WO2019060837A1 - Procédé de production de céramidase acide humaine recombinante - Google Patents

Procédé de production de céramidase acide humaine recombinante Download PDF

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WO2019060837A1
WO2019060837A1 PCT/US2018/052463 US2018052463W WO2019060837A1 WO 2019060837 A1 WO2019060837 A1 WO 2019060837A1 US 2018052463 W US2018052463 W US 2018052463W WO 2019060837 A1 WO2019060837 A1 WO 2019060837A1
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disease
cells
subject
capto
disorder
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PCT/US2018/052463
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Alexander Standish TRACY
Patrick Guertin
Steve TURBAYNE
Amy BELLIVEAU
Chris Mccoy
Ed HINES
Brian Bergeron
Joe MAKOWIECKI
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Enzyvant Farber Gmbh
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/32—Bonded phase chromatography
    • B01D15/325—Reversed phase
    • B01D15/327—Reversed phase with hydrophobic interaction
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
    • B01D15/1864—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using two or more columns
    • B01D15/1871—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using two or more columns placed in series
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • B01D15/361—Ion-exchange
    • B01D15/362—Cation-exchange
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • B01D15/361—Ion-exchange
    • B01D15/363—Anion-exchange
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types

Definitions

  • Farber disease is a rare autosomal recessive lysosomal storage disease where deficiency in acid ceramidase (AC) enzyme results in accumulation of sphingolipids. This results in painful abnormalities, such as painful nodules, in the joints, liver, throat, and various tissues, as well as central nervous system (CNS) symptoms. Mutations in the AC enzyme have been identified in patients with Farber disease (Koch et al. J. Biol. Chem. 271 : 33110-33115 (1996)). Currently, the only treatments available for Farber disease are pain management with corticosteroids and bone marrow transplantation. Enzyme replacement therapy with recombinant human AC (rhAC) has been proposed as an alternative to bone marrow transplantation.
  • AC acid ceramidase
  • AC The activity of AC is regulated by cleavage of the inactive precursor polypeptide into the active enzyme consisting of an alpha and beta subunit linked via disulfide bonds (Shtraizent et al., /. Biol. Chem. 283: 11253-11259 (2008)).
  • Recombinant AC produced in Chinese Hamster ovary ("CHO") cells and secreted into the media is a mixture of inactive precursor and active (cleaved) enzyme (He et al., /. Biol. Chem. 278: 32978-32986 (2003)).
  • the purification process to obtain rhAC may have a large effect on the amount of functional protein obtained based on the relative presence of active and inactive AC.
  • compositions that comprises a combination of inactive and active rhAC, where the composition does not comprise acid sphingomyelinase (ASM) activity (US 2016/0038574).
  • ASM acid sphingomyelinase
  • This application describes use of heat inactivation to remove ASM activity from the preparation.
  • the processes described in this application may not be ideal for the preparation of protein for human therapeutic use, e.g., because the process lacked a viral removal step and utilized columns that could potentially leach toxic substances into the end product. As such, improved processes for purification of AC are needed.
  • Described herein is a process for purification of rhAC.
  • this purification process does not require heat inactivation of a co-purifying protein.
  • Described herein is a method for purifying recombinantly produced acid ceramidase, comprising subjecting the recombinantly produced acid ceramidase to at least two chromatography steps selected from i) cation exchange chromatography; ii) hydrophobic interaction chromatography (HIC); and iii) anion exchange chromatography; and subjecting the recombinantly produced acid ceramidase to one or more viral inactivation steps, thereby obtaining a purified recombinantly produced acid ceramidase.
  • chromatography steps selected from i) cation exchange chromatography; ii) hydrophobic interaction chromatography (HIC); and iii) anion exchange chromatography
  • the recombinantly produced acid ceramidase is subjected to each of cation exchange chromatography, hydrophobic interaction
  • cation exchange chromatography comprises CaptoS ImpAct.
  • hydrophobic interaction chromatography comprises Capto Butyl HIC.
  • anion exchange chromatography comprises Capto Q.
  • the one or more viral inactivation steps comprises contacting the recombinantly produced acid ceramidase with citric acid.
  • the recombinantly produced acid ceramidase is titrated to pH 3.7 with citric acid.
  • the one or more viral inactivation steps is conducted before one of the at least two chromatography steps. In some embodiments, the one or more viral inactivation steps is conducted before at least two of the at least two chromatography steps. In some embodiments, the one or more viral inactivation steps is conducted after the at least two chromatography steps. [0011] In some embodiments, the method further comprises one or more filtration steps. In some embodiments, the one or more filtration steps comprises viral filtration. In some embodiments, the viral filtration comprises tangential flow filtration.
  • the purified recombinantly produced acid ceramidase has a purity of at least 90%, 93%, 95%, 98%, or 99%, or a purity of 100%.
  • the purified recombinantly produced acid ceramidase has no detectable acid sphingomyelinase activity.
  • Also described herein is a purified recombinantly produced acid ceramidase produced by any of the methods described herein.
  • composition comprising the purified recombinantly produced acid ceramidase produced by any of the methods described herein.
  • the therapeutic composition further comprises a pharmaceutically acceptable carrier.
  • the therapeutic composition further comprises one or more pharmaceutically acceptable adjuvants, excipients, or stabilizers.
  • the one or more pharmaceutically acceptable adjuvants, excipients, or stabilizers comprise one or more of trisodium citrate, citric acid, human serum albumin, mannitol, sodium phosphate monobasic , sodium phosphate dibasic, polysorbate, sodium chloride, histidine, sucrose, trehalose, glycine , and water.
  • the salts are hydrates (e.g., trisodium citrate dihydrate, citric acid monohydrate, sodium phosphate monobasic monohydrate, and/or sodium phosphate dibasic heptahydrate).
  • the therapeutic composition further comprises one or more additional agents that reduce ceramide levels.
  • Also described herein is a method of treatment of a disease or disorder associated with reduced or absent acid ceramidase, comprising administering an effective amount of any of the therapeutic compositions described herein in vivo to a subject in need thereof or in vitro to a population of cells.
  • the therapeutic compositions described herein in vivo to a subject in need thereof or in vitro to a population of cells.
  • composition is administered in vivo to a subject in need thereof.
  • therapeutic composition is administered in vitro to a population of cells.
  • the method of treatment further comprises: selecting a population of cells having the potential to differentiate into chondrocytes; and treating the selected cell population of cells with an effective amount of the therapeutic composition to transform one or more cells in the selected population into chondrocytes.
