhASA

(255) Dose/Response Experiment:

(256) Experimental Design

(257) Fibroblasts from a MLD patient with null-mutation (GM00243, purchased from Coriell Cell Repository, USA) are grown almost to confluency in 25 cm.sup.2 flasks with medium containing heat-inactivated fetal calf serum (FCS). Cells are loaded with the natural substrate, .sup.14C-palmitoyl sulfatide (15 M). Following incubation for 40 h the medium is changed to rhASA containing medium (0, 25, 50 and 100 mU/ml affinity-purified rhASA, respectively). After 24 h the cells are harvested and lipid extracts are prepared from the cells by a chloroform-methanol extraction. The lipid fractions are analysed by TLC-chromatography by comparing to radioactively labelled references. The TLC plate is exposed to X-ray film and the different lipid fractions from the TLC plate are quantified using liquid scintillation counting. The data is expressed as percent of radioactivity of remaining and metabolised sulfatides.

(258) Results

(259) The data from this experiment (Table 3 and FIG. 4) shows that all dose levels of rhASA used (0.25, 2.5, 25, 50 and 100 mU/ml) metabolize approximately 40-70% of the .sup.14C labelled sulfatide loaded into the MLD fibroblasts. The background degradation of the substrate is approximately 15% in cells not incubated with rhASA. This background may be explained by a low residual activity of sulfatases in the MLD cells, or some sulfatase activity from the heat-inactivated serum, even though no ASA activity can be detected in the cells or the FCS. This can also explain the low sulfatide metabolism in the control cells in which no rhASA is added.

(260) TABLE-US-00013 TABLE 7 Degradation of radiolabelled sulfatide in MLD fibroblasts with or without the addition of recombinant human arylsulfatase A (rhASA). The results are given as percent of recovered radioactivity in the cellular lipid fraction. Experiment A B Mean C D Mean Added arylsulfatase A 0 0 25 25 (mU/ml) Metabolised sulfatide 17.3 15.8 16.6 68.7 67.6 68.2 (%) Remaining sulfatide 82.7 84.2 83.4 31.3 32.4 31.8 (%) E F Mean G H Mean 50 50 100 100 69.0 68.7 68.9 68.5 69.1 68.8 31.0 31.3 31.1 31.5 30.9 31.2
Time-Course Experiment:
Experimental Design

(261) Cells are loaded with .sup.14C-palmitoyl sulfatide (15 M) as described above. The medium is changed to medium containing 25 mU/ml affinity-purified rhASA and harvested at 6, 24 and 48 hours. Lipid extracts are prepared and analysed as a described above. The data is expressed as percent of radioactivity of remaining and metabolised sulfatides.

(262) Results

(263) The data from this experiment illustrate that the metabolism of the .sup.14C labelled sulfatide loaded into the MLD fibroblasts increases over 48 hours after addition of affinity-purified rhASA. Data are shown in FIG. 5.

(264) Conclusion:

(265) From these data it can be concluded that rhASA is efficiently taken up by fibroblasts from a MLD patient and that sulfatides loaded into these fibroblasts can be efficiently metabolised by the exogenous rhASA even at low doses and after incubation for a few hours.

Example 4

Characterisation and Use of CHO-rhASA Produced and Purified in Large Scale

(266) Characterization of the CHO-rhASA

(267) Human ASA was purified from secretions of Chinese hamster ovary (CHO) cells overexpressing the human ASA from the expression plasmid pASAExp1 (Zymenex A/S, Hillerd, Denmarkformer HemeBiotech A/S). The specific activity of the enzyme preparation was above 60 U/mg. The CHO-rhASA was rebuffered in 1TBS pH 7.4 to a concentration of 2.5-4.3 mg/ml and analysed by SDS-PAGE and MALDI-TOF spectroscopy. MALDI mass spectra were collected using a Voyager-DE STR BioSpectrometry workstation (Perspective Biosystems, Inc., Framingham, USA) equipped with a 337 nm nitrogen laser. Measurements were taken manually in linear, positive ion mode at a 20-24 kV acceleration voltage, 90% grid voltage and 200 ns delayed ion extraction. Each mass spectrum obtained was the sum of 300 unselected laser profiles on one sample preparation. Sinapinic acid was used as matrix. For partial or complete deglycosylation of CHO-rhASA 1 g enzyme was reacted with 1 or 500 mU PNGase F (Roche Diagnostics, Mannheim, Germany) for 20 h at 37 C. The endocytosis assay was done with 1 g CHO-rhASA per ml medium for 20 hr as described (Matzner, U. et al. Gene Ther., 7, 805-812). ASA was measured by an indirect sandwich ELISA and an activity assay (Matzner U, et al. (2000) Gene Ther. 7(14):1250-7, Baum, H. et al. (1959). Clin. Chim. Acta., 4, 453-455).

