ES2824623T3 - Process for the production and purification of recombinant lysosomal alpha-mannosidase - Google Patents

Abstract

A process for the purification of recombinant human lysosomal alpha-mannosidase from a cell culture, wherein a fraction of said cell culture comprising recombinant human lysosomal alpha-mannosidase is subjected to chromatography on a resin comprising a multimode ligand, wherein said ligand Resin-bound multimode is a substance that has a carboxylic acid or sulfonic acid group, wherein said process results in a composition comprising purified alpha-mannosidase characterized in that at least 80% of the alpha-mannosidase is present as a glycoprotein 130 kDa.

Description

DESCRIPTION

Process for the production and purification of recombinant lysosomal alpha-mannosidase

Technical field of the invention

The present invention relates to a process for the purification of recombinant human lysosomal alpha-mannosidase, to a process for the production of alpha-mannosidase, to a composition comprising alpha-mannosidase, to the use of the composition as a medicine and to its use as a medicine for the treatment of alpha-mannosidosis.

Background of the invention

Alpha-mannosidosis

Alpha-mannosidosis is an autosomal recessive disease that occurs worldwide with a frequency of between 1 / 1,000,000 and 1 / 500,000. Mannosidosis occurs in all ethnic groups in Europe, America, Africa, and also Asia. It is detected in all countries with a good diagnostic service for lysosomal storage disorders, with a similar frequency. Patients are born apparently healthy, although the symptoms of the disease are progressive. Alpha-mannosidosis presents clinical heterogeneity, ranging from very severe to very mild forms. The clinical symptoms are: mental retardation, skeletal changes, immune system alterations resulting in recurrent infections, hearing impairment, and the disease is usually associated with typical facial features, such as a thick face, a prominent forehead, a flattened nasal bridge, a small nose and a wide mouth. In the most severe cases (type I mannosidosis), children suffer from hepatosplenomegaly and die during the first years of life. This early death is possibly caused by serious infections due to the immunodeficiency caused by the disease. In milder cases (mannosidosis type 2), patients usually reach adulthood. The skeletal weakness of patients causes patients to need a wheelchair when they reach the age of 20 to 40 years. This disease causes diffuse dysfunction in the brain that normally results in weak mental performance that excludes anything but the most basic literacy skills. These problems associated with hearing impairments and other clinical manifestations prevent patients from developing an independent life, with the consequence that they require lifelong care.

Lysosomal alpha-mannosidase

Alpha-mannosidosis is the result of deficient activity of lysosomal alpha-mannosidase (LAMAN, EC3.2.1.24). The disease is characterized by a massive intracellular accumulation of mannose-rich oligosaccharides, that is, oligosaccharides that carry α1,2-, α1,3-, and α1,6-mannosyl residues at their non-reducing ends. These oligosaccharides originate mainly from the intralisosomal degradation of glycoproteins with N-linked oligosaccharides. However, some originate from the catabolism of dolichol-linked oligosaccharides and misfolded glycoproteins that are redirected to the cytosol for degradation by the proteasome. (Hirsch et al. EMBO J. 22, 1036-1046, 2003 and Saint-Pol et al. J. Biol. Chem. 274, 13547-13555, 1999). Lysosomal storage is observed in a large number of cell types and tissues, including neurons in all brain regions. LAMAN is an exoglycosidase that hydrolyzes these non-reducing terminal alpha-D-mannose residues from the non-reducing end of alpha-D-mannosides during the orderly degradation of N-linked glycoproteins (Aronson and Kuranda FASEB J 3: 2615-2622. 1989). The human precursor enzyme is synthesized as a single 1011 amino acid polypeptide that includes a 49 residue signal peptide. The precursor is proteolytically processed into three major 15, 42, and 70 kD glycopeptides in the mature enzyme in the lysosome. The 70 kD glycopeptide is further processed into three disulfide-linked subunits. (Berg et al. Mol. Gen. and Metabolism 73, 18-29, 2001, Nilssen et al. Hum.Mol.Genet. 6, 717-726. 1997).

The lysosomal alpha-mannosidase gene

The gene encoding LAMAN (MANB) is located on chromosome 19 (19cen-q12), (Kaneda et al. Chromosoma 95: 8-12, 1987). MANB consists of 24 exons, spanning 21.5 kb (GenBank reference numbers U60885-U60899; Riise et al. Genomics 42: 200-207, 1997). The LAMAN transcript is »3,500 nucleotides (nt) and contains an open reading frame that encodes 1,011 amino acids (GenBank U60266.1).

The cloning and sequencing of the human cDNA encoding LAMAN has been published in three articles (Nilssen et al . Hum.Mol.Genet. 6, 717-726. 1997; Liao et al. J.Biol.Chem. 271, 28348-28358 1996; Nebes et al. Biochem.Biophys.Res.Commun. 200, 239-245. 1994). Interestingly, the three sequences are not identical. When compared with the sequence of Nilssen et al (reference # U60266.1) Liao et al observed a change from TA to AT at positions 1670 and 1671 resulting in a substitution of valine to aspartic acid and Nebes et al. to the. they also observed a change from C to at position 1152 that did not result in changes in the amino acid sequence.

Diagnosis

Currently, the diagnosis of alpha-mannosidase is based on clinical evaluation, detection of oligosaccharides rich in mannose in the urine, and on direct measurements of alpha-mannosidase activity in various cell types, such as leukocytes, fibroblasts, and amniocytes (Chester et al. ., In: Durand P, O'Brian J (eds) Genetic errors of glycoprotein metabolism. Edi-Ermes, Milan, pp. 89-120. 1982; Thomas and Beaudet. In: Scriver CR, Beaudet al, Sly WA, Valle D (eds) The metabolic and molecular bases of inherited disease, Vol 5. McGraw-Hill, New York, pp.

2529-2562. nineteen ninety five).

Because symptoms are usually mild initially and biochemical diagnosis is difficult, the diagnosis is usually made late in the course of the disease. It is clear that patients and their families would benefit substantially from an early diagnosis.

Animal models

Alpha-mannosidosis has been described in cattle (Hocking et al. Biochem J 128: 69-78. 1972), cats (Walkley et al. Proc. Nat. Acad. Sci. 91: 2970-2974, 1994) and guinea pigs ( Crawley et al. Pediatr Res 46: 501-509, 1999). Recently, a mouse model was generated by targeted disruption of the mannosidase gene (Stinchi et al. Hum Mol Genet 8: 1366-72, 1999).

As in humans, alpha-mannosidosis appears to be caused by specific mutations in the gene encoding lysosomal alpha-mannosidase. Berg et al. (Biochem J. 328: 863-870.1997) reported purification of feline hepatic lysosomal alpha-mannosidase and determination of its cDNA sequence. The active enzyme consists of 3 polypeptides, communicating that their molecular masses are 72, 41 and 12 kD. In a similar way to the human enzyme, it was shown that the feline enzyme is synthesized as a single-chain precursor with a putative signal peptide of 50 amino acids followed by a polypeptide chain of 957 amino acids, which is cleaved into the 3 polypeptides of the mature enzyme. The deduced amino acid sequence was 81.1% and 83.2% identical to the human and bovine sequences, respectively. A 4 bp deletion was identified in an affected Persian cat; deletion resulted in a phase shift from codon 583 and premature termination at codon 645. No enzyme activity could be detected in the cat liver. A long-haired domestic cat expressing a milder phenotype had an enzyme activity of 2% of normal; this cat did not possess the 4 bp deletion. Tollersrud et al. (Eur J Biochem 246: 410-419. 1997) purified the bovine kidney enzyme to homogeneity and cloned the gene. The gene was organized into 24 exons spanning 16 kb. Based on the gene sequence, they identified two mutations in cattle.

Medical necessity for the treatment of alpha-mannosidosis

In view of the severe clinical manifestations resulting from the accumulation of glucose-rich oligosaccharides, the lack of an effective treatment for alpha-mannosidosis is well recognized. Currently, the main therapeutic option for the treatment of the disease is bone marrow transplantation, however, the objective of the present invention is to promote enzyme replacement therapy as a potential future alternative.

Bone marrow transplant

In 1996, Walkley et al. (Proc. Nat. Acad. Sci. 91: 2970-2974, 1994) published an article about 3 kittens with mannosidosis that were treated with a bone marrow transplant (BMT) in 1991. In the 2 animals that were euthanized , a normalization was observed, not only in the body, but more importantly, also in the brain. The 3rd cat was fine after 6 years. Typically, an untreated cat dies within 3-6 months. In 1987, a child with mannosidosis was treated with BMT (Will et al. Arch Dis Child, 1987 Oct; 62 (10): 1044-9). He died after 18 weeks due to complications related to the procedure. Little enzyme activity was observed in the brain. This unsatisfactory result could be explained by strong immunosuppressive treatment before death or because it takes time for enzyme activity to increase in the brain after BMT. The donor was the mother (who could have been expected as a carrier to have less than 50% enzyme activity) or it could be the case that, in humans, BMT has no effect on enzyme function in the brain . Despite variable results, few attempts at bone marrow transplantation have indicated that a successful graft could correct the clinical manifestations of alpha-mannosidosis, at least partially. However, it is still not possible to reduce the serious complications related to the procedure when bone marrow transplantation is applied in treatments for humans.