  • the selected cell population comprises mammalian cells.
  • the selected cell population comprises bone marrow cells, stem cells, and/or fibroblasts.
  • the stem cells are mesenchymal stem cells.
  • the subject has a joint disease or disorder, a neurodegenerative disease or disorder, a cardiac disease or disorder, diabetes, a pathogenic infection in combination with cystic fibrosis, COPD, and/or an open wound, a ceramide accumulation infection, or Farber disease.
  • the subject has a joint disease or disorder.
  • the joint disease or disorder comprises osteoarthritis, rheumatoid arthritis, mucopolysaccharidosis, degenerative joint disease, joint injury, or Farber lipogranulomatosis.
  • the subject has a neurodegenerative disease or disorder.
  • the neurodegenerative disease or disorder is selected from the group consisting of Alzheimer's disease, Frontotemporal Dementia, Dementia with Lewy Bodies, Prion disease, Parkinson's disease, Huntington's disease, Progressive Supranuclear Palsy, Corticobasal Degeneration, Multiple System Atrophy, amyotrophic lateral sclerosis (ALS), inclusion body myositis, degenerative myopathy, spinocerebellar atrophy, metabolic neuropathy, diabetic neuropathy, endocrine neuropathy, orthostatic hypotension, brain injury, spinal cord injury, stroke, or a motor neuron disease.
  • motor neuron disease is spinal muscular atrophy.
  • the subject has a cardiac disease or disorder.
  • the cardiac disease or disorder comprises heart disease, cardiac injury, atherosclerosis, thrombosis, or cardiomyocyte apoptosis.
  • the subject has diabetes.
  • the subject has a pathogenic infection in a subject having cystic fibrosis, COPD, and/or an open wound.
  • the pathogenic infection comprises a viral, fungal, prionic, or bacterial infection.
  • subject has a ceramide accumulation infection.
  • the subject has Farber disease.
  • the method of treatment further comprises
  • an antipyretic, an antihistamine, a corticosteroid or any combination thereof prior to, concurrently with, or after administration of the composition.
  • the therapeutic composition is administered orally, by inhalation, by intranasal instillation, topically, transdermally, parenterally, subcutaneously, by intravenous injection, by intra- arterial injection, by intramuscular injection, intraplurally, intraperitoneally, intrathecally, or by application to a mucous membrane.
  • the method of treatment further comprises one or more repeat administrations of the therapeutic composition.
  • the method of treatment further comprises
  • FIG. 1 shows a flow diagram of one embodiment of the purification process.
  • FIG. 2 shows the CaptoS ImpAct elution process chromatogram.
  • UV131 refers to ultraviolet absorption, indicating the presence of protein.
  • Cond_101 refers to the conductivity, which will be affected by solutions of different salt concentrations running through the column.
  • pH_121 refers to pH measurement.
  • FIG. 3 shows the Capto Butyl elution process chromatogram.
  • FIG. 4 shows the Capto Q flow through process chromatogram.
  • FIG. 5 shows AC activity ("act") in the process steps of the third 10L run. Post
  • FIGs. 6a and 6b show the viable cell density of different clones (6A) and product titer (6B) tested under 4 different conditions.
  • Cell boost 7a was always 10X of cell boost 7b.
  • the conditions were as follows: Condition 1: Clone 47 - Ratio of feeds 5%, 0.5% Cell Boost 7a & b (HyClone); Condition 2: Clone 09 - Ratio of feeds 5%, 0.5% Cell Boost 7a & b; Condition 3: Clone 77 - Ratio of feeds 5%, 0.5% Cell Boost 7a & b; and Condition 4: Clone 47 - Repeat (1) with different feed ratio 4%, 0.4% Cell Boost 7a & b.
  • FIGs. 7a and 7b show the viable cell density of different clones (7 A) and product titer (7B) tested under 6 different fed-batch conditions with three 125mL shake flasks per condition.
  • the conditions were as follows: Condition 1: Cell BoostTM Feeds 7a and 7b, with 5%: 0.5% bolus additions; Condition 2: Efficient Feed BTM with 5% bolus additions; Condition 3: Cell Boost Feed 5, with 5% bolus additions; Condition 4: Cell Boost 6, with 5% bolus additions; Condition 5: Repeat (1) Cell Boost Feeds 7a and 7b, with 5%:0.5% bolus additions and temperature reduction on Day 4; and Condition 6: Cell Boost Feeds 7a and 7b, with 4%:0.4% bolus additions.
  • FIG. 8 shows ammonia concentrations for the different conditions described for FIGs. 7 A and 7B.
  • FIGs. 9A and 9B show viable cell density (FIG. 9A) and product titer (FIG.
  • FIGs. 10A and 10B show viable cell density (FIG. 10A) and product titer
  • FIG. 10B for different culture runs. Runs were as follows: 10L: engineering run XDR 10; 200L: engineering run XDR 200; cGMP 200L: first cGMP XDR 200 run; and cGMP200L2: second cGMP XDR 200 run.
  • FIG. 11 shows effect of excipients on AC function.
  • PBS rhAC in PBS; PBS
  • FIG. 12 shows effects of excipients of Fig. 11 on AC function over time when measured at 4°C (@4C) or at room temperature (@RT). DESCRIPTION OF THE SEQUENCES
  • Table 1 provides a listing of certain sequences referenced herein.
  • any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Ranges are approximate and may vary by more than an integer.
  • SI Unites
  • acid ceramidase or “AC” refers to the protein encoded by the ASAH1 gene (NCBI UniGene GenelD No. 427). AC hydrolyzes the amide bond linking the sphingosine and fatty acid moieties of the lipid ceramide (Park and Schuchman, Biochim. Biophys. Acta. 1758(12): 2133-2138 (2006); Nikolova-Karakashian et al., Methods Enzymol. 311: 194-201 (2000); Hassler et al., Adv. Lipid Res. 26:49-57 (1993)). Mutations of both ASAH1 alleles can lead to Farber' s disease.
  • AC (N-acylsphingosine deacylase, I.U.B.M.B. Enzyme No. EC 3.5.1.23) protein has been purified from several sources, and the human and mouse cDNAs and genes have been obtained. See Bernardo et al., /. Biol. Chem. 270: 11098-102 (1995); Koch et al., /. Biol. Chem. 2711:33110-5 (1996); Li et al., Genomics 50:267-74 (1998); Li et al., Genomics 62:223-31 (1999). It is produced through cleavage of the AC precursor protein (see Ferlinz et al., /. Biol. Chem.