(268) Results

(269) SDS-PAGE and MALDI-TOF analysis of CHO-rhASA preparations detected a compound of correct size and the absence of contaminants (FIGS. 7A and 8A). Wildtype human ASA has three N-linked carbohydrates with a combined molecular mass of approximately 5-6 kDa (Gieselmann, V. et al. (1992), J. Biol. Chem., 267, 13262-13266). Treatment with PNGase F reduced the apparent molecular mass of CHO-rhASA indicating glycosylation of the recombinant enzyme (FIG. 7A). After reacting the CHO-rhASA with low concentrations of PNGase F MALDI-TOF analysis revealed four deglycosylation intermediates (FIG. 8B), possibly representing the polypeptide linked to three, two, one and no N-linked glycan(s). The mass difference between the products with the highest and the lowest mass is in the range of 5 kDa suggesting full glycosylation of the enzyme. To evaluate the mannose phosphorylation of CHO-rhASA, the mannose 6-phosphate (M6P)-dependent endocytosis of the enzyme was evaluated by an in vitro feeding assay. The assay revealed efficient endocytosis of CHO-rhASA by BHK cells (FIG. 7B). Furthermore, uptake could be completely blocked by M6P, but not by glucose 6-phosphate. It can be concluded that CHO-rhASA bears M6P residues.

(270) Discussion

(271) Cell culture experiments revealed that CHO-rhASA bears M6P residues and uses the M6P receptor-dependent pathway for cell entry (FIG. 7B). The BHK cells which were utilized in this in vitro assay do not express other receptors for lysosomal enzymes. It is therefore possible that other receptors such as the mannose receptor or the asialoglycoprotein receptor compete for the binding and endocytosis of substituted CHO-rhASA under in vivo conditions.

Example 6

Administration of Recombinant Human rhASA to Arylsulfatase a Deficient Mice

(272) Materials and Methods

(273) Human recombinant arylsulfatse A was produced as described in example 1. The batches of rhASA used for animal studies included G0301 (concentration was 4 mg/ml and the enzyme activity was 166 U/ml) and G0302 (concentration was 4.3 mg/ml and the enzyme activity was 242 U/ml). The rhASA was stored at 20 C. Before start of experiment the enzyme batches were thawed and pooled and the protein content and enzyme activity was analysed. The rhASA in this pool is diluted with TBS so the injection volume was 250-300 l in all animal groups. The dilutions were made immediately before injection. The body weight and the dose volume were noted for each animal.

(274) Treatment of the Mice

(275) ASA knockout mice and wildtype controls with the mixed genetic background C57BI/6J129ola (Hess B, et al. (1996) Proc Natl Acad Sci USA. 93(25):14821-6) were kept under standard housing conditions in accordance with the current German law on the protection of animals. All experiments were approved by the local committee for animal welfare (Bezirksregierung Kln, reference number 50.203.2-BN 24, 18/04). Experiments were done with 8-12 mo old animals. Depending on the animal weight and the concentration of the CHO-rhASA stock, 200-300 l enzyme solution (CHO-rhASA in 1TBS pH 7.4) was administered by an intravenous bolus injection into the tail vein. Control animals were injected with 250 l 1TBS pH 7.4.

(276) Analysis of Mice

(277) During the treatment period blood was taken from the tail vein. For the final analysis m ice were deeply anaesthetized using an intraperitoneal injection of tribromoethanol and transcardially perfused. For histological investigations mice were first perfused with PBS and then with 6% glutaraldehyde in 100 mM phosphate buffer pH 7.4. Tissues were then dissected and processed as described below. For biochemical analyses, mice were perfused with PBS alone. Kidneys, liver, brain, brachial plexus and sciatic nerves were dissected, weighed and frozen. Tissue samples were homogenized in 1TBS pH 7.4. Aliquots of the homogenates were used for lipid extraction (see below), protein determination (BioRad Dc assay, BioRad, Hercules, USA) and measurements of ASA by ELISA (9).