Enzyme replacement therapy

When lysosomal storage diseases were discovered, hopes rose that they could be treated by enzyme replacement. Enzyme replacement therapy has been shown to be effective in Gaucher disease. When exogenous lysosomal glucocerebrosidase is injected into the patient, enzyme-deficient cells take up this enzyme (Barton et al. N Engl J Med 324: 1464-1470). Said uptake is regulated by certain receptors on the cell surface, such as, for example, the mannose-6-phosphate receptor, which is practically ubiquitous on the surface of cells, and other receptors, such as the asialoglycoprotein receptor and the mannose receptor, They are restricted to certain cell types, such as cells of the monocyte / macrophage cell lineage and hepatocytes. Therefore, the cellular uptake of the enzyme is highly dependent on its glycosylation profile. If properly designed, the defective enzyme could be replaced by regular injections of exogenous enzyme in the same way that diabetic patients take insulin. In vitro studies with purified active lysosomal alpha-mannosidase added to the medium of enzyme-deficient fibroblasts demonstrated correction of substrate lysosomal accumulation. On the other hand, in vivo treatment has been hampered, in part, by the problem of producing a sufficient quantity of enzymes, due to complicated large-scale production and purification processes and complications resulting from immune reactions against the exogenous enzyme.

However, and more importantly, special considerations apply in relation to lysosomal storage diseases with a major neurological component, such as alpha-mannosidosis, where the clinical manifestations are related to increased lysosomal storage within the nervous system. central. Therefore, enzyme replacement therapy has not been shown to be effective against the acute neuropathic variant of Gaucher disease (Prows et al. Am J Med Genet 71: 16-21).

The delivery of therapeutic enzymes to the brain is impeded by the absence of transport of these large molecules across the blood-brain barrier. Based on the general notion that the blood-brain barrier must be circumvented to observe the effect of therapeutic agents in the brain, the use of a wide variety of delivery systems has been contemplated. These include invasive techniques, such as osmotic opening of the blood-brain barrier with, for example, mannitol, and non-invasive techniques, such as chimeric enzyme receptor-mediated endocytosis. Since enzyme replacement is expected to require administration of the enzyme on a regular basis, the use of invasive techniques should be avoided. Only recently, the use of non-invasive techniques has provided promising results in animal models (for alpha-mannosidosis see below, for other lysosomal disorders see, for example: Grubb et al. ^P N ^a S 2008, 105 (7) pp 2616-2621). It has been contemplated that reduced storage in the visceral organs and meninges could reduce the amount of oligosaccharides that are transported to the brain. However, these considerations are not believed to apply to lysosomal disorders, in which neurological damage is primary and severe (Neufeld, EF Enzyme replacement therapy, in "Lysosomal disorders of the brain" (Platt, FM Walkley, S. V: eds Oxford University Press) However, as described in Roces et al. Human Molecular Genetics 2004, 13 (18) pp. 1979-1988, Blanz et al. Human Molecular Genetics 2008, 17 (22) pp. 3437-3445 and WO 05/094874, it has been shown that it is possible to increase LAMAN levels in the central nervous system of animals using, for example, intravenous injection of a formulation comprising alpha-mannosidase, thereby reducing the intracellular levels of mannose-rich neutral oligosaccharides in one or more regions of the central nervous system. This indicates that recombinant alpha-mannosidase is useful in enzyme replacement therapy of patients suffering from alpha-mannosidosis. Therefore, one of the main The problem in providing effective treatment of alpha-mannosidosis using hormone replacement is to provide sufficient amounts of pure recombinant alpha-mannosidase in an inexpensive manner.

Production and purification of alpha-mannosidase

WO 02/099092 discloses a process for the small scale production of rhLAMAN in CHO cells using serum medium at 37 ° C. Also described is a small-scale purification process that involves diafiltration of the crude enzyme and weak anion exchange chromatography using DEAE sepharose FF columns in the capture step, followed by a series of chromatographic purification steps involving chromatography of hydrophobic and mixed mode interaction.

Forsee et al. discloses the purification of a 1,2-alpha-mannosidase isolated from rabbit liver. Rabbit mannosidase is a 52 kDa monomeric enzyme. Several chromatography steps are carried out, using Cibracon Blue agarose resin in one of the latter. However, Forsee et al. do not disclose the use of multimode resins (such as Cibracon Blue) for the purification of recombinant human LAMAN.

WO 2007/112757 discloses a method for the concentration of a peptide and suitable types of resin for chromatography are described. However, WO 2007/112757 as a whole focuses on PBGD and does not disclose the use of multimode resins for the purification of rhLAMAN.

WO 2009/007451 discloses the purification of Factor VIII protein using mixed or multimode resins. The purification of rhLAMAN is not disclosed.

Zhao et al. disclose a review of a series of mixed mode resins useful for protein chromatography. The purification of mannosidase is not disclosed.

WO 05/094874 discloses a process for the small-scale production of rhLAMAN in ovarian cells Chinese hamster (CHO) using serum medium at 37 ° C. A small-scale purification process analogous to that of WO 02/099092 is also described.

WO 05/077093 describes the manufacture of highly phosphorylated lysocymic enzymes. Example IV describes a purification method for acid alpha-glucosidase (GAA) using a multimode resin (Bluesepharose). Although GAA is a lysocymic enzyme, it is completely different from rhLAMAN. GAA is highly phosphorylated, while rhLAMAN has a low degree of phosphorylation. Furthermore, the sequence identity score is less than 12% between GAA and rhLAMAN and finally, their theoretical isoelectric points differ by more than one pH unit (5.42 and 6.48, respectively). Therefore, the method described in WO 05/077093 for purifying GAA cannot be applied to rhLAMAN.

A small-scale production process of rhLAMAN in CHO cells using 0.25% serum (V / V) and addition of DMSO has been reported (Berg et al. Molecular Genetics and Metabolism, 73, pp. 18-29, 2001 It also describes two purification processes involving a) a three-step process involving ultrafiltration, anion exchange chromatography, and gel filtration or b) single-step immunoaffinity chromatography. Furthermore, it is disclosed how method a) results in complete fragmentation of the 130 kDa enzyme into 55 kDa and 72 kDa fragments, while method b) results in partial fragmentation of the 130 kDa precursor in significant amounts. 55 and 72 kDa fragments.

Thus, an improved process for the production and purification of recombinant alpha-mannosidase could be advantageous. In particular, an improved process for large-scale cultivation of a cell line capable of expressing alpha-mannosidase and a more efficient large-scale purification process for isolating pure alpha-mannosidase with high enzyme activity from a culture could be advantageous. mobile.

Summary of the invention

Therefore, an object of the present invention relates to a production and purification process for recombinant human lysosomal alpha-mannosidase.

In particular, an object of the present invention is to provide a scalable production and purification process that solves the aforementioned problems of the prior art by providing sufficient amounts of high purity human lysosomal alpha-mannosidase with high enzymatic activity, thereby provided a treatment for patients suffering from alpha-mannosidosis.

Therefore, one aspect of the invention relates to a process for the purification of recombinant human lysosomal alpha-mannosidase from a cell culture, wherein a fraction of said cell culture comprising recombinant human lysosomal alpha-mannosidase is subjected to chromatography on a resin containing a multimode ligand, wherein said multimode ligand bound to the resin is a substance that has a carboxylic acid or sulfonic acid group and wherein said process results in a composition comprising purified alpha-mannosidase characterized in that at least one 80% of the alpha-mannosidase is present as a 130 kDa glycoprotein. The surprising finding that this purification process resulted in higher persistent percentages (such as greater than 80%) of the non-fragmented 130 kDa glycoprotein after purification than previously achieved is advantageous, as this provides a more uniform compared to a fragmented enzyme, which in turn enhances the ability to obtain a pharmaceutical grade product

Another aspect of the present invention relates to a process for the batch fed or continuous production of recombinant human lysosomal alpha-mannosidase, comprising the following steps:

to. inoculating a production reactor comprising a base medium with cells capable of producing recombinant human lysosomal alpha-mannosidase on day 0, to provide a cell culture;

b. adding a feeding medium to said cell culture at least once from day 1;

c. adjust the temperature of said cell culture to a maximum of 35 ° C, either after day 3 or when the density of viable cells is greater than 2.1 MVC / ml, whichever comes first,

d. a purification process as described herein.

The inventors surprisingly found that the above production process resulted in a cell culture comprising recombinant human lysosomal alpha-mannosidase in high yields that could be easily transferred to the purification column of the present invention without any dilution.

Yet another aspect of the present invention is to provide a composition comprising recombinant human lysosomal alpha-mannosidase purified by a purification process as described herein, wherein at least 80% of the alpha-mannosidase is present as a 130 kDa glycoprotein.

Another aspect of the present invention is a composition comprising human lysosomal alpha-mannosidase purified recombinant for use in the treatment of alpha-mannosidosis.

Brief description of the figures

Figure 1 shows a general outline of the currently preferred purification process design for alpha-mannosidase from harvest to drug substance fill.

Figure 2 shows an example of a Capto ™ MMC column chromatogram for alpha-mannosidase. Figure 3 shows an example of a butyl Sepharose ™ FF column chromatogram for alpha-mannosidase. Figure 4 shows an example of a CHT type 1 column chromatogram for alpha-mannosidase.

Figure 5 shows an example of a Q Sepharose ™ HO column chromatogram for alpha-mannosidase. Figure 6 shows an SDS-PAGE chromatogram of the purified alpha-mannosidase composition indicating the distribution of the 130 kDa, 75 kDa and 55 kDa glycoprotein species.

Figure 7 shows three HPLC diagrams for purified alpha-mannosidase, illustrating the amount of the 130 kDa species compared to the 55 and 75 kDa species. The first peak from the left is the 55 kDa species, followed by the 130 kDa and 75 kDa species, respectively. The 2-step process is without the use of a multimodal ligand chromatography step, while the 3 and 4-step processes use a multimodal ligand chromatography step.

Next, the present invention will be described in more detail.