  • the AC alpha subunit begins at the amino acid at position 22 and continues through position 142 (as shown in bold in SEQ ID NO: 1 in the Table of Sequences), while the beta subunit of the AC begins with the amino acid at position 143 and continues through position 395 (as shown in italics in SEQ ID NO: 1).
  • active acid ceramidase or “active AC” refers to AC precursor proteins that has undergone autoproteolytic cleavage into the active form
  • inactive acid ceramidase As used herein, “inactive acid ceramidase,” “inactive AC,” or “inactive acid ceramidase precursor,” “inactive AC precursor,” or (AC preprotein) refers to AC precursor protein that has not undergone autoproteolytic cleavage into the active form.
  • Inactive AC precursors and active ACs suitable for use in the recombinant acid ceramidase of this and all aspects of the present invention can be homologous (i.e., derived from the same species) or heterologous (i.e., derived from a different species) to the tissue, cells, and/or subject being treated.
  • Acid ceramidase (e.g., AC) precursor proteins undergo autoproteolytic cleavage into the active form (composed of a- and ⁇ -subunits). The mechanism of human AC cleavage and activation is reported in Shtraizent et al., /. Biol. Chem. 283(17): 11253-11259 (2008)).
  • ceramidase as used herein includes both active ceramidases and ceramidase precursor proteins, where ceramidase precursor proteins are converted into active ceramidase proteins through autoproteolytic cleavage.
  • the precursor protein is taken up by the cell of interest and converted into active ceramidase thereby, as well as embodiments in which the precursor protein is converted into active ceramidase by a different cell or agent (present, for example, in a culture medium), are both contemplated.
  • Active ACs and inactive AC precursor proteins that can be used in this and all aspects of the present invention include, without limitation, those set forth in Table 1 of US 2016/0038574.
  • the recombinant acid ceramidase of the therapeutic composition may, in some embodiments, contain a greater amount of the inactive AC precursor than active AC.
  • the purified recombinant acid ceramidase of the therapeutic composition may, in some instances, contain a lesser amount of inactive AC precursor than active AC.
  • the amount of the inactive AC precursor compared to the active AC in the mixture ranges from 5 to 95 wt % of the inactive AC precursor and 95 to wt % of the active AC; 20 to 80 wt % of the inactive AC precursor and 80 to 20 wt % of the active AC; 30 to 70 wt % of the inactive AC precursor and 70 to 30 wt % of the active AC; 40 to 60 wt % of the inactive AC precursor and 60 to 40 wt % of the active AC; 55 to 95 wt % of the inactive AC precursor and 45 to 5 wt % of the active AC; or 70 to 95 wt % of the inactive AC precursor and 30 to 5 wt % of the active AC; and may alternatively range from 80 to 90 wt % of the in
  • the amount of the inactive AC precursor is 90 wt % while the active ceramidase is 10 wt % of the mixture.
  • An alternative embodiment may include 80 wt % of the inactive ceramidase precursor and 20 wt % of the active AC in the purified recombinant acid ceramidase.
  • the purified recombinant acid ceramidase may contain 60 wt % inactive ceramidase precursor and 40 wt % active ceramidase.
  • the composition of the invention contains more of the active AC, such as at least 90%, 80%, 70%, 60% or more than 50% is active AC.
  • rhAC recombinant human acid ceramidase
  • ASM activity refers to a related lipid hydrolase that tightly binds to AC and co-purifies with it (Bernardo et al., /. Biol. Chem. 270: 11098-11102 (1995)).
  • production refers to the production and purification of a biotherapeutic. Production can encompass
  • chromatography refers to any laboratory technique for separation of mixtures.
  • anion exchange chromatography or "AIEX” refers to a process that separates substances based on their charges using an ion-exchange resin containing positively charged groups. Capto Q is an exemplary AIEX media.
  • CIEX ation exchange chromatography
  • Capto S Imp Act is an exemplary CIEX media.
  • hydrophobic interaction chromatography or “HIC” refers to a process that separates substances based on their hydrophobicity.
  • Capto Butyl HIC is an exemplary HIC media.
  • Ceramide accumulation inflammation or "ceramide accumulation infection” refers to inflammation or infection caused by higher than normal levels of ceramide. Ceramide accumulation inflammation or infection may occur based on changes in pH that result in an imbalance between ASM cleavage of sphingomyelin to ceramide and AC consumption of ceramide. Ceramide accumulation inflammation or infection has been described in models of cystic fibrosis and may lead to pulmonary inflammation, respiratory epithelial cell death, DNA deposits in bronchi, and increased susceptibility to Pseudomonas aeruginosa infections (see Teichgraber, V. et al., Nature Medicine 14:382-391 (2008)).
  • the therapeutic composition may also include pharmaceutically acceptable adjuvants, excipients, and/or stabilizers, and can be in solid or liquid form, such as tablets, capsules, powders, solutions, suspensions, or emulsions.
  • additional pharmaceutically acceptable ingredients have been used in a variety of enzyme replacement therapy compositions and include, without limitation, trisodium citrate, citric acid, human serum albumin, mannitol, sodium phosphate monobasic, sodium phosphate dibasic, polysorbate, sodium chloride, histidine, sucrose, trehalose, glycine, and/or water for injections.
  • the salts are hydrates (e.g., trisodium citrate dihydrate, citric acid
  • a second aspect of the present invention relates to a method of AC treatment, including formulating the AC used in said treatment as a purified recombinant acid ceramidase, where the purified recombinant acid ceramidase includes an inactive AC precursor and an active AC.
  • Treatment according to this aspect of the present invention is carried out using methods that will be apparent to the skilled artisan.
  • AC for a discussion of AC in the context of human disease, see Park et al., Biochim. Phiophys. Act. 1758:2133-2138 (2006) and Zeidan et al., Curr. Drug Targets 9(8):653-661 (2008)).
  • treatment with the human recombinant AC produced by the methods of the present invention may be accompanied with a pre-treatment, or treatment with an antipyretic, antihistamine and/or corticosteroid to reduce occurrence of adverse effects.
  • treatment is carried out by introducing a ceramidase protein into the cells.