(278) Lipid Analysis

(279) Aliquots of tissue homogenates (see above) were centrifuged at 100,000g for 1 h and the pellet was first extracted with 5 ml chloroform/methanol (C/M) 2:1 (v/v) and then with 5 ml C/M 1:1 at 60 C. for 4 h in each case. Following evaporation of the solvent the dry lipids were redissolved in 5 ml MeOH. Alkaline methanolysis was started with 125 l 4 N NaOH at 37 C. and stopped after 2 h with 20 l 100% acetic acid. Lipids were dried and dissolved in 1 ml MeOH. For desalting by reverse phase chromatography Lichroprep RP-18 columns (Merck, Darmstadt, Germany) with a bed volume of 1 ml were equilibrated with C/M/0.1M KCl 6:96:94. After adding 1 volume of 0.3 M ammonium acetate to the lipid solution the mixture was loaded onto the column. After washing with 6 ml H.sub.2O, lipids were eluted with 1 ml MeOH and then with 6 ml C/M 1:1. Aliquots of the lipid extracts were sprayed onto silica gel 60 plates (Merck) using the Automatic TLC Sampler 4 from CAMAG (Muttenz, Switzerland). Loading volumes were normalized on the protein concentration of the crude homogenates used for lipid extraction. Different amounts (0.5-8 g) of lipid standards (cholesterol, sphingomyelin, sulfatide, all standards from Sigma) were loaded on separate lanes. After thin-layer chromatography (TLC) with C/M/H.sub.2O 70:30:4 as a solvent system lipids were visualized according to Yao and Rastetter (33). The plates were scanned with a flat bed scanner (PowerLook III from UMAX Data Systems, Hsinchu, Taiwan) and the intensities of lipid bands were determined with the analysis software Aida 2.11 (Raytest, Straubenhardt, Germany). The amount of cholesterol, sphingomyelin and sulfatide are expressed as arbitrary units representing the intensities of the respective TLC band after background correction. Statistical analysis was performed using Student's t-test.

(280) Histology

(281) Kidneys, spinal cord and brain were dissected from perfusion-fixed mice. For the detection of sulfatides, tissue slices (100 m thick) were prepared with a vibratome and incubated with alcian blue (Alcec Blue, Sigma-Aldrich, Taufkirchen, Germany) as described (Wittke, D. et al. Acta Neuropathol, (Berl.), 108, 261-271). The histochemical conditions (pH 5.7, 300 mM MgCl.sub.2) were such as to warrant specific staining of sulfatides (Scott, J. E. and Dorling, J. (1965), Histochemie, 5, 221-233). Paraffin sections from kidney blocks were prepared after pre-embedding incubation with alcian blue. Sciatic nerves and kidney samples were embedded in araldite according to routine methods for preparing semithin sections, either with or without pre-embedding incubation in alcian blue.

(282) Results:

(283) Pharmacokinetics and Biodistribution of CHO-rhASA after Single Dosing

(284) ASA knockout mice were first treated by a single injection of CHO-rhASA into the tail vein. To determine the rate of rhASA clearance from the circulation, plasma levels of enzyme were analysed at different times after infusion of 20 or 40 mg enzyme per kg body weight (FIG. 9A). For both doses the plasma levels reached a maximum in the first minutes after injection and declined from then on. Irrespective of the administered dose, rhASA was cleared from plasma with a half time of approximately 40 min. To evaluate the kinetics of tissue uptake, mice were perfused at different times after infusion and several organs were analysed for rhASA concentrations. Immunoreactivity for rhASA could be detected already 10 min after a single treatment with 40 mg/kg in all tissues (FIGS. 9B and C). In liver which acquired the highest enzyme concentration (see below) the enzyme levels increased around 4-fold within the next five hours and dropped thereafter until day 14 to approximately 4% of the maximum level (FIG. 9B). The kinetics were similar for kidney, sciatic nerve and brain (FIG. 9C). Independent of the differences between the tissue-specific uptake rates, the enzyme was eliminated from all tissues within a comparable time course. Thus, the half life of immunologically detectable rhASA in liver, kidney, sciatic nerve and brain ranged around 4 days (FIGS. 9B and C). It is striking that the maximum concentration of rhASA differed by more than three orders of magnitude between liver and brain 5 h after infusion (FIGS. 9B and C). To analyse the biodistribution of rhASA in more detail, ASA knockout mice were infused with increasing doses of rhASA and tissues were analysed 8 days later by ELISA. A roughly linear, dose-dependent increase of the enzyme concentration could be detected in kidney and peripheral nerves (FIG. 10A). The majority of the infused enzyme was found, however, in liver (shown for 20 mg/kg in FIG. 10B). Compared to liver the rhASA concentrations were around 7% in kidney, <0.05% in brain and 12-15% in sciatic nerve and brachial plexus. Taking the different masses of these tissues into consideration it can be calculated that around 97% of the retrievable enzyme was found in liver, around 3% in kidneys and below 0.1% in the CNS and peripheral nerves.