Detailed description of the invention

Definitions

Before describing the present invention in more detail, the following terms and conventions will first be defined:

Recombinant alpha mannosidase

In the context of the present invention, recombinant alpha-mannosidase is defined as an alpha-mannosidase that by virtue of its origin or manipulation is not equal to all or part of the wild-type alpha-mannosidases found in nature. Therefore, it is constructed using recombinant techniques involving recombinant DNA molecules, that is, hybrid DNA sequences comprising at least two fused DNA sequences, the first sequence fused to the second sequence not normally found in nature. The recombinant alpha-mannosidase protein can be of human or non-human origin. In particular, it can be a recombinant human lysosomal alpha-mannosidase (rhLAMAN). The alpha-mannosidase product can be a single polypeptide or a mixture of an individual polypeptide and fractions thereof. Likewise, alphamanosidase can undergo post-translational modifications and therefore can be in the form of a glycoprotein.

Cell culture

A cell culture is the process by which cells are grown under controlled conditions. In the present context, cells in culture are specially designed to express a protein of interest, such as recombinant alpha-mannosidase. Cell culture can be housed in a bioreactor, which is specially designed to allow control of chemical and physical conditions.

Fraction

In the present context, a fraction refers to a fraction from a cell culture. The fraction may constitute the entire cell culture, but is usually a treated fraction of the culture, such as a clarified, filtered, concentrated, diluted or partially purified fraction.

Resin

In the context of the present invention, a resin forms the basis of a stationary phase in a chromatography system, on which various groups or chemicals are attached to provide a specific amount of affinity for a given molecule or protein of interest. The resins are normally polymeric beads with covalently bound ligands, said resins being insoluble in the liquid mobile phases used.

Multimodal ligand

By multimodal ligand is meant any ligand that is designed to interact with a molecule or protein of interest in at least 2 different ways. The individual interactions can be, independently, hydrophobic, hydrophilic, ionic, Van der Waals interactions, hydrogen bonding, or any other chemical or physical intermolecular interaction. In the present context, a ligand is an organic chemical bound to a resin as defined above. A multimodal ligand will have different affinities for different substances that are passed through the chromatography column dissolved in a mobile phase. The affinity differences give rise to variations in the retention time of the different substances on the chromatography column, allowing the substances to be separated. Retention times also depend on other factors such as, for example, mobile phase constituents, pH and temperature. Resins comprising multimodal ligands are sometimes also referred to as "mixed mode" resins, but in the present context, resins comprising a multimodal ligand should not be confused with so-called "mixed mode ion exchange resins", which comprise several different "ligands" on the same resin that may have opposite charges, such as, for example, OH, -Ca + and -PO ^{42 "} in the case of ceramic hydroxyapatite (CHT) resin. In these resins, the individual ligands they are not multimodal.

Load

In the present context, loading refers to the transfer of a collected fraction, eluate or other solution to a chromatography system, such as a chromatography column comprising a resin as the stationary phase.

Tampon

The term buffer is well known as a general description of a solution that contains a weak acid and / or its corresponding salt or a weak base and / or its corresponding salt, which is resistant to changes in pH. In the context of the present invention, the buffers used are suitable for use in chromatography systems, including but not limited to: Phosphate buffers, for example disodium phosphate (Na ² HPO ⁴ ), sodium phosphate or phosphate of potassium, acetate buffers, eg sodium acetate or potassium acetate, sulfate buffers, eg sodium sulfate or potassium sulfate, ammonium sulfate or Hepes or other buffers, eg sodium borate or tris-buffer. HCl.

Ultrafiltration

Ultrafiltration is a method of separation in which hydraulic pressure is used to force molecules and solvent through a membrane that comprises pores of a specific size, also known as a size or cut-off value. Only molecules that have a molecular weight lower than the cut-off value of the membrane are capable of crossing the membrane, while those that have a higher molecular weight do not cross the membrane and form what is called retained. Therefore, the molecules present in the retentate can be concentrated as the solvent flows through the membrane.

In a particular embodiment, the concentration of a solution or composition comprising a polypeptide, such as alpha-mannosidase, can be carried out by tangential flow filtration (TFF). This method is particularly useful for large-scale concentration, that is, for the concentration of solutions with a volume of one liter to several hundred liters. Therefore, this method is particularly useful for the production of concentrated solutions of a polypeptide of interest on an industrial scale.

The TFF technique is based on the use of a specific device that makes the solution to be filtered flow through a semi-permeable membrane; only molecules that are smaller than the pores of the membrane will pass through the membrane, forming the filtrate, leaving behind the matter to be collected (retained). With the TFF method, two different pressures are applied; one to pump the solution into the system and circulate it through the system (inlet pressure) and another pressure is applied on the membrane (membrane pressure) to force the small molecules and solvent through the membrane. The inlet pressure can typically be in the range of 1-3 bar, such as between 1.5-2 bar. The transmembrane pressure (TMP) can typically be greater than 1 bar. The concentrated composition of a polypeptide of interest can be collected as a retentate when TFF is used to concentrate the composition. Membranes useful for TFF can typically be made from regenerated cellulose or polyethersulfone (PES).

Diafiltration

In the present context, diafiltration is a filtration process where a species of interest is found in the retentate, that is, it is not allowed to pass through the filter, while other components such as, for example, buffers and salts pass through the filter. Thus, diafiltration can be used, for example, to exchange one buffer for another or to concentrate solutions containing a species of interest, such as recombinant alpha-mannosidase.

A first aspect of the present disclosure is to provide a process for the purification of recombinant alpha-mannosidase from a cell culture, wherein a fraction of said cell culture comprising recombinant alpha-mannosidase is subjected to chromatography on a resin containing a ligand multimode. The advantage of using resins comprising a multimodal ligand in the present context is that these resins allow the binding of alpha-mannosidase species in solutions having high levels of conductivity. This has the advantage that undiluted collected fractions with high levels of conductivity can be used and there is no need to exchange the collected fraction buffer. Therefore, the chromatographic step comprising a multimodal ligand may preferably be the first chromatography step after isolating the fraction from the cell culture. Such a chromatography step comprising a multimodal ligand can usually be referred to as a "capture step", since the protein of interest is initially retained on the column (ie, captured), while various impurities pass through the column during the washing stages. Subsequently, the protein is eluted using a specific elution buffer.

Therefore, in one embodiment of the invention a process is provided wherein the fraction of the cell culture comprising the recombinant human lysosomal alpha-mannosidase is a clarified undiluted collected fraction. In the context of the present invention, the term "clarified undiluted collected fraction" means a fraction collected from a cell culture, which is free of undissolved material or solids, ie it is a clear solution. The collected fraction may have been treated to make it a clear solution. Such treatments may include, but are not limited to: Filtration and centrifugation. Furthermore, the collected fraction is not significantly diluted before being subjected to chromatography steps. Therefore, the collected fraction is diluted less than 10%, such as less than 7%, less than 5%, less than 2%, less than 1%, less than 0.5%, such as less than 0.1%. In the most preferred embodiment, the collected fraction is undiluted.

In another embodiment, a process is provided wherein the clarified undiluted collected fraction has a conductivity of 10-20 mS / cm, such as 12-17 mS / cm, preferably 15 mS / cm. Conductivity is measured before loading the collected fraction into a chromatography system.

In one embodiment, said chromatography is carried out on a resin comprising a multimode ligand having a carboxylic acid or sulfonic acid group. The carboxylic and / or sulfonic acids comprised in these ligands can be in the protonated or deprotonated (salt) form depending on the conditions in the chromatographic system, in particular the pH of the mobile phase.

In yet another embodiment, a process is provided wherein the multimodal ligand bound to the resin is a substance of formula (I), (II) or (III):

where R of the substances of formula (II) and (III) is a functional group of formula (IV):

The multimode ligand represented by the functional group of formula (IV) is generally referred to as "Cibracon Blue 3G" and examples of commercial products represented by the substances of formula (I), (II) and (III) are "Capto ™ MMC "," Capto ™ Blue "and" Blue sepharose ™ fast flow ", respectively. Other useful multimode type resins include: Capto ™ Adhere, MEP HyperCel ™, HEA HyperCel ™ and PPA HyperCel ™. In the context of the present invention, said resins have proven to be especially effective in the initial purification of an undiluted collected fraction comprising recombinant alpha-mannosidase.

A further embodiment of the disclosure provides a process wherein the fraction of said cell culture loaded on the resin comprising a multimode ligand, is subjected to at least one washing step with a solution comprising isopropanol, preferably isopropanol to at least the 1% (V: V), such as isopropanol at least 2%, 3%, 4%, 4.5% (V: V), preferably isopropanol at least 5% (V: V). The advantage of using a solution comprising isopropanol is that it provides better removal of unwanted host cell proteins (HCP), specifically, it helps to remove a protease responsible for proteolytic degradation of the desired 130 kDa rhLAMAN species. HCPs are to be understood as endogenous host cell proteins used in cell culture during production. Although isopropanol is preferred, other useful alcohols for this process include ethanol, n-propanol, and n-butanol.

In yet another embodiment, a process is provided wherein the pH of the solution used for the washing steps is in the range of pH 3.5-6.5, such as pH 4.0-6.0, pH 4 5-5.5, preferably pH 4.7-5.0.

Another embodiment provides a process wherein the solution used for the washing step comprises an acetate buffer, preferably, in a concentration in the range of 0.05-1.6 M, such as 0.1-1.5 M, 0 , 5-1.4 M, 0.7 1.3 M, 0.8-1.2 M, 0.9-1.1 M, preferably 0.95 M. The acetate buffer can preferably be selected from the group that It consists of sodium acetate, potassium acetate, lithium acetate, ammonium acetate.