  • proteins or polypeptide agents e.g., active ceramidase, inactive ceramidase precursor proteins
  • conjugation of the desired protein or polypeptide to a polymer that is stabilized to avoid enzymatic degradation of the conjugated protein or polypeptide. Conjugated proteins or polypeptides of this type are described in U.S. Pat. No. 5,681,811 to Ekwuribe.
  • the chimeric protein can include a ligand domain and the polypeptide agent (e.g., rAC, active AC, other ceramidase, inactive AC precursor protein, other ceramidase precursor proteins).
  • the ligand domain is specific for receptors located on a target cell.
  • Further embodiments of the present aspect relate to methods of treatment for a certain disease or disorder.
  • Such methods comprise administering to the patient the purified recombinant AC produced by the methods of the invention ("purified recombinant AC").
  • the recombinant AC purified by the methods of the invention maybe formulated into a powder or cake to be dissolved and administered as an injection or an infusion or maybe formulated directly as a liquid composition.
  • the composition may also be formulated into an inhaled formulation.
  • the disease or disorder is a joint disease or disorder and the purified recombinant AC according to the methods of the present invention is administered to a subject to treat the subject for the joint disease or disorder.
  • exemplary types of joint disease or disorders include, without limitation, osteoarthritis, rheumatoid arthritis, mucopolysaccharidosis, degenerative joint disease, joint injury, and Farber
  • the disease or disorder is a neurodegenerative disease or disorder and the purified recombinant AC according to the methods of the present invention is administered to a subject to treat the subject for the neurodegenerative disease or disorder.
  • neurodegenerative diseases or disorders include, without limitation, Alzheimer's disease, Frontotemporal Dementia, Dementia with Lewy Bodies, Prion disease, Parkinson's disease, Huntington's disease, Progressive Supranuclear Palsy, Corticobasal Degeneration, Multiple System Atrophy, amyotrophic lateral sclerosis, inclusion body myositis, degenerative myopathy, spinocerebellar atrophy, metabolic neuropathy, diabetic neuropathy, endocrine neuropathy, orthostatic hypotension, brain injury, spinal cord injury, stroke, and motor neuron diseases such as spinal muscular atrophy.
  • the disease or disorder is a cardiac disease or disorder and the purified recombinant AC according to the methods of the present invention is administered to a subject to treat the subject for the cardiac disease or disorder.
  • cardiac diseases or disorders include, without limitation, heart disease, cardiac injury, atherosclerosis, thrombosis, cardiomyocyte apoptosis, hypercardia, heart infarction, mitral regurgitation, aortic regurgitation, septal defect, and tachycardia-bradycardia syndrome.
  • the disease or disorder is diabetes and the purified recombinant AC according to the methods of the present invention is administered to a subject to treat the subject for diabetes.
  • the disease or disorder is a pathogenic infection in a subject having cystic fibrosis, chronic obstructive pulmonary disease (COPD), and/or an open wound, and the purified recombinant AC according to the methods of the present invention is administered to a subject to treat the subject for the pathogenic infection.
  • pathogenic infections include, without limitation, viral, fungal, prionic, and bacterial.
  • Subjects suffering from cystic fibrosis, COPD, and/or an open wound may possess a high susceptibility for acquiring acute and/or chronic pathogenic infections, such as, e.g., bacterial, viral, fungal, protozoan, and/or prionic pathogenic infections.
  • Bacterial pathogens include, without limitation, Bacillus anthracis, Bordetella pertussis, Borrelia burgdorferi, Campylobacter jejuni, Chlamydia trachomatis, Clostridium botulinum,
  • Mycobacterium leprae Mycobacterium tuberculosis, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus spp., Pseudomonas aeruginosa, Rickettsia, Salmonella spp., Shigella spp., Staphylococcus spp., Staphylococcus aureus, Streptococcus spp., Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus B, Group A beta hemolytic
  • the pathogenic infection is a Pseudomonas infection.
  • Viral pathogens include, without limitation, RNA viruses, DNA viruses, adenovirdiae (e.g., mastadeno virus and aviadeno virus), herpesviridae (e.g., herpes simplex virus 1, herpes simplex virus 2, herpes simplex virus 5, and herpes simplex virus 6), leviviridae (e.g., levivirus, enterobacteria phage MS2, allolevirus), poxyiridae (e.g., chordopoxyirinae, parapoxvirus, avipoxvirus, capripoxvirus, leporipoxvirus, suipoxvirus, molluscipox virus, and entomopoxyirinae), papovaviridae (e.g., polyomavirus and papillomavirus), paramyxoviridae (e.g., paramyxovirus, parainfluenza virus 1, mobill
  • immunodeficiency virus 1 and human immunodeficiency virus 2; and spuma virus flaviviridae (e.g., hepatitis C virus), hepadnaviridae (e.g., hepatitis B virus), togaviridae (e.g., alphavirus-such as Sindbis virus and rubivirus, such as rubella virus), rhabdoviridae (e.g., vesiculovirus, lyssavirus, ephemera virus, cytorhabdovirus, and necleorhabdovirus), arenaviridae (e.g., arenavirus, lymphocytic choriomeningitis virus, Ippy virus, and lassa virus), and coronaviridae (e.g., coronavirus and toro virus), Cytomegalovirus
  • flaviviridae e.g., hepatitis C virus
  • hepadnaviridae e.g.,
  • Dengue virus Dengue fever, shock syndrome
  • Poliovirus paralysis
  • Rhinovirus common cold
  • Rubella virus fetal malformations
  • Vaccinia virus generalized infection
  • Yellow fever virus jaundice, renal and hepatic failure
  • Varicella zoster virus chickenpox
  • Pathogenic fungi include, without limitation, the genera Aspergillus (e.g., Bacillus subtilis, Bacillus, Bacillus, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus, Bacillus, Bacillus, Bacillus, Bacillus, Bacillus, Bacillus, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus
  • Pathogenic protozoan include, without limitation, Trypanosome spp., Leishmania spp., Plasmodium spp., Entamoeba spp., and Giardia spp., such as Giardia lamblia.
  • an "open wound” refers to a type of injury in which an epithelial layer, i.e., skin, is torn, cut, and/or punctured.
  • an open wound refers to a sharp injury which damages the dermis of the skin and concomitantly increases the chance of acquiring an infection.
  • the term "open wound” also encompasses burns.
  • the disease or disorder is an infection caused by ceramide accumulation and the purified recombinant AC according to the methods of the present invention is administered to a subject to treat the subject for the ceramide
  • the present invention may, in other embodiments, be used to treat Farber disease in a subject, such as a human subject, with the purified recombinant AC of the invention.