(285) Reduction of Sulfatide Levels after Single Dosing of CHO-rhASA

(286) To evaluate the therapeutic potential of CHO-rhASA treatment, ASA knockout mice were intravenously infused with a single dose of 40 mg CHO-rhASA per kg body weight and lipids were extracted from kidney 8 days later. TLC of the lipid extracts revealed a prominent decline of sulfatide levels compared to mock-treated controls (FIG. 11A). The time dependence of sulfatide reduction was investigated in a second experiment. For that purpose sulfatide levels were determined in kidneys at different times after injection of 40 mg/kg (FIG. 11B). Significant clearance of sulfatide could be detected already 5 h after infusion and the extent of reduction increased until day 8. At that time around two thirds of the excess sulfatide were cleared from kidney. Six days later sulfatide reappeared and the residual mean sulfatide level rose by approximately 22%. To determine the dose-dependence of sulfatide reduction, mice were treated with different doses of CHO-rhASA and analysed 8 days later. Already 10 mg/kg resulted in a significant decline of sulfatide storage in kidney (FIG. 11C). The extent of sulfatide clearance increased with increasing doses and a roughly linear relation between dose and loss of sulfatide was detectable. To evaluate effects of enzyme replacement on the lipid catabolism of the nervous system, total brain, sciatic nerve and brachial plexus from the differently treated animals was also analysed. Compared to kidney, where sulfatide levels increased around 10-fold in aged ASA knockout mice (FIGS. 11B and C), the nervous system showed only a roughly 2-fold elevation of sulfatide levels (FIG. 12A-C and FIG. 13B-D). Single dosing with 40 mg/kg had no effect on sulfatide storage in the brain (FIG. 12A). A significant decline was, however, detectable in the sciatic nerve (FIG. 12B) and the brachial plexus (FIG. 12C) after administration of 40 mg/kg. Also enzyme levels of 10 and 20 mg/kg reduced the mean sulfatide storage in peripheral nerves, but the difference to control tissues was statistically not significant (FIGS. 12B and C).

(287) Reduction of Sulfatide Storage after Repeated Dosing of CHO-rhASA

(288) The unexpected high efficacy of single enzyme doses in reducing sulfatide levels in peripheral tissues provided the rationale to evaluate the therapeutic potential of repeated injections. We chose a treatment schedule based on up to four injections of 20 mg CHO-rhASA/kg once a week. Sulfatide levels were analysed 8 days after the last injection in kidney, peripheral nerves and brain of mice treated by one, two, three or four injections (FIG. 13).

(289) TLC revealed that sulfatide declined progressively with an increasing number of infusions in all peripheral tissues. After the fourth treatment 65% of excess sulfatide was cleared from kidney and brachial plexus (FIGS. 13A and D) and 82% from the sciatic nerve (FIG. 13C), respectively. In brain no decline of sulfatide was detectable after the first, second and third treatment (FIG. 13B). After the fourth treatment, however, the sulfatide level was significantly reduced by 13% on average. This represents a clearance of 30% of excess sulfatide from brain.

(290) To verify the sulfatide reduction in the central nervous system of mice treated by four injections, histological analysis of brain and spinal cord was performed. In the CNS white and gray matter of mock-treated knockout mice, the sulfatide storage pattern was identical to the pattern previously described for ASA knockout mice (Wittke, D. et al. Acta Neuropathol. (Berl.), 108, 261-271) and two morphological types of storage material could be distinguished (FIG. 14). Large (>20 m) deposits which are typical for phagocytes and neurons and small storage granules characteristic of oligodendroglia (FIGS. 14C and G). The phagocytes have been previously identified as activated microglial cells (Hess B, et al. (1996) Proc Natl Acad Sci USA. 93(25):14821-6). Storage in phagocytes and neurons can be distinguished by the less compact and more ring-shaped appearance of alcianophilic material in neurons (FIG. 14C inset). In the brain and spinal cord of rhASA-treated mice sulfatide was largely cleared from phagocytes both in the white and gray matter (FIG. 14 A to H) whereas staining of neurons and oligodendrocytes was unaltered and similar as in mock-treated mice (shown for brain in FIGS. 14D and H).