Yet another embodiment provides a process wherein a first eluate comprising recombinant alpha-mannosidase is eluted from the resin comprising a multimodal ligand using an aqueous solution comprising ethylene glycol or propylene glycol. The addition of ethylene glycol to the elution buffer was found to significantly improve the performance of the eluted recombinant alpha-mannosidase. Propylene glycol also improved performance, but ethylene glycol is preferred.

One embodiment provides a process where the concentration of ethylene glycol or propylene glycol in the aqueous solution is 20-60%, 20-50%, 25-50%, 30-50%, 35-45%, such as 40%.

In a preferred embodiment, a process is provided wherein the aqueous solution comprising ethylene glycol or propylene glycol comprises sodium chloride. The addition of sodium chloride to this solution was found to significantly improve performance by promoting elution of the rhLAMAN enzyme.

In another embodiment, the concentration of sodium chloride in the aqueous solution comprising ethylene glycol or propylene glycol is in the range of 0.2 to 2.4 M, such as in the range of 0.4 to 2.2 M. , 6 to 2.0 M, 0.8 to 1.9 M, 1.0 to 1.8 M, 1.2 to 1.7 M, 1.4 to 1.6 M, preferably 1.5 M Alternatively, the sodium chloride concentration may be in the range of 0.2 to 1.6 M or in the range of 1.4 to 2.4 M.

In a preferred embodiment, the aqueous solution comprising ethylene glycol or propylene glycol comprises a buffer. Said buffer can preferably be a phosphate buffer, such as sodium phosphate or potassium phosphate. Although phosphate buffers are preferred, additional useful buffers for the aqueous solution include citrate and borate buffers, Tris, MES, MOPS, and Hepes buffers.

In another preferred embodiment, the concentration of the buffer salts in the aqueous solution comprising ethylene glycol or propylene glycol is 50-350 mM, 55-300 mM, 65-280 mM, 70-250 mM, 75-200, 80-200 mM , 85-150 mM, preferably 90 mM.

In yet another preferred embodiment, the pH of the aqueous solution comprising ethylene glycol or propylene glycol is pH 7.0-9.0, such as pH 7.1-8.5, pH 7.2-8.3, pH 7.5 -8.0, preferably pH 7.7.

In one embodiment, a process is provided wherein a first eluate comprising alpha-mannosidase obtained from the resin comprising a multimode ligand is further subjected to a process that comprises the stages of:

i) applying a fraction comprising alpha-mannosidase to a hydrophobic interaction chromatography resin to provide an eluate comprising the recombinant alpha-mannosidase,

ii) passing a fraction comprising alpha-mannosidase through a mixed-mode ion exchange resin to allow retention of contaminants to provide a flow-through comprising the recombinant alpha-mannosidase; Y

iii) subjecting a fraction comprising alpha-mannosidase to chromatography on an anion exchange resin to provide an eluate comprising the recombinant alpha-mannosidase.

In one embodiment, a process is provided that involves steps i) -iii) as described above, wherein the fraction in step i) has been subjected to purification on said resin comprising a multimode ligand, the fraction proceeding from stage ii) from the eluate from stage i) and the fraction from stage iii) proceeding from the flow-through of stage ii). In other words, steps i) to iii) are carried out in the order listed, although without ruling out intermediate steps between steps i) to iii). These can be intermediate purification steps and / or virus reduction or virus elimination steps.

In a preferred embodiment, the hydrophobic interaction chromatography resin of step i) is an alkyl substituted resin, preferably butyl sepharose resin. Alkyl substituted resins can include ethyl, butyl, and octyl sepharose resins. In addition, phenyl sepharose resins can also be applied. Examples of such resins are Butyl-S Sepharose ™ 6 Fast Flow, Butyl Sepharose ™ 4 Fast Flow, Octyl Sepharose ™ 4 Fast Flow, Phenyl Sepharose ™ 6 Fast Flow (high substitution) and Phenyl Sepharose ™ 6 Fast Flow (low substitution). , High Performance Butyl Sepharose ™, High Performance Phenyl Sepharose ™. The advantage of a purification step involving hydrophobic interacting resins and in particular butyl sepharose resin is the efficient removal of host cell proteins and DNA debris, while maintaining a good performance of the rhLAMAN enzyme. .

In yet another embodiment, step i) comprises at least one washing step, wherein the solution used for washing comprises a phosphate buffer and an acetate buffer, preferably sodium phosphate and sodium acetate. This dual buffer wash step has proven to be especially effective in removing impurities, such as host cell proteins and DNA debris.

In yet another embodiment, the phosphate buffer concentration in the dual buffer wash of step i) is in the range of 5-40 mM, such as 10-30 mM, 15-25 mM, preferably 20 mM and the concentration of acetate buffer is in the range 0.9-1.5 M, such as 1.0-1.4 M, 1.1-1.3 M, preferably 1.2 M.

In another embodiment, step i) comprises at least one washing step, wherein the solution used for washing comprises no more than one buffer, preferably a phosphate buffer, preferably sodium phosphate.

In another embodiment, the buffer from at least one wash step comprising not more than one buffer is present at a concentration in the range 0.4-0.8 M, such as 0.5-0.7 M, preferably 0.6 M.

In one embodiment, a process is provided wherein the mixed mode ion exchange resin from step ii) is a hydroxyapatite or fluoroapatite ceramic resin, preferably, type I hydroxyapatite (CHT I) ceramic resin. The application of this chromatography step has been shown to efficiently remove a significant amount of DNA impurities from the recombinant alpha-mannosidase composition and bind to host cell proteins while the rhLAMAN enzyme product passes through the column. without joining.

In another embodiment, the anion exchange resin of step iii) is a strong anion exchange resin, such as a quaternary ammonium strong anion exchange resin. Such resins include, but are not limited to, the following examples: Q-sepharose ™ HP, Q-sepharose ™ FF, DEAE-sepharose ™, Capto ™ Q, Uno ™ Q, ANX sepharose ™.

In yet another embodiment, a process is provided wherein a virus inactivation step is carried out, preferably between step ii) and step iii).

In a preferred embodiment, the virus inactivation step comprises mixing the flow-through of step ii) with an aqueous isopropanol solution (1: 1 V / V flow-through / aqueous isopropanol) for at least 2 hours, preferably followed concentration by ultrafiltration and removal of isopropanol using diafiltration. Aqueous isopropanol during inactivation may be in the range of 10-50% isopropanol, such as 20-40%, 25-35%, 28-32%, preferably 30% isopropanol. The 1: 1 V / V flow-through aqueous isopropanol solution therefore has a final isopropanol concentration of 15%.

Another preferred embodiment is a process where a virus reduction step is carried out, preferably after chromatography step iii).

In one embodiment, the virus reduction step comprises filtering a solution comprising recombinant alpha-mannosidase, preferably the eluate from step iii), through a filter, preferably a virus removal filter, such as an ultipor filter. DV20 grade VF ™ or a 15N or 20N planova ™ filter, preferably a 15N Planova ™ filter is used.

The purification process of the present disclosure can advantageously be carried out on a large scale, therefore, in preferred embodiments, the process is carried out in chromatography columns having a column volume of at least 0.5 L, such such as at least 1.0L, 2.0L, 5.0L, 10L, preferably at least 13.0L.

In another embodiment of the present invention, a purification process is provided as described above, wherein the alpha-mannosidase has a sequence selected from:

A) the sequence set forth in SEQ ID NO: 2

B) an analog of the sequence in A

C) a subsequence of the sequence in A) or B)

Where the sequence described by SEQ ID NO: 2 represents the amino acid sequence for recombinant human lysosomal alpha-mannosidase (rhLAMAN) provided in WO 02/099092.

By "subsequence" is meant a fragment of the parental sequence that has a size of not less than 50% of the parental sequence, such as not less than 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or no less than 95% of the parental sequence. Accordingly, the subsequences in question may be 505-1009 consecutive amino acid residues in length, such as 525-1009, 550-1009, 575-1009, 600-1009, 625-1009. 650 1009, 675-1009, 700-1009, 725-1009, 750-1009, 775-1009, 800-1009, 825-1009. 850-1009, 875-1009, 900-1009, 925-1009, 950-1009, 975-1009, 980-1009, 990-1009 or such as 1000-1009 consecutive amino acid residues. Furthermore, the relevant subsequences of SEQ ID NO: 2 or analogs thereof have to conserve the catalytic site. Although the 3D structure of human LAMAN is unknown, the 3D structure of bovine LAMAN has been reported and based on these data, it has been concluded that the following amino acids participate in the active site and / or are responsible to coordinate the Zn2 + atom necessary for activity in human LAMAN: AA 72 = H, AA 74 = D, AA 196 = D, AA 446 = H (UniProtKB / Swiss-Prot Database: 000754, MA2B1_HUMAN_, Heikinheimo et al . J. Mol. Biol. 327, 631-644, 2003). Mutations of AA 72 and 196 in human LAMAN have been shown to result in a virtually complete loss of enzyme activity (Hansen et al., Biochem. J. (2004), 381, pp. 537-567). To show activity, a subsequence of rhLAMAN must conserve at least the regions containing the four amino acids above.

Preferably, the rhLAMAN subsequences also comprise one or more additional conformational parts, including, for example, binding sites, beta turns, disulfide bridges, stop codons, and others. In the human form of LAMAN, there are several disease-causing mutations that indicate importance for that particular amino acid, for example AA 53, 72, 77, 188, 200, 355, 356, 359, 402, 453, 461, 518, 563 , 639, 714, 750, 760, 801, 809, 916 (The human gene mutation database, HMDG® professional, Cardiff University, 2009) and there are also amino acids that are important for glycosylations, including AA 133, 310, 367, 497, 645, 651, 692, 766, 832, 930 and 989 and amino acids involved in disulfide bridges, such as AA 55 + 358, 268 + 273, 412 + 472 and 493 + 501.