  • Mammalian subjects include, for example, human subjects, rodent subjects, equine subjects, porcine subjects, feline subjects, and canine subjects.
  • any combination of active ceramidase, ceramidase precursor protein, and/or nucleic acid encoding ceramidase/ceramidase precursor protein can be administered. Administration can be accomplished either via systemic administration to the subject or via targeted administration to affected tissues, organs, and/or cells.
  • the purified recombinant AC may be administered to a non-targeted area along with one or more agents that facilitate migration of the purified recombinant AC to (and/or uptake by) a targeted tissue, organ, or cell.
  • the purified recombinant AC itself can be modified to facilitate its transport to (and uptake by) the desired tissue, organ, or cell, as will be apparent to one of ordinary skill in the art.
  • the purified recombinant AC will be administered to a subject in a vehicle that delivers the ceramidase to the target cell, tissue, or organ.
  • routes of administration include, without limitation, orally, by inhalation, intratracheal inoculation, aspiration, airway instillation, aerosolization, nebulization, intranasal instillation, oral or nasogastric instillation, intraperitoneal injection, intravascular injection, topically, transdermally, parenterally, subcutaneously, intravenous injection, intra-arterial injection (such as via the pulmonary artery), intramuscular injection, intrapleural instillation, intraventricularly, intralesionally, intrathecally, by application to mucous membranes (such as that of the nose, throat, bronchial tubes, genitals, and/or anus), or implantation of a sustained release vehicle.
  • the purified recombinant AC is administered orally, topically, intranasally, intraperitoneally, intravenously, subcutaneously, or by aerosol inhalation. In some embodiments, the purified recombinant AC is administered via aerosol inhalation. In some embodiments, the purified recombinant AC can be incorporated into pharmaceutical compositions suitable for administration, as described herein.
  • the purified recombinant AC may be orally administered, for example, with an inert diluent, or with an assimilable edible carrier, or they may be enclosed in hard or soft shell capsules, or they may be compressed into tablets, or may be incorporated directly with the food of the diet.
  • the purified recombinant AC may be incorporated with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like.
  • Such compositions and preparations should contain at least 0.1% of ceramidase.
  • the percentage of purified recombinant AC in these compositions may, of course, be varied and may conveniently be between 2% to 60% of the weight of the unit.
  • the amount of the purified recombinant AC in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • the tablets, capsules, and the like may also contain a binder such as gum tragacanth, acacia, corn starch, or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, or alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose, or saccharin.
  • a binder such as gum tragacanth, acacia, corn starch, or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, or alginic acid
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose, or saccharin.
  • a liquid carrier such as fatty oil.
  • the purified recombinant AC may also be administered parenterally.
  • Solutions or suspensions of ceramidase can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils.
  • Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil.
  • Liquid carriers include, but are not limited to, water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycol or polyethylene glycol, for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
  • the purified recombinant AC may also be administered directly to the airways in the form of an aerosol.
  • ceramidase in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
  • suitable propellants for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
  • the purified recombinant AC may also be administered in a non-pressurized form.
  • Exemplary delivery devices include, without limitation, nebulizers, atomizers, liposomes (including both active and passive drug delivery techniques) (Wang et al., Proc. Nat'l Acad. Set USA 84:7851-5 (1987); Bangham et al., /. Mol. Biol. 13:238-52 (1965); U.S. Pat. No. 5,653,996 to Hsu; U.S. Pat. No. 5,643,599 to Lee et al.; U.S. Pat. No. 5,885,613 to Holland et al.; U.S. Pat. No. 5,631,237 to Dzau et al.; and U.S. Pat. No.
  • Administration can be carried out as frequently as required and for a duration that is suitable to provide effective treatment.
  • administration can be carried out with a single sustained-release dosage formulation or with multiple daily doses.
  • Treatment according to this and all aspects of the present invention may be carried out in vitro or in vivo.
  • In vivo treatments include, for example, embodiments in which the population of cells is present in a mammalian subject.
  • the population of cells can be either autologous (produced by the subject), homologous, or heterologous.
  • Suitable subjects according to these embodiments include mammals, e.g., human subjects, equine subjects, porcine subjects, feline subjects, and canine subjects.
  • one or more additional agents which reduce ceramide levels may be administered with the purified recombinant AC.
  • the effective amount of a therapeutic agent/cell population of the present invention administered to the subject will depend on the type and severity of the disease or disorder and on the characteristics of the individual, such as general health, age, sex, body weight, and tolerance to drugs. It will also depend on the degree, severity, and type of disease or disorder. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
  • the method includes treating one or more mammalian cells ex vivo with said purified recombinant AC to promote cell survival.
  • Cells whose survival can be promoted according to this aspect of the present invention include, without limitation, those that utilize the ceramidase apoptosis pathway, which includes a wide variety of cells (Obeid et al., Science 259: 1769-71 (1993)), e.g., hepatocytes (Arora et al., Hepatol. 25:958-63 (1997)), skin fibroblasts (Mizushima et al., Ann. Rheum. Dis.
  • chondrocytes MacRae et al., /. Endocrinol. 191(2):369-77 (2006)
  • lung epithelium Choan & Goldkorn, Am. J. Respir. Cell Mol. Biol. 22(4):460-8 (2000)
  • erythrocytes Liang et al., Cell. Physiol. Biochem. 15: 195-202 (2005)
  • cardiomyocytes Parra, V. et al., Cardiovasc. Res. 77(2): 387-97 (2007)
  • lymphocytes Gombos et al., Immunol. Lett. 104(l-2):59-69 (2006)
  • the cell types are eggs (fertilized or
  • ceramide apoptosis pathway appears to be conserved across mammalian species (Lee & Amoscato, Vitam. Horm. 67:229-55 (2004); see also, Samadi, Mol. Vis. 13: 1618-26 (2007) (humans); Parra, V. et al., Cardiovasc. Res. 77(2):387- 97 (2007) (rat); de Castro E Paula et al., Mol. Reprod. Devel., DOI No. 10.1002/mrd.20841 (2007) (cows)).
  • suitable cells include those of humans, monkeys, mice, rats, guinea pigs, cows, horses, sheep, pigs, dogs, and cats. This method may also be used to prolong the survival of eggs and/or embryos during in vitro fertilization procedures, facilitating the identification and selection of healthy embryos for reimplantation, especially for older human women and for veterinary breeding procedures.