(291) Apart from the nervous system, the kidney was also histologically analysed. Kidneys of mock-treated mice displayed the same sulfatide storage patterns as previously described (Lullmann-Rauch, R. et al. (2001), Histochem. Cell Biol., 116, 161-169). Storage was intense in thin limbs and thick ascending limbs of Henle's loop and moderate in distal convoluted tubules and collecting ducts (FIG. 15). After four enzyme injections the storage material was almost completely cleared from the distal convoluted tubules (FIG. 15J) and storage was clearly reduced in the upper portions of the thick ascending limbs (FIG. 15G). The storage in thin limbs and collecting ducts, however, persisted and resembled that of mock-treated mice (FIG. 15B to E).

(292) The analysis of the kidney also revealed a significant 1.4-fold increase of the kidney wet weight in 9-months-old ASA knockout mice compared to wildtype controls (data not shown). Interestingly, enzyme replacement reduced and partially normalized the increased kidney size. The extent of reduction was statistically significant after the third and after the fourth treatment (student's t-test, p<0.05) and the kidney weight declined to 1.2-fold of normal after four injections (not shown). In a second, independent experiment the mean kidney weight of 12-months-old knockout mice was 1.5-fold increased compared to wildtype mice (not shown). It declined significantly (student's t-test, p<0.05) to 1.1-fold of normal after 4 injections of enzyme (not shown). Liver and brain were weighed as controls and no significant differences were detectable between the experimental groups for these organs (not shown).

(293) Discussion

(294) Approximately 30% of the total amount of injected rhASA could be retrieved from dissected mouse organs 5 h after intravenous injection of 40 mg/kg (not shown). Among the retrievable fraction more than 90% was localized to liver while kidney and peripheral nerves shared the vast majority of the remaining enzyme (FIG. 10B). A comparison with previous data about ASA activities in wildtype mice (Matzner U, et al. (2000) Gene Ther. 7(14):1250-7) suggests that enzyme levels after rhASA treatment were on average 95 fold (liver), 1.2-fold (kidney), 0.6-fold (peripheral nerves) and 0.001-fold (brain) of normal.

(295) Already one intravenous injection of rhASA led to a pronounced time- and dose-dependent decline of sulfatide storage in kidney and peripheral nerves (FIG. 11). This is the first proof that ERT using ASA is effective in reducing the sulfatide storage in vivo. Notably, already 5 h after injection of 40 mg/kg a significant decline of sulfatide storage in kidney was detectable (FIG. 11B). Eight days after treatment storage was diminished to a minimum and up to 70% of the excess sulfatide had vanished from kidney (FIG. 11B) and peripheral nerves (FIGS. 12B and C).

(296) Compared to previous gene therapy experiments, in which TLC did not reveal a significant decline of the mean concentration of sulfatide in total kidney (Matzner U, et al. (2002) Gene Ther 9(1):53-63), both the velocity and the extent of storage reduction was surprising. The difference between the two studies is striking since the steady state level of hASA in kidney, which was achieved by transplantation was 1.3-fold of normal on average and thus virtually the same as the maximum level reached by a single injection of 40 mg CHO-rhASA/kg (see above). Thus, CHO-rhASA which was present in kidney only for a couple of days eliminated more than two thirds of excess sulfatide, while the same amount of enzyme did not reduce the mean storage when it was stably expressed from cells of the hematopoietic system for almost one year. The dependence of the therapeutic efficacy on the cell type which expresses the enzyme points to cell type-specific differences in the biosynthesis of human ASA. Cell culture studies suggested that human ASA is inefficiently phosphorylated by cells of the hematopoietic system, but efficiently phosphorylated by CHO and BHK cells (Muschol, N. et al. 2002, Biochem. J., 368, 845-853, and FIG. 7B). Phosphorylation appears, however, to be important for therapeutic efficacy in ASA knockout mice. A low phosphorylation of human ASA by hematopoietic cells and a high phosphorylation by CHO cells may thus explain the partial failure of bone marrow stem cell gene therapy and the success of Exyme Replacement Therapy in ASA knockout mice.