By "analog" is meant a sequence with a certain percentage of sequence identity with respect to the parental sequence, this can be a sequence identity of at least 60%, such as at least 70%, 80%, 85%, 90 %, 95%, 98% or preferably 99% sequence identity. It will be understood that the analogs and subsequences set forth above are preferably functionally equivalent to alpha-mannosidase having the amino acid sequence set forth in SEQ ID NO: 2 in the sense that they are capable of exerting substantially the same enzymatic activity.

The term "substantially the same enzyme activity" refers to an equivalent or analog part that is at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 75%. , more preferably, at least 80%, more preferably, at least 85%, more preferably, at least 90%, more preferably, at least 95%, and most preferably, at least 97%, when less 98% or at least 99% of the enzymatic activity of the natural enzyme. An example of a functionally equivalent analog of the enzyme could be a fusion protein that includes the catalytic site of the enzyme in a functional form, but it can also be a homologous variant of the enzyme from another species. Likewise, fully synthetic molecules that mimic the specific enzymatic activity of the relevant enzyme could constitute the "functionally equivalent analogs". Generally, non-human LAMAN analogs cannot be applied in therapy as they can potentially induce antibody formation in the patient and cause disease. However, human analogs could be useful for enzyme replacement therapies, when the mutations are not disease-causing and do not significantly reduce the desired enzyme activity. Examples of such mutations They are: His70Leu, Gln240Arg, Ser250Ala, Leu278Val, Ser282Pro, Thr312Ile, Ala337Arg, Ser413Asn, Ser481Ala, Gln582Glu, Arg741Gly, Thr873Pro (source: http://www.ensembl.org; transcript ID EN000456935). Also, the inventors know that Pro669Leu and Asp402Lys do not cause disease. In general, the person skilled in the art will easily be able to design suitable assays for the determination of enzymatic activity. For LAMAN, a suitable enzyme activity assay is disclosed in WO 02/099092, page 26, lines 8-28. Briefly, the following procedure can be carried out for screening purposes using flat-bottomed 96-well plates: Add 75 μl of 4X Assay Buffer (8 mM p-nitrophenyl-alpha-D-mannopyranoside, 2 mg / ml BSA , 0.4 M Na acetate (pH 4.5) to 75 µl of sample or an appropriate dilution thereof (in 10 mM Tris, pH 7.4 containing 150 mM 10% Superblock NaCl). incubate at 37 ° C for 30 min and stop with 75 μl of Na2CO31.8 M and record the absorbance at 405 nm in a plate reader. 96-well plates are read in a spectrophotometer. Specific activity is defined as pmol of hydrolyzed p-nitrophenyl-alpha-D-mannopyranoside per minute per mg of protein.

As commonly defined, "identity" is defined herein as sequence identity between genes or proteins at the nucleotide or amino acid level, respectively. Thus, in the present context, "sequence identity" is a measure of identity between proteins at the amino acid level and a measure of identity between nucleic acids at the nucleotide level. Protein sequence identity can be determined by comparing the amino acid sequence at a given position in each sequence when the sequences are aligned. Similarly, nucleic acid sequence identity can be determined by comparing the nucleotide sequence at a given position in each sequence when the sequences are aligned.

To determine the percent identity of two amino acid sequences or two nucleic acids, the sequences are aligned in order to make an optimal comparison (for example, gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence to achieve optimal alignment with a second amino acid or nucleic acid sequence). The amino acid or nucleotide residues are then compared at corresponding amino acid positions or nucleotide positions. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e.% identity = # of identical positions / total # of positions (e.g. overlapping positions) x 100) . In one embodiment, the two sequences are the same length.

Sequences can be manually aligned and the number of identical amino acids counted. Alternatively, alignment of two sequences for percent identity determination can be achieved using a mathematical algorithm. This algorithm is incorporated into the NBLAST and XBLAST programs. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, W (wordlength) value = 12, to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, W (wordlength) value = 3 to obtain amino acid sequences homologous to a protein molecule of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used. Alternatively, PSI-Blast can be used to carry out an iterated search, which detects distant relationships between molecules. When using the NBLAST, XBLAST and Gapped BLAST programs, the default parameters of the respective programs can be used. See http://www.ncbi.nlm.nih.gov. Alternatively, sequence identity can be calculated after the sequences have been aligned, for example, using the BLAST program in the EMBL database (www.ncbi.nlm.gov/cgi-bin/BLAST). In general, for alignment, the default settings for, for example, "scoring matrix" and "gap penalty" can be used. In the context of the present invention, the default BLASTN and PSI BLAST settings may be advantageous.

The percent identity between two sequences can be determined using techniques similar to those described above, allowing or not allowing gaps. When calculating percent identity, only exact matches are counted.

Another embodiment of the present disclosure is a composition comprising alpha-mannosidase that can be obtained by the purification process described above.

In a second aspect of the present disclosure, a process for the fed batch or continuous production of recombinant alpha-mannosidase is provided, comprising the following steps:

to. inoculating a production reactor comprising a base medium with cells capable of producing recombinant alpha-mannosidase on day 0, to provide a cell culture;

b. adding a feeding medium to said cell culture at least once from day 1;

c. adjust the temperature of said cell culture to a maximum of 35 ° C, such as 34 ° C, 33 ° C, 32 ° C, preferably a maximum of 31 ° C, either after day 3 or when the density of viable cells is greater than 2.1 MVC / ml, whichever comes first.

In the above process, the inoculation day is defined as day 0 and the next day is day 1 and so on. The initial temperature used from day 0 to the setting described in point c. it is in the range of 36-37 ° C, preferably 36.5 ° C. The aforementioned temperatures are to be understood to be actual measured temperatures, not set points, ie, in the bioreactor configuration used for the present invention, the 31 ° C temperature mentioned above required a 32 ° C temperature set point. Similarly, a temperature of 36.5 ° C requires a temperature set point of 37 ° C.

Suitable host cells for the expression and production of recombinant alpha-mannosidase are derived from multicellular organisms, preferably mammals. In particular, cells used to produce recombinant alpha-mannosidase can be selected from the group consisting of the SV40 transformed monkey kidney line CVI (COS-7); the human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK); Chinese Hamster Ovary Cells / -DHFR (CHO); mouse Sertoli cells (TM4); monkey kidney cells (CV I); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); dog kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2, HB 8065); mouse breast tumor (MMT 060562); TRI cells; MCR 5 cells; FS4 cells; and a human hepatoma line (Hep G2). Human fibroblast or insect cell lines are also available as suitable host cells.

The production of recombinant alpha-mannosidase is obtained using cells transfected with a suitable nucleic acid construct using techniques known to those of skill in the art. In particular, the nucleic acid construct may comprise a nucleic acid sequence selected from the group consisting of: i) the nucleic acid sequence set forth in SEQ ID NO: 1; Y

ii) a nucleic acid sequence encoding a subsequence or analog of the sequence set forth in SEQ ID NO: 2 provided above.

The cells may preferably be a rhLAMAN Chinese Hamster Ovary (CHO) cell line specifically developed for the purpose of producing recombinant enzyme as described in WO 02/099092. A culture of this cell line, DSM ACC2549, which was deposited at DSMZ GmbH, Maschroderweg 1b, D-38124 Braunschweig, Germany can be used for the purpose of patent filing as provided in the Budapest Treaty on June 6, 2002. This cell can be obtained using the expression plasmid pLamanExp1 having the sequence shown in SEQ ID NO: 1.

The process of stages a-c may further comprise the following stage:

d. A process for the purification of recombinant alpha-mannosidase from said cell culture, wherein a fraction of said cell culture comprising recombinant alpha-mannosidase is subjected to chromatography on a resin containing a multimode ligand having a carboxylic acid or sulfonic acid group , as described above.

In yet another embodiment, the cell culture used in the production process is essentially free of any supplements of animal origin, such as cod liver oil supplements. Avoiding the use of such supplements reduces the risk of viral contamination in the final enzyme product.

In a preferred embodiment of the production process, the undiluted collected fraction from fed batch or continuous production has an alpha-mannosidase concentration of at least 0.1 g / l, such as at least 0.2 g / l, 0.3 g / l, 0.4 g / l, preferably at least 0.5 g / l.

In another embodiment, the undiluted collected fraction from the fed batch or continuous production has an enzyme activity in the range of 3-35 U / ml, such as 5-35 U / ml, 7-35 U / ml, preferably, in the range of 10-35 U / ml. It should be noted that, after further optimization processes, an enzyme activity even higher than 35 U / ml can be obtained.

The production process can advantageously be carried out on a large scale. Therefore, in one embodiment, the fed batch or continuous production process is carried out in a volume of at least 30L, such as at least 50L, 75L, 100L, 150L, 200L, preferably, at least 250 l.

In another embodiment of the present invention, a production process is provided as described above, wherein the alpha-mannosidase has a sequence selected from:

A) the sequence set forth in SEQ ID NO: 2

B) an analog of the sequence in A

C) a subsequence of the sequence in A) or B)

Another embodiment of the present disclosure is a composition comprising alpha-mannosidase that can be obtained through the production process described above. In preferred embodiments, said compositions comprise additional active ingredient (API) ingredients, adjuvants, and / or excipients.

In a third aspect, the present disclosure provides a composition comprising purified recombinant alpha-mannosidase, wherein at least 80% of the alpha-mannosidase is present as a 130 kDa glycoprotein.