  • Cells according to this aspect of the present invention can be provided by methods that will be apparent to the skilled artisan.
  • the cells can be obtained from an animal or from an existing ex vivo source (e.g., a tissue sample, a cell culture, etc.) using standard techniques.
  • Treating cells ex vivo includes treating cells present in a homogeneous culture, as well as cells present in a heterogenous culture (e.g., a tissue sample).
  • the purified recombinant AC in all aspects of the invention can be produced using ACs set forth in Table 1 of US 2016/0038574, as noted above.
  • the AC can be homologous (i.e., derived from the same species) or heterologous (i.e., derived from a different species) to the one or more cells being treated.
  • the human AC is preferred in methods for human therapy.
  • One embodiment of the present aspect of AC treatment relates to a method of producing chondrocytes with the purified recombinant AC. This method involves selecting a population of cells having the potential to differentiate into chondrocytes and treating the selected cell population with the purified recombinant AC to transform one or more of the cells in the selected population into chondrocytes.
  • Cells having the potential to differentiate into chondrocytes include, without limitation, bone marrow cells, fibroblasts, mesenchymal stem cells, and/or fibroblasts (see Mizushima et al., Ann. Rheum. Dis. 57:495-9 (1998)).
  • Chondrocytes include, without limitation, articular chondrocytes, nasal chondrocytes, tracheal chondrocytes, meniscal chondrocytes, and aural chondrocytes. These include, for example, mammalian chondrocytes, e.g., human chondrocytes, equine chondrocytes, porcine chondrocytes, feline chondrocytes, and canine chondrocytes. In some embodiments, the chondrocytes are primary chondrocytes.
  • Suitable cells include mammalian cells, e.g., human cells, equine cells, porcine cells, feline cells, and/or canine cells.
  • the purified recombinant AC and methods of treating the populations of cells with purified recombinant AC include all those set forth supra.
  • Another embodiment of the present aspect of AC treatment relates to a method of promoting chondrogenesis with the purified recombinant AC.
  • this method further includes selecting a population of stem cells in need of differentiation into chondrocytes, treating the population of stem cells with the purified recombinant AC to enrich mesenchymal stem cells within the stem cell population, and treating the population of enriched mesenchymal stem cells with the purified recombinant AC to promote
  • Suitable cells populations include mammalian cells populations, e.g., human cell populations, equine cell populations, porcine cell populations, feline cell populations, rodent cell populations, and/or canine cell populations.
  • Suitable stem cells include, but are not limited to, bone marrow cells, adipocytes, and skin cells.
  • Additional stem cells include, without limitation, embryonic stem cells, somatic stem cells, induced pluripotent stem cells, totipotent stem cells, pluripotent stem cells, and multipotent stem cells.
  • Exemplary stem cells include, for example, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, endothelial progenitor cells, epithelial stem cells, epidermal stem cells, adipocytes, and cardiac stem cells.
  • Suitable stem cells include, but are not limited to, mammalian cells, e.g., human, equine, porcine, feline, rodent, and canine bone marrow cells, adipocytes, and skin cells.
  • Suitable chondrocytes are consistent with those described supra.
  • the differentiated mesenchymal stem cells may, alternatively, be primary cells such as, but not limited to, neurons, hepatocytes, bone cells, lung cells, and cardiac cells.
  • the number of differentiated cells in the cell population is maintained. In at least one embodiment, the number of differentiated cells in the cell population is increased. As will be apparent to the skilled artisan, maintaining or increasing the overall number of differentiated cells in the population can be achieved by decreasing or preventing de-differentiation of cells in the population that are already differentiated, by stimulating the differentiation of undifferentiated cells in the population, or both.
  • the purified recombinant AC and methods of treating the populations of cells with purified recombinant AC include all those set forth supra.
  • the recombinant protein of the present invention may be prepared for use in the above described methods of the present invention using standard methods of synthesis known in the art, including solid phase peptide synthesis (Fmoc or Boc strategies) or solution phase peptide synthesis.
  • proteins of the present invention may be prepared using recombinant expression systems.
  • the human recombinant AC is produced in CHO cells using the methods described in WO2014/118619, incorporated herein by reference in its entirety.
  • the use of recombinant expression systems involves inserting the nucleic acid molecule encoding the amino acid sequence of the desired peptide into an expression system to which the molecule is heterologous (i.e., not normally present).
  • One or more desired nucleic acid molecules encoding a peptide of the invention may be inserted into the vector.
  • the multiple nucleic acid molecules may encode the same or different peptides.
  • the heterologous nucleic acid molecule is inserted into the expression system or vector in proper sense (5' ⁇ 3') orientation relative to the promoter and any other 5' regulatory molecules, and correct reading frame.
  • nucleic acid constructs can be carried out using standard cloning procedures well known in the art as described by Sambrook, J., et al., Molecular Cloning: A laboratory manual (Cold Springs Harbor 1989). U.S. Pat. No.
  • a variety of genetic signals and processing events that control many levels of gene expression can be incorporated into the nucleic acid construct to maximize peptide production.
  • mRNA messenger RNA
  • any one of a number of suitable promoters may be used. For instance, when cloning in E.
  • promoters such as the T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the PR and PL promoters of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
  • trp-lacUV5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
  • Common promoters suitable for directing expression in mammalian cells include, without limitation, SV40, MMTV, metallothionein-1, adenovirus Ela, CMV, immediate early, immunoglobulin heavy chain promoter and enhancer, and RSV- LTR.
  • Mammalian cells that may be used for manufacture of the recombinant protein of the present invention include, for example, Chinese Hamster Ovary (CHO) cells, plant cells, chicken eggs, and human fibroblasts.
  • nucleic acid construct there are other specific initiation signals required for efficient gene transcription and translation in prokaryotic cells that can be included in the nucleic acid construct to maximize peptide production.
  • suitable transcription and/or translation elements including constitutive, inducible, and repressible promoters, as well as minimal 5' promoter elements, enhancers or leader sequences may be used.
  • Roberts and Lauer Methods in Enzymology 68:473-82 (1979).