(297) The single-dose experiments indicated that clearance of storage is only transient and sulfatide reaccumulated in the second week after treatment (FIG. 11B). The partial reaccumulation of storage can be explained by the limited half life of the internalized rhASA, which was in the range of only 4 days (9B and C). The reaccumulation of sulfatide necessitated a regimen based on repeated enzyme injections in order to maintain or even enhance storage reduction over the long range. Repeated treatment with 20 mg CHO-rhASA per kg resulted in a step-wise decline of sulfatide storage in peripheral tissues (FIGS. 13A, C and D). Up to 65% and 82% of excess sulfatide could be eliminated from kidney and peripheral nerves by four injections, respectively. The histological analysis of kidney revealed the most prominent decline of sulfatide storage in the cortex (FIG. 15). Here storage was abolished or greatly reduced in distal convoluted tubules and the upper portion of the thick ascending limbs of Henle's loop, respectively. Presently, it is unclear why these segments of the nephron respond more clearly to ERT than other segments. Possibly, the region specificity is determined by the expression pattern of the receptor(s) which endocytose rhASA.

(298) Surprisingly, repeated dosing did not only reduce storage in peripheral tissues, but also in the CNS. This was first evidenced by TLC of brain lipids which showed a decline of sulfatide by 13% after the fourth treatment (FIG. 13B). Reduction of CNS storage was, however, more clearly seen in the morphological analysis. Histology of brain and spinal cord revealed a strikingly reduced frequency of the enlarged sulfatide-storing phagocytes throughout the CNS (shown for brain stem in FIG. 14). Thus, the reduction of sulfatide levels in the CNS appears to be mainly due to clearance of lipid from these phagocytes representing activated microglial cells (Hess B, et al. (1996) Proc Natl. Acad Sci USA. 93(25):14821-6) rather than from neurons or oligodendroglia. It has been shown recently in animal models for Krabbe and Sandhoff disease that microglial activation plays a major role in the pathogenesis. of these related sphingolipid storage diseases (Matsushima, G. K. et al. (1994), Cell, 78, 645-656., Wada, R. et al. Proc. Natl. Acad. Sci. U.S.A., 97, 10954-10959). Microglial activation is also prominent in ASA knockout mice in the second year of life (Hess B, et al. (1996) Proc Natl Acad Sci USA. 93(25):14821-6). Reduction of sulfatide storage in microglial cells can therefore be expected to be beneficial even in the absence of detectable clearance of sulfatide from other glial cells and neurons.

(299) Since the blood-brain barrier prevents transfer of rhASA from the circulation to the CNS, the brain did not acquire enzyme levels exceeding 0.1% of normal during the treatment period of 4 weeks (FIGS. 9C and 10B and not shown). due to the low concentrations, arylsulfatase A present in the CNS is unlikely to be responsible for all of the observed clearance of sulfatides from phagocytes. A second mechanism which may contribute to the clearance involves endocytosis of the enzyme into phagocytes prior to immigration into the CNS. However, the main part of the population of CNS phagocytes (i.e. microglia) are believed to represent an autonomous, self-renewing cell population derived from macrophage progenitors immigrated into the CNS early during life, and being distinct from the blood monocytes/macrophages present in non-neural tissues. A third contributory mechanism exists, involving the export of sulfatides from the brain cells to peripheral cells. The driving force for this export appears to be an increasing imbalance of the equilibrium between sulfatide storage in the CNS and peripheral tissues due to the ASA-catalyzed hydrolysis of sulfatide in the periphery.

Example 6

Studies on Neurologic ParametersRotarod Studies

(300) ASA knockout mice develop nerve conduction impairments and a number of neurologic symptoms. To measure putative therapeutic effects on neurologic parameters the rotarod performance was examined.

(301) Previous behavioral tests revealed progressive deficits of ASA knockout mice in balancing on a slowly rotating rod (D'Hooge, R. et al. Brain Res., 907, 35-43, Matzner U, et al. (2002) Gene Ther 9(1):53-63). To determine effects of treatment on motor coordination, mice were tested before the first and after the third infusion of CHO-rhASA by rotarod experiments. In a first study mice at a mean age of about 9 months were analysed. In the test before treatment, wildtype mice were successful in 32 of 40 trials (80%), whereas the two groups of (yet untreated) ASA knockout mice were successful in 22 of 40 (55%) and 25 of 40 (63%) trials (not shown). Thus, the data confirmed that behavioral deficits of ASA knockout mice are already detectable, but still comparably mild at 9 months of age (D'Hooge, et al. Brain Res., 907, 35-43). After treatment of one group of knockout mice with three weekly doses of 20 mg CHO-rhASA/kg the same three groups were reanalyzed 4 weeks later. Compared to the first test the mean success of rhASA-treated mice was improved by 27% and reached 82%. In contrast to this group the mean performance of wildtype and mock-treated controls was only improved by 10% and 5%, respectively (FIG. 16B). Thus, by a combination of treatment and training 9 months old ASA knockout mice acquired the ability to stay on the rod with a higher frequency than untrained wildtype mice of the same age.