In a preferred embodiment, the composition comprising purified recombinant alpha-mannosidase is provided, wherein the recombinant alpha-mannosidase remains stable in liquid solution for at least 4 days when stored at 5 ° C or for at least 24 months when stored at -20 ° C.

The currently preferred composition for the buffer solution formulation for the rhLAMAN enzyme product is described below and the stabilities achieved are also listed:

Na2HPO43.50 mM (dibasic sodium phosphate)

NaH2PO40.17 mM (monobasic sodium phosphate)

Glycine 27 mM

Mannitol 250 mM

pH 7.70, 290 mOsm / kg (isotonic solution)

Stability in use: Stable solution 5 ° C - 4 days

20 ° C - 6 hours

-20 ° C -24 months

Freeze-dried 5 ° C - 24 months

In another embodiment of the present disclosure, a composition is provided as described above, wherein the alpha-mannosidase has a sequence selected from:

A) the sequence set forth in SEQ ID NO: 2

B) an analog of the sequence in A

C) a subsequence of the sequence in A) or B)

Another preferred embodiment is the above composition comprising recombinant alpha-mannosidase for use as a medicine.

In a further embodiment, the composition comprising purified recombinant alpha-mannosidase is for use in treating alpha-mannosidosis.

Yet another embodiment is the use of the above composition comprising recombinant alpha-mannosidase for the preparation of a medicament for the treatment of alpha-mannosidosis.

Another embodiment is a method of treating alpha-mannosidosis and / or reducing or alleviating symptoms associated with alpha-mannosidosis, said method comprising a step of administering a composition comprising a purified recombinant alpha-mannosidase as provided above to a subject in need thereof. . It should be noted that the embodiments and features described in the context of one of the aspects of the present invention also apply to other aspects of the invention.

Now, the invention will be described in more detail in the following non-limiting examples.

Examples

Abbreviations used:

CIP: Cleaning in Place

CV: Column Volume

Cv: density of viable cells

DF: Diafiltration

DO: Dissolved oxygen

IPA: Isopropanol

MVC / ml: 106 viable cells / ml

NaPi: Sodium phosphate

NaAc: Sodium acetate

DO: Optical Density

EG: ethylene glycol

TFF: Tangential Flow Filtration

TMP: Transmembrane pressure

UF: Ultrafiltration

Example 1 - Currently Preferred General Purification Procedure

The purification procedure to obtain the optimum yields and purities for alpha-mannosidase is described below in the context of the present invention. Standard conditions are used for regeneration and cleaning of the resin, as prescribed for the individual resin (see also Examples 2-5). The resins used are available from GE Healthcare Life Sciences and BioRad.

• Provide a fraction of a collected fraction from an alpha-mannosidase production and clarify the fraction without significant dilution. Preferably, no dilution is carried out.

• Carry out a capture step that involves column chromatography of the above fraction on a resin that comprises a multimode ligand. These resins are selected from the group consisting of: Capto ™ MMC, Capto ™ Adhere, PlasmidSelect ™ Xtra, Capto ™ Blue, Blue Sepharose ™ Fast Flow resin, MEP HyperCel ™, HEA HyperCel ™ and PPA HyperCel ™. Several washes are carried out at pH 4.5-8.5 and the wash buffers are selected from the group consisting of: sodium acetate, potassium acetate, ammonium acetate, sodium phosphate, potassium phosphate, sulfate sodium, potassium sulfate, ammonium sulfate, MES, MOPS, Hepes, sodium borate, tris-HCl, citrate buffer or combinations thereof (hereinafter group A buffer). However, in at least one wash, the wash solution comprises isopropanol and the pH is between pH 4-6. The elution buffer is selected from a group A buffer and the elution solution comprises ethylene glycol. Elution pH is maintained at pH 7.0-8.5.

• Carry out an intermediate active step that involves column chromatography of a composition comprising alpha-mannosidase on a hydrophobic interaction resin. These resins are selected from the group consisting of: Butyl-S Sepharose ™ 6 Fast Flow, Butyl Sepharose ™ 4 Fast Flow, Octyl Sepharose ™ 4 Fast Flow, Phenyl Sepharose ™ 6 Fast Flow (high substitution) and Phenyl Sepharose ™ 6 Fast Flow (low substitution), High Yield Butyl Sepharose ™, High Yield Phenyl Sepharose ™.

Several washes are carried out at pH 7-8. The wash buffers are selected from group A buffers. The elution buffer is also selected from group A buffers and the elution pH is between pH 7-8.

• Carrying out a passive intermediate step that involves column chromatography of a composition comprising alpha-mannosidase on a mixed-mode ion exchange resin to provide a flow-through. These resins are selected from the group consisting of: type I or II hydroxyapatite ceramic resin (preferably type I) or fluoroapatite. A wash is carried out to provide a flow through at pH 7-8. The wash buffer is selected from group A buffers.

• Carry out a refining step that involves column chromatography of a composition comprising alpha-mannosidase on an anion exchange resin. These resins are selected from the group consisting of: Q-sepharose ™ HP, Q-sepharose ™ FF, DEAE-sepharose ™, Capto ™ Q, Uno ™ Q, ANX sepharose ™.

Several washes are carried out at pH 7-8. Wash buffers are selected from group A buffers and TRIS-HCl buffer. The elution buffer is also selected from group A buffers and the elution pH is kept between pH 7-8.

• Carry out a virus inactivation step by contacting a solution of alpha-mannosidase with isopropanol.

• Carry out a virus elimination step using nanofiltration.

The yields and the proportions of product components for the purified composition comprising alpha-mannosidase according to the present invention are shown in Table 1 below, with a comparison with the previous methods, where:

Method 1 is: The presently preferred method according to the present invention.

Method 2 is: Similar to Method 1, without a refining stage. It also lacks wash stages comprising isopropanol for the capture stage and with fewer washes in the intermediate active stage and is finally without virus inactivation / elimination stages.

Method 3 is: As described in WO 02/099092 (multimode ligands are not used).

See also Example 10 and Figure 7.

Example 2 - Chromatographic capture step using a multimode ligand

The clarified undiluted collected fraction comprising alpha-mannosidase is bound by mixed mode interaction to a multimode ligand type resin, such as Capto ™ MMC as used in this example. Increasing salt and adding ethylene glycol elutes the product. The Capto ™ MMC capacity was 260 U / ml resin. The capture stage was carried out using the following stages:

Regenerate the column with 1-2 column volumes (CV) of 3M NaCl, pH 10-12 at 300 cm / h.

Equilibrate with 5 CV of 50 mM sodium phosphate (NaPi) buffer, 0.15 M NaCl, pH 7.5 at 300 cm / h.

Load the collected clarified undiluted fraction (conductivity ~ 15 mS / cm) at 300 cm / h.

Wash 1: 4 CV of equilibration buffer.

Wash 2: 3 CV of 0.95 M NaAc, 5% isopropanol (v: v), pH 4.9 at 300 cm / hr.

Equilibration Buffer 3: 4 CV wash (until a stable baseline reading, ~ 0.06 Au with 5 mm flow cell) is obtained at 300 cm / hr.

• Elute the product with 6 CV of 1.5 M NaCl, 40% ethylene glycol in 90 mM NaPi, pH 7.7 at a maximum of 120 cm / h. Collection starts when absorbance increases (approximately 10 mAu from new baseline reading). ~ 4 CV is collected.

• Regenerate the column, as in the previous case, with 3 CV, downward flow direction, maximum 120 cm / h. • Apply clean- in-place (CIP) and sanitize, preferably in an upward flow direction, with 3 CV of H2O, 3 CV of 1 M NaOH (~ 60 minutes of contact time), 2-3 CV of phosphate buffer, pH ~ 7.3 CV of 20 mM sodium phosphate 20% ethanol. Store in 20 mM sodium phosphate 20% ethanol.

Table 2 shows an example of a purification scheme. Table 3 summarizes the stage. Figure 2 shows an example of a chromatogram for this step.

Table 2: Capture Step Purification Scheme: Capto ™ MMC packed on a 135 x 2 ml 27 ml XK 16 column continues in the next area

Table 3: Summary of conditions for the Capto ™ MMC capture stage

Example 3 - Active intermediate chromatographic stage using hydrophobic interaction

The product of the capture step comprising alpha-mannosidase binds by hydrophobic interactions after addition of sodium sulfate to hydrophobic interaction type resins, such as Butyl Sepharose ™ 4 FF, as used in this example. Reducing the salt concentration elutes the product. The capacity was 195 U / ml of resin. The following stages were used in the intermediate active stage:

• Regenerate the column with 1 CV of 20 mM sodium phosphate buffer (NaPi), pH 7.5 at 100 cm / h.

• Equilibrate the column with 5 CV of Na2SO40.5 M, NaPi 20 mM, pH 7.5 at 150 cm / h.

• Mix the combined product from step 1 with the same volume of 20 mM NaPi, Na2SO40.8 M, pH 7.5 and load on the column at 70 cm / h. Mixing can be done online or up to 3 hours before charging starts. The 1: 1 volume: volume (v: v) mixture corresponds to approximately 1.11: 1, weight: weight (p: w) (eluate: sodium sulfate buffer). If necessary, the conditioned load should be filtered through a 0.45 µm filter (hydrophilic PES or PVDF) prior to loading.

• Wash the column with 3 CV of equilibration buffer at 70 cm / h to remove the ethylene glycol, in addition to the host cell proteins, from the previous step.

• Wash with 3.5 CV of 20 mM NaPi, 1.2 M NaAc, pH 7.5 at 100 cm / h.