  • a nucleic acid molecule encoding a recombinant protein of the present invention, a promoter molecule of choice, including, without limitation, enhancers, and leader sequences; a suitable 3' regulatory region to allow transcription in the host, and any additional desired components, such as reporter or marker genes, are cloned into the vector of choice using standard cloning procedures in the art, such as described in Sambrook, J., et al., Molecular Cloning: A laboratory manual (Cold Springs Harbor 1989); Ausubel, F. M., Short Protocols in Molecular Biology (Wiley 1999), and U.S. Pat. No. 4,237,224 to Cohen and Boyer.
  • nucleic acid molecule encoding the peptide Once the nucleic acid molecule encoding the peptide has been cloned into an expression vector, it is ready to be incorporated into a host. Recombinant molecules can be introduced into cells, without limitation, via transfection (if the host is a eukaryote), transduction, conjugation, mobilization, or electroporation, lipofection, protoplast fusion, mobilization, or particle bombardment, using standard cloning procedures known in the art, such as described by Sambrook, J., et al., Molecular Cloning: A laboratory manual (Cold Springs Harbor 1989).
  • host-vector systems may be utilized to express the recombinant protein or polypeptide.
  • the vector system must be compatible with the host used.
  • Host-vector systems include, without limitation, the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria.
  • Purified peptides may be obtained by several methods readily known in the art, including ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, gel filtration, and reverse phase chromatography.
  • the peptide is produced in purified form (for example, at least 80%, 85%, 90% or 95% pure) by conventional techniques. Depending on whether the recombinant host cell is made to secrete the peptide into growth medium (see U.S. Pat. No.
  • the peptide can be isolated and purified by centrifugation (to separate cellular components from supernatant containing the secreted peptide) followed by sequential ammonium sulfate precipitation of the supernatant.
  • cells may be transformed with DNA encoding AC and then cultured under conditions effective to produce the medium containing inactive AC precursor.
  • the fraction containing the peptide is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the peptides from other proteins. If necessary, the peptide fraction may be further purified by other chromatography.
  • the incubation is carried out under conditions effective to reduce the transformation rate of inactive AC precursor to active AC compared to the transformation rate achieved when said incubating is carried out at a pH of 4 and a temperature of 4°C or 37°C, for 24 hours, under otherwise consistent conditions.
  • the incubating may be carried out under conditions effective to enhance the transformation rate of inactive AC precursor to active AC compared to those same conditions.
  • the purified recombinant AC during the incubating may have a pH over 4.0 and up to 6.5.
  • the mixture may, for example, have a pH of 4.0, 4.5, 5.0, 5.5, 6.0, or 6.5.
  • the temperature of the purified recombinant AC during said incubating may be at least -30°C and under 37°C
  • the temperature of the mixture may, for example, be -30°C, -25°C, -20°C, -15°C, -10°C, -5°C, 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, or 35°C
  • the mixture may be incubated under conditions of -30°C with a pH of 4.0, 4°C with a pH of 4.0 or 6.5, 25°C with a pH of 4.0, or 37°C with a pH of 4.0.
  • the mixture may be incubated for a period of time such as, but not limited to, approximately 30 minutes, 1 hour, 3 hours, 30 hours, or 300 hours.
  • Example 1 Process design of 10L purification protocol [00138] A number of needs were identified for improving biomanufacturing of rhAC. These included to convert from hollow fiber to single-use stirred tank bioreactors and to scale-up from shake flasks to 10L and 200L reactors.
  • Other methods to purify rhAC comprise columns that select by mannose content and size exclusion chromatography (see US2016/0038574). This downstream process cannot be used to produce recombinant AC intended for therapeutic uses for example, because it contains contaminants leaching from the purification process itself, such as Con A chromatography. Therefore, steps were undertaken to improve the AC purification process such that it could result in a product suitable for therapeutic uses.
  • FIG. 1 shows one embodiment of the process flow used for improved purification of rhAC. This process does not include heat inactivation to remove ASM activity and other contaminating proteins from the preparation as previously described in US
  • FIG. 1 incorporates cation exchange chromatography (using Capto S Imp Act, GE Healthcare), anion exchange chromatography (using Capto Q, GE Healthcare), and hydrophobic interaction chromatography (Capto Butyl HIC, GE Healthcare).
  • CHO clones expressing rhAC were developed using technologies to enhance expression of AC (see, e.g., WO2014118619).
  • Table 2 describes buffers used in the purification process in the 10L process.
  • the 200L non-current good manufacturing process (Non-cGMP Protocol) described in the footnotes refers to a process not performed under GMP standards, and all changes suggested for the Non-cGMP protocol were incorporated in that process, which will be described below.
  • Sodium Phosphate Dibasic and Sodium Phosphate Monobasic was used in all instances where Sodium Phosphate Dibasic and Sodium Phosphate Monobasic was called for in the 10L protocol.
  • Sodium Phosphate Dibasic and Sodium Phosphate Monobasic chemicals will be updated to Di-Sodium Hydrogen Phosphate and Sodium Dihydrogen Phosphate, respectively, for the 200L Non-cGMP Protocol due to availability and long lead times of the chemicals.
  • the g/L of all phosphate buffers will be updated with the same proportions of the monobasic and dibasic due to change in the molecular weight of the chemicals.
  • the Di- Sodium Hydrogen Phosphate molecular weight is 178.0 g/mol and the Sodium Dihydrogen Phosphate molecular weight is 156.0 g/mol.
  • the Di-Sodium Hydrogen Phosphate and Sodium Dihydrogen Phosphate will be USP Multicompendial grade.
  • Injection Buffer N-test Buffer (100 mM Sodium Phosphate, 1.5 M Sodium Chloride, pH respectively, instead of 25.84 g/L and 2.65 g/L. This buffer will also not be required for the 200L Non-cGMP Protocol as columns will be prepacked.
  • Capto S ImpAct Elution Buffer 110 mM MES, 45 mM Sodium Chloride, pH 6.4 density is 1.00. Additionally, after the 2.8 g/L of NaOH pellets were added additional 10N NaOH was used to titrate the buffer into the correct pH range. The 200L NoncGMP Protocol will continue to specify the 2.8 g/L but also allow for additional 10N NaOH to be used as needed for pH titration.
  • Capto S ImpAct Storage, Capto Butyl Storage, and Capto Q Storage Buffer used 200 mL/L of 100% ethanol instead of 210 mL/L of 95% Ethanol due to the availability of stocks of 100% Ethanol already in house.
  • the final buffer composition was unaffected.
  • the 200L Non-cGMP Protocol will use 100% Ethanol due to its lower cost.