(302) To investigate effects on more advanced motor coordination disabilities, 12 months old mice (3 months older than the above) were analysed in a second experiment. Now the percentages of successful mice before treatment were 43% (wildtype controls), 18% (ASA knockouts destined for mock treatment) and 10% (ASA knockouts destined for treatment with rhASA) on average (not shown). After treatment 65% of wildtype controls and 13% of mock-treated ASA knockout mice were successful (FIG. 16C). The mean percentage of rhASA-treated knockout mice, however, increased to 31%. Thus, also in elder mice with progressed coordination impairments motor coordination could be substantially improved by three treatments with CHO-rhASA.

Example 7

Studies on Nerve Motor Conduction Velocity

(303) To further measure putative therapeutic effects on neurologic parameters the compound motor action potential (CMAP) nerve conduction of sciatic nerves was studied under anaesthesia by established electrophysiological methods (Zielasek, J. et al. Muscle Nerve, 19, 946-952). In brief, the compound motor action potential (CMAP) was recorded with two needle electrodes in the foot muscles after distal stimulation of the tibial nerve at the ankle and proximal stimulation of the sciatic nerve at the sciatic notch. Statistical analysis was performed using Student's t-test.

(304) Neurophysiological studies of the sciatic nerve were done 6 days after the fourth treatment of 12 months old mice. After distal stimulation, age-matched wildtype control animals showed a normal CMAP with an amplitude of 19.01.7 mV (meanSD, n=8), a latency of 0.840.11 msec and a duration of 3.30.36 msec (FIG. 16A and FIG. 17). Mock-treated ASA-deficient animals showed a less compact motor response with significantly reduced mean amplitude (15.63.9 mV, p<0.05) and increased duration (4.10.31 msec, p<0.01). The mean latency and the nerve conduction velocity was 0.810.09 msec and 45.48.8 m/sec, respectively, and not significantly different from that of wildtype mice (FIG. 16A, FIG. 17 and not shown). Treatment with CHO-rhASA resulted in an increase of the amplitude to normal values (20.45.9 mV, p<0.05) and a significant decrease of the duration (3.80.35 msec, p<0.05). Thus the impaired conduction of the sciatic nerve was virtually normalized after treatment with rhASA. Similar data were obtained after proximal stimulation and the data were reproduced in an independent experiment using mice of another treatment series (not shown).