• Wash with 3.5 CV of 0.6 M NaPi, pH 7.0 at 150 cm / h.

• Elute the product with 4 CV of NaPi 60 mM, pH 7.5 at 150 cm / h. The peak is collected from the initial increase in absorbance until the baseline value, ~ 2 CV, is reached.

• Regenerate the column with 2 CV of 20 mM NaPi, pH 7.5 followed by 3 CV of H2O at 150 cm / h.

• Clean and sanitize with 3 CV of 1 M NaOH (60 min contact time), 1 CV of H2O, 1-3 CV of phosphate buffer and 2 CV of 20 mM sodium phosphate 20% ethanol. Store in 20 mM sodium phosphate 20% ethanol.

Table 4 shows an example of a purification scheme. Table 5 summarizes the stage. Figure 3 shows an example of a chromatogram for this step.

Table 4: Intermediate active purification scheme using Butyl Sepharose ™ 4FF packed on a 135 cm x 2 cm2 27 ml XK 16 column continues in the next area.

™

Example 4 - Passive intermediate chromatographic stage using ion exchange in mixed mode

Two eluates comprising alpha-mannosidase from the active intermediate stage were pooled and mixed 1: 1 (weight: weight) with water to reduce conductivity and loaded onto an ion exchange resin in mixed mode, such as in this example, a ceramic resin of hydroxyapatite I (CHT I). The product passes unbound, while the host cell proteins bind to the column. The flow-through containing the product was collected. The capacity was 550 U / ml of resin. In this example the following single stage caps were used passive intermediate:

• Regenerate the column with 2 CV of 0.6 M NaPi pH 7.0 at 300 cm / h.

• Equilibrate the column with 5 CV of 60 mM NaPi, pH 7.5 at 300 cm / h.

• Load the conditioned eluate from the cap 2 to 300 cm / h and collect the flow through, which contains the product. The conductivity and pH of the load will be ~ 10 mS / cm and 7.3, respectively. The flow-through, containing the product, is collected from when there is an increase in OD of 20 mAu until the OD returns to 20 mAu, approximately the volume of loading and washing with 2 CV. At the end of the stage, the pooled product is filtered through a 0.45 pm hydrophilic PES or PVDF filter.

• Wash the column with 4 CV of equilibration buffer at 300 cm / h.

• Regenerate the column with 3 CV of 0.6 M NaPi, pH 7.0 at 300 cm / h.

• Clean and sanitize with 3 CV of 1 M NaOH (60 min contact time), 1 CV of 60 mM NaPi, pH 7.5 and 2 CV of 20 mM sodium phosphate 20% ethanol. Store in 20 mM sodium phosphate 20% ethanol.

Table 6 shows an example of a purification scheme. Table 7 summarizes the stage. Figure 4 shows an example of a chromatogram for this step.

Table 6: Purification scheme for the passive intermediate step using CHT I packed on a 10 cm x 2 cm2 20 iml XK 16 column.

Example 5 - Virus inactivation step

Virus inactivation can be carried out at different stages of the process. In this example, virus inactivation was carried out after the intermediate passive stage and before the refining stage. Virus inactivation of the passive intermediate pool comprising alpha-mannosidase was obtained by incubation for 135 615 min at 21 ± 5 ° C with 15% isopropanol (1: 1 mixture with 30% aqueous isopropanol). The tank can be cooled by means of a cooling jacket to 4 ° C to keep the process at 21 ± 5 ° C. Tangential flow filtration (TFF), with a 100 kDa polyethersulfone membrane, Screen A (from Millipore or Sartorius) was used to remove isopropanol and switch to sodium phosphate buffer. The following steps were used to inactivate viruses in this example:

• Mix the product (flow through) of the passive intermediate stage with 30% isopropanol in 60 mM sodium phosphate, 1: 1 (v: v), which corresponds to 1: 0: 94 (w / w). Mix, for example, by recirculating pumping. The concentration of the protein product will be ~ 0.3-1 mg / ml.

• Incubate the pooled solvent / product mixture at room temperature for 135 ± 15 min.

• Equilibrate the TFF membrane with 60 mM sodium phosphate buffer.

• Concentrate the pooled fraction to a target concentration of 2 mg / ml (0.5-3 mg / ml) by ultrafiltration at a transmembrane pressure (TMP) of 1.1 bar, at 21 ± 5 ° C, inlet pressure = ~ 1.4-1.5 bar and outlet pressure = 0.7 ~ 0.8 bar.

• Exchange ~ 6 volumes by diafiltration against 60 mM sodium phosphate buffer. It starts at a TMP of 1 bar (1.4 bar inlet / 0.6 bar outlet). After the first volume has been exchanged, the TMP can be increased to 1.1.

• Collect the retained fraction. The membrane is rinsed with 2-3 volumes of dilution buffer system to remove weakly bound product. The clarified fraction is collected together with the retained fraction. The final target protein concentration is 2 mg / ml (0.5-3 mg / ml).

• Clean the membrane with H2O, followed by 0.5 M NaOH (60 min contact time). It is stored in 0.1 M NaOH.

Table 10 shows the conditions for the virus / TFF inactivation step.

Example 6 - Chromatographic refining step using anion exchange

The conductivity of the retained fraction comprising alpha-mannosidase from the passive intermediate step was reduced by diluting 6 times with conditioning buffer (20 mM Tris-HCl, 10 mM NaCl, 75 mM mannitol, 0.005% tween ™ 80, pH 7 , 5) to be bonded by ion interaction to an anion exchange resin, such as in this example, a high yield quaternary ammonium strong anion exchange resin (Q Sepharose ™ HP resin). The retained fraction was diluted directly prior to loading or by on-line dilution. The product was eluted, in a vessel pre-filled with 1 CV of elution buffer, by adding sodium chloride. The capacity is 400 U / ml of resin. The following steps were used for the refining step in this example:

Regenerate the column with 1 CV of 50 mM NaPi, 1 M sodium chloride, pH 7.5 at 120 cm / h.

Equilibrate the column with 5 CV of 20 mM Tris-HCl, 10 mM sodium chloride, pH 7.5 at 120 cm / h.

Load the diluted retentate from step 4 at 120 cm / h.

Wash the column with 5 CV of equilibration buffer and 1 CV of 20 mM sodium phosphate, pH 7.5 at 120 cm / h. Elute the product with 4 CV of 50 mM sodium phosphate, 0.2 M sodium chloride, pH 7.5 at 120 cm / h in the pre-filled container (1 CV of elution buffer). The peak is collected from the initial increase (collection starts at 10-20 mAu) in absorbance to 30-50 mAu (2mm flow cell), 0.5-1.5 CV.

• Regenerate the column with 3 CV of 50 mM NaPi, 1 M sodium chloride, pH 7.5 at 120 cm / h.

• Clean and sanitize with 3 CV of 1 M NaOH (60 min contact time) and 3 CV of 10 mM NaOH. It is stored in 10 mM NaOH.

Table 8 shows an example of a purification scheme. Table 9 summarizes the stage. Figure 5 shows an example of a chromatogram.

Table 8: Example purification scheme for the refining step using Q Sepharose ™ HP resin, 19 cm x 2

™

Example 7 - Virus reduction stage

Virus reduction can be done at different stages of the process. In this example, virus reduction was carried out after the refining step. The eluted fraction from the refining stage was nanofiltered, after a previous filtration through a Planova ™ 15N 0.1 pm filter. The following stages were used:

• The eluate from the refining stage was previously filtered through a 0.1 pm filter. The filter was rinsed with a small volume of 50 mM sodium phosphate, 0.2 M sodium chloride, pH 7.5 to remove weakly bound product.

• The eluted fraction is filtered at a pressure of 0.8 bar, at room temperature. Filtration is followed by a subsequent wash of approximately three volumes of the Planova 15 system with 50 mM sodium phosphate, 0.2 M sodium chloride, pH 7.5.

Example 8 - Formulation and storage

Tangential Flow Filtration (TFF), with a 100 kDa polyethersulfone membrane, Screen A (Sartorius ™ or Millipore ™) changes the buffer to formulation buffer. The tank can be cooled by means of a cooling jacket to 4 ° C to keep the process at 21 ± 5 ° C. Estimated capacity is 100 l / m2 The following steps were used for formulation and storage: •

• Equilibrate the membrane with 3.5 mM Na2HPO4, 0.17 mM NaHPO4, 250 mM mannitol, 27 mM glycine, pH 7.7 (formulation buffer).

• Dilute the purified product comprising alpha-mannosidase with approximately 1 volume of formulation buffer to a target concentration of 2-3 mg / ml. In case the protein concentration is low in the product, it is possible (but not necessary) to concentrate up to 4-6 mg / ml to reduce the volume before diluting with formulation buffer.

• Concentrate up to ~ twice to a target concentration of 6 mg / ml by ultrafiltration at a TMP of 0.8 and at 21 ± 5 ° C.

• Exchange 6 volumes by diafiltration against formulation buffer at a TMP of 0.8, 21 ± 5 ° C.

• Concentrate ~ 1.5 times by ultrafiltration and collect the retained fraction. The membrane is rinsed with 1 volume of formulation buffer system to remove weakly bound product. The clarified fraction is collected together with the retained fraction. An alternative is to measure OD 280 in the rinsed fraction and pool only if it contains product. The final target protein concentration is 7 ± 2 mg / ml.

• Clean the membrane with H2O, followed by 0.5 M NaOH (60 min contact time). It is stored in 0.1 M NaOH.

^

The product was diluted to 5 mg / ml and sterilized by filtration. The filtered drug substance is filled into bottles and frozen.

Example 9 - Feed batch culture process for alpha-mannosidase

After the cells were thawed, the cells were expanded in shake flasks, 10 L seed bioreactor, and 50 L seed bioreactor before transferring to a production bioreactor (250 L). On the day of inoculation of the production bioreactor, the cell density in the 50 L seed bioreactor was between 2 and 2.5 MVC / ml. Cells were inoculated into the production reactor from the seed bioreactor at a cell density of 0.5 MVC / ml such that the volume of the cell suspension was 100 µl when the inoculation was complete. The day of inoculation is called day 0, the day after is called day 1, and so on. From day 1 to the end of the cycle, booster feed medium was added daily according to a predefined schedule (see below). From day 1 to the end of the cycle, glutamine and glucose booster were added daily according to predefined guidelines and rules (see below). When the viable cell density was> 2.2 MVC / ml or on day 3, whichever comes first, the temperature was reduced to production temperature.

Table 12: Tem eratures real experimental conditions.

continuation

• Medium base

ACF medium (ExCell302, SAFC) supplemented with 2 mM glutamine and containing 11.1 mM glucose (2 g / l). The base medium without glutamine can be transferred to the production bioreactor for up to 3 days and stored at 36.5 ° C, ie during the sterility test of the bioreactor. Otherwise, the medium is stored at 4 ° C.

• Means of feeding

The feed medium, E35, was CHO CD Efficient Feed B feed concentrate (InVitrogen cat. # SKU # A10240-01) 35% diluted in ACF medium without glutamine and containing 11.1 mM glucose. The feed medium was stored in the dark at 4 ° C. The feed medium can be left in the dark for up to 96 hours, ie four days, at room temperature during cultivation.

Table 13: Volume of feed medium added per day

Days 0 1 to 5 6 to 16

Volume added / day in (l) 0 12 4

• Additives

a) 2500 mM glucose stock solution. b) 200 mM glutamine stock solution. c) Alkali, Na2CO30.5 M. • Supply of the feeding medium, glucose and glutamine

Boost daily feed medium was pumped in. Glucose and glutamine were added daily to maintain their concentrations within the given parameters by adding, as a booster, stock solutions of glucose and glutamine in amounts as discussed below.

Glutamine addition rules:

• Day 1 to day 3: a volume of glutamine stock solution is added in such a way that the glutamine concentration in the bioreactor is 2 mM.

• From day 4: a volume of glutamine stock solution is added in such a way that the glutamine concentration in the bioreactor is 1 mM.

Glucose addition rules:

• Day 0 to day 8: glucose stock solution is not added

• From day 9, in case the glucose concentration in the production bioreactor is <8 mM, a volume of glucose stock solution is added in such a way that the glucose concentration in the bioreactor is 8 mM.

General outline of the process:

Example 10 - Characterization of the purified product comprising alpha-mannosidase

Table 16 below shows how the purification scheme of the present invention provided alphamanosidase with a high proportion of the 130 kDa glycoprotein species, compared to the 75 and 55 kDa degradation products, respectively. It also shows how a 250L process using a 4-stage purification process with improved washing stages for both the capture stage comprising multimode ligands and the active intermediate stage, as well as for a Q Sepharose HP refining stage. outperformed the 30L process using a 3-step process, with respect to yield, overall purity, and yield of the 130kDa species.

-

The distribution of the species is observed in the HPLC diagram in figure 7 for three processes, where those in the upper part represent the processes in 4 stages and 3 stages, respectively. The 2-step process shown is without using a multimode ligand step. The first peak from the left is the 55 kDa species, followed by the 130 kDa and 75 kDa species, respectively.

References

Hirsch et al. EMBO J. 22, 1036-1046, 2003 and Saint-Pol et al. J. Biol. Chem. 274, 13547-13555, 1999

Aronson and Kuranda FASEB J 3: 2615-2622. 1989

Nilssen et al. Hum.Mol.Genet. 6, 717-726. 1997

Kaneda et al. Chromosoma 95: 8-12. 1987

Riise et al. Genomics 42: 200-207, 1997

Nilssen et al. Hum.Mol.Genet. 6, 717-726. 1997

Liao et al. J.Biol.Chem. 271, 28348-28358. nineteen ninety six

Nebes et al. Biochem.Biophys.Res.Commun. 200, 239-245. 1994

Claims (18)

1. A process for the purification of recombinant human lysosomal alpha-mannosidase from a cell culture, wherein a fraction of said cell culture comprising recombinant human lysosomal alpha-mannosidase is subjected to chromatography on a resin comprising a multimode ligand, wherein said resin-bound multimode ligand is a substance having a carboxylic acid or sulfonic acid group, wherein said process results in a composition comprising purified alpha-mannosidase characterized in that at least 80% of the alpha-mannosidase is present as a 130 kDa glycoprotein. 2. A process according to claim 1, wherein the cell culture comprises cells specifically designed to express recombinant alpha-mannosidase, wherein said cells are selected from the group consisting of the SV40-transformed CVI monkey kidney line (COS-7); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK); Chinese Hamster Ovary Cells / -DHFR (CHO); mouse Sertoli cells (TM4); monkey kidney cells (CV I); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); dog kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2, HB 8065); mouse breast tumor (MMT 060562); TRI cells; MRC 5 cells; FS4 cells and a human hepatoma line (Hep G2). 3. A process according to any one of claims 1 or 2, wherein the recombinant alpha-mannosidase is obtained using cells transfected with a nucleic acid construct, and wherein said nucleic acid construct comprises a selected nucleic acid sequence among the group consisting of: i) the nucleic acid sequence set forth in SEQ ID NO: 1; Y ii) a nucleic acid sequence encoding the sequence set forth in SEQ ID NO: 2 or a subsequence or analog of the sequence set forth in SEQ ID NO: 2. 4. A process according to any one of claims 1-3, wherein said fraction of the cell culture comprising recombinant human lysosomal alpha-mannosidase is a clarified undiluted collected fraction. 5. A process according to any of claims 1-4, wherein the multimodal ligand bound to the resin is a substance of formula (I), (II) or (III):

where R of the substances of formula (II) and (III) is a functional group of formula (IV):

6. A process according to any of claims 1-5, wherein a first eluate comprising recombinant human lysosomal alpha-mannosidase is eluted from the resin comprising a multimodal ligand using an aqueous solution comprising ethylene glycol or propylene glycol. 7. A process according to any of claims 1-6, wherein a first eluate comprising recombinant human lysosomal alpha-mannosidase obtained from the resin comprising a multimodal ligand is further subjected to a process comprising the steps of i) applying a fraction comprising recombinant human lysosomal alpha-mannosidase to a hydrophobic interaction chromatography resin to provide an eluate comprising recombinant human lysosomal alpha-mannosidase, ii) passing a fraction comprising recombinant human lysosomal alpha-mannosidase through a mixed-mode ion exchange resin to allow retention of contaminants to provide a through-stream comprising recombinant human lysosomal alpha-mannosidase; Y iii) subjecting a fraction comprising recombinant human lysosomal alpha-mannosidase to chromatography on an anion exchange resin to provide an eluate comprising recombinant human lysosomal alpha-mannosidase. 8. A process according to any of claims 1-7, wherein the recombinant human lysosomal alpha-mannosidase has a sequence selected from: A) the sequence set forth in SEQ ID NO: 2, B) a sequence that has at least 80% sequence identity with SEQ ID NO: 2, C) a subsequence of the sequence in A) or B). 9. A composition comprising purified recombinant lysosomal alpha-mannosidase obtainable by the purification process according to claim 8, wherein at least 80% of the alpha-mannosidase is present as a 130 kDa glycoprotein. 10. A process for the fed batch or continuous production of recombinant human lysosomal alpha-mannosidase, comprising the following steps: to. inoculating a production reactor comprising a base medium with cells capable of producing recombinant human lysosomal alpha-mannosidase on day 0, to provide a cell culture; b. adding a feeding medium to said cell culture at least once from day 1; c. adjusting the temperature of said cell culture to a maximum of 35 ° C, either after day 3 or when the density of viable cells is greater than 2.1 MVC / ml, whichever comes first; d. a purification process according to any of claims 1-8. A process according to claim 10, wherein the cell culture is essentially devoid of any supplements of animal origin, such as cod liver oil supplements. 12. A process according to any of claims 10-11, wherein the cells are selected from the group consisting of the SV40 transformed monkey kidney line CVI (COS-7); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK); Chinese Hamster Ovary Cells / -DHFR (CHO); mouse Sertoli cells (TM4); monkey kidney cells (CV I); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); dog kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2, HB 8065); mouse breast tumor (MMT 060562); TRI cells; MRC 5 cells; FS4 cells and a human hepatoma line (Hep G2). 13. The process according to claim 12, wherein step d) is a purification process as defined in claim 7. 14. A composition comprising alpha-mannosidase obtainable by the production process of according to claim 13, wherein at least 80 % of the alpha-mannosidase is present as a 130 kDa glycoprotein. 15. A composition according to claim 9 or claim 14, wherein the recombinant alpha-mannosidase remains stable in liquid solution for at least 4 days when stored at 5 ° C or for at least 24 months when stored at - 20 ° C. 16. A composition according to any of claims 9, 14 or 15, wherein the alpha-mannosidase has a sequence selected from: A) the sequence set forth in SEQ ID NO: 2, B) a sequence that has at least 80% sequence identity with SEQ ID NO: 2, C) a subsequence of the sequence in A) or B). 17. The composition according to any of claims 9 and 14-16 for use as a medicine. 18. The composition according to any of claims 9 and 14-16 for use in the treatment of alpha-mannosidosis.