(305) Determination of Electrophysiological Parameters

(306) TABLE-US-00014 TABLE 8 Raw data distal nerve mot dura- ampl f- prox dur ampl dis- cond lat tion distal wave lat prox prox tance vel wild- type number 43 0.84 3 19.7 4.88 1.48 2.9 17.9 24 37.50 44 1 3.6 21.6 5.72 1.6 2.9 19.7 24 40.00 40 0.96 2.9 * 5.48 1.52 3.2 * 26 46.43 37 0.88 3.7 17.4 6.04 1.6 3.2 15.8 26 36.11 45 0.68 2.9 19.8 5.2 1.36 3 16.5 26 38.24 39 0.76 3.2 19 5.68 1.44 3.2 15.9 25 36.76 38 0.76 3.8 16.3 6 1.4 3.3 13.8 25 39.06 mean 0.84 3.30 17.40 5.57 1.49 3.10 15.41 25.14 39.16 SD 0.11 0.36 4.15 0.39 0.09 0.15 3.37 0.83 3.21 ko TBS number 6 0.76 3.9 10.3 5.68 1.36 3.1 6.9 25 41.67 4 0.84 4 11.3 5.64 1.44 3.3 11.7 24 40.00 2 1 4.3 12.4 5.32 1.4 nd 13.6 27 67.50 3 0.8 4.8 15.7 5.64 1.44 3.6 14.8 25 39.06 7 0.76 4 16.8 4.76 1.32 nd 11.7 25 44.64 1 0.84 4.2 17 5.48 1.36 nd 12.8 25 48.08 5 0.8 4 23 5.08 1.4 nd 18.1 26 43.33 8 0.68 3.7 18.1 5.04 1.32 nd 16.2 25 39.06 mean 0.81 4.11 15.58 5.33 1.38 3.33 13.23 25.25 45.42 SD 0.09 0.31 3.89 0.32 0.04 0.21 3.16 0.83 8.83 ko rhASA number 26 0.76 4.3 28.5 5.2 1.36 nd 24 25 41.67 11 0.84 4.3 25.3 5.88 1.36 nd 22.6 24 46.15 27 0.68 4 12.7 5.08 1.36 nd 12.9 26 38.24 18 0.8 3.6 22.2 5.16 1.32 nd 17.2 25 48.08 14 0.72 3.4 17.7 4.88 1.32 nd 17.1 26 43.33 30 0.68 3.5 16.8 4.68 1.24 nd 14.3 25 44.64 24 0.76 3.6 12.7 5 1.32 nd 11.4 27 48.21 25 0.84 3.5 27.5 5 1.44 nd 11.9 24 40.00 mean 0.76 3.78 20.43 5.11 1.34 nd 16.43 25.25 43.79 SD 0.06 0.35 5.94 0.33 0.05 nd 4.46 0.97 3.43 * wildtype mouse #40 yielded low amplitudes due to technical problems (ampl dist = 8.0; ampl prox = 8.3); ndduration after proximal stimulation not determinable

(307) TABLE-US-00015 TABLE 9 statistical evaluation using Student's t-test P values ampl prox dur ampl dml duration distal f-wave lat prox prox distance NLG wildtype 0.2930 0.0004 0.0449 0.1203 0.0073 nd 0.0258 nd 0.0611 untreated vs knockout mock-treated knockout 0.1149 0.0375 0.0461 0.1121 0.0745 nd 0.0720 nd 0.3282 mock- treated vs knockout rhASA-treated bold - statistically significant difference (P < 0.05)
Results the following changes in the electrophysiological pattern of knockout mice are statistically significant (wildtype vs mock-treated knockouts): duration (of amplitude after distal stimulation) is increased amplitude (height) after distal stimulation is decreased latency after proximal stimulation is decreased amplitude (height) after proximal stimulation is decreased treatment results in the following statistically significant changes of the pattern (mock-treated knockouts vs rhASA-treated knockouts) duration (of amplitude after distal stimulation) is decreased towards normal values amplitude (height) after distal stimulation is increased towards normal values

(308) (1) The amplitude is the result (sum) of individual axon potentials. If all axon potentials pass the recording electrode at the same time point the amplitude would be short and high. If the potentials pass it at different time points (because some axons conduct fast and others slow) the amplitude would be broad and low. Compared to wildtype mice the amplitude of ASA knockout mice is more flattened and extended.

(309) (2) For the determination of the nerve conductance velocity the time between stimulation and begin of the amplitude is measured. This time is virtually the same for knockout and wildtype mice. It can be concluded that the knockout mice possess nerve fibers with normal conductance velocity.

(310) From (1) and (2) it can be concluded that knockout mice have fast conducting fibers (normal nerve conductance velocity), but also a substantial fraction of fibers which conductance velocity is more or less reduced (flattened and extended amplitude).

(311) Treatment results in a significant improvement of the duration and height of the amplitude. Recordings of the CMAP in the sciatic nerve of untreated ASA knockout mice suggested an impaired conduction of a subset of axonal fibers. This was indicated by a significantly lower and broader amplitude in the presence of a normal nerve conduction velocity (FIG. 16A and FIG. 17). Treatment decreased the duration and increased the height of the flattened amplitude demonstrating the abrogation of inhibitory effects. This corrective effect might be associated with the critical role of sulfatide in the organisation of paranodal axoglial junctions and the correct clustering of voltage-gated Na.sup.+ and K.sup.+ channels along the axolemma. The possibility to reverse changes of the CMAP by a comparably short exposure to recombinant enzyme might have great implications for the treatment of MLD. Since PNS symptoms prevail before the end stage of MLD, ERT might substantially retard the disease progression and improve the quality of life. This notion is supported by the rotarod data in mice indicating improvement of the motor coordination both at an early as well as at a more advanced stage of the disease (FIGS. 16B and C).

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