  • Capto Butyl Chromatography Elution Buffer (5 mM Tris, pH 9.0) density is 0.99 g/mL.
  • the 200L Non-cGMP Protocol will not include the addition of 0.01 g/L of NaOH. It will be added as an option if needed for titration.
  • Non-cGMP protocol 10 mM Sodium Phosphate, 185 mM Sodium Chloride, pH 7.4 to optimize and improve recovery for the Capto Q process step.
  • the pH and conductivity target and acceptable range for this new buffer will be 7.4 ⁇ 0.1 and 20 + 1.0 mS/cm in the 200L Non-cGMP Protocol.
  • the Viral Inactivation Base Adjustment and Capto S ImpAct eluate pH adjustment buffer will be changed from 2M Tris pH 9.5 to 1 M Tris Base for the 200L Non-cGMP Protocol to both reduce the number of buffers and since 1M Tris Base is stronger this will also reduce the volumes of buffer required.
  • Chromatography resins used in the purification protocol are described in Table 3. These resins are exemplary. Packed chromatography columns used in the purification protocol are described in Table 4. Table 3: Chromatography Resins
  • Capto S ImpAct column was packed in NaCl solution using a Compression factor of 1.225 and a 1.17 Packing Factor at 150 cm hr. Updated based on GE manual supplied with the resin. Height equivalent to the theoretical plate (HETP) and Asymmetry were within specifications and performed as expected.
  • Capto Butyl HIC column Bed height was chosen to be 20.0cm based on using a 20cm bed height at the 200L scale. The column was packed to a compression factor of 1.19 not 1.14. Bed volume was 1570mL not 1180mL based on increasing the bed height to 20.0cm.
  • Capto Q column was packed in a HiScale 50/20 column due to availability of equipment. Bed height was packed to 15.4cm not 10cm as the desired bed height for the future 200L run was 20cm but there was only enough resin to pack the smaller column. Column volume was 302. 4mL instead of 200mL based on increasing bed height to 15.4cm.
  • Clarification process media used in the purification protocol are described in Table 5.
  • the clarification process is described in Table 6.
  • Each 0.102m filter was flushed individually at the specified L/m *h flow rate to sufficient flushing of filters.
  • the 10SP02A and 90ZB08A depth filters were loaded at 27.3 L/m 2 and 41.0 L/m 2 instead of 26.1 L/m 2 and 39.2 L/m 2 , respectively. No issues were seen while processing over the 10L protocol amount and the L/m 2 volume will be updated for the 200L Non-cGMP protocol.
  • the 0.2 ⁇ filter used was a Sartopore 2 300 0.45/0.2 ⁇ PES filter instead of a GE Healthcare ULTA Capsule HC Filter.
  • the filter size was 0.03m 2 and loaded at 278.7 L/m 2 instead of 0.2 m 2 and 80 L/m 2 .
  • the 200L Non-cGMP Protocol will use a Sartopore 2 0.8m 2 0.8/0.2 ⁇ PES filter with a loading of ⁇ 250 L/m 2 (200L Harvest / 0.8m 2 ).
  • the change in the filter's prefilter pore size should not affect the filter loading capacity.
  • stage 1 and stage 2 depth filters were blown down with 8 psig (5-10 psig) post equilibration which was not included in the 10L protocol. This reduces the total volume of the clarification process.
  • the blow down post initial USP flush of membranes will be removed from the 200L Non-cGMP Protocol as equilibration will be performed on the same day.
  • Product hold duration after clarification will be updated to 18-25 °C ⁇ 6 hours for the 200L Non-cGMP Protocol because of limited data for hold duration on this process step.
  • the 200L Non-cGMP Protocol will proceed directly into the Viral Inactivation step like the 10L batch.
  • Virus inactivation used in the purification protocol is described in Table 7.
  • Virus inactivation process parameters are described in Table 8.
  • Post VI Filter used was a Sartopore 2 0.05m 2 0.45/0.2 ⁇ PES dual layer filter instead of a single layer 0.2 ⁇ filter. This was changed to add a prefilter to the filter. The original filter loading specification was too optimistic. The Sartopore 2 filter clogged after 13.4 L (10.854 g of Neutralized Post VI low pH hold material). The filter was changed out and replaced with a new Sartopore 2 0.1m 2 0.45/0.2 ⁇ PES filter. The final 9.17L of Neutralized Post VI low pH hold was filtered with no issues.
  • the 200L Non-cGMP Protocol will be using a Sartopore 2 2.4m 2 0.8/0.2 ⁇ PES filter which will be loaded at estimated 242 L/m 2 and the loading will be based on L/m 2 instead of g/m 2 .
  • the 200L Non-cGMP Protocol will have an acceptable range of ⁇ 260 L/m 2 and a target of 240 L/m 2 .
  • the post viral inactivation product Hold duration and temperature will be changed to 18-25 °C for ⁇ 24 hrs.
  • Table 9 describes the Capto S ImpAct cation ion exchange chromatography (CIEX) process.
  • Table 10 describes the Capto S ImpAct CIEX parameters. The profile found with the Capto S ImpAct column shows that it is a high-capacity column that is able to be eluted in a small volume. Both of these characteristics may improve results and ease of use as an initial purification column in some embodiments.
  • the Pre-Use Sanitization was performed at reduced flowrate of 200 cm/hr for the 1st column volume (CV) due to high back pressure while removing the column storage buffer (50 mM Sodium Phosphate, 20% Ethanol, pH 7.2). Once the Ethanol was removed the flowrate was increased to the target flowrate of 300 cm/hr.
  • the 200L confirmation batch will be performed at 200 cm/hr for the 1st CV then 300 cm/hr for the remaining 2 CV's to match the process performed for the 10L confirmation batch.
  • the Equilibration volume used was 6.3 CV's which was over the 5 CV's specified.
  • the extra volume used was due to troubleshooting the online pH instrument as it was reading 0.5 pH units above the offline reading.
  • the offline pH readings were used for testing of the equilibration column effluent which was within specifications. Going over the 5 CV's is not an issue.
  • the Capto S ImpAct Column Load was run at 4 minute residence time instead of the specified 2 minutes.
  • the Capto S ImpAct Column was run at the specified 300 cm/hr and 20cm bed height which equates to a 4 minute residence time.
  • the residence time and flowrate specifications conflict.
  • the process step yield of 76.28% was equivalent to the process development experiment yield of 73%.
  • the Column Storage flow rate was reduced from 300 cm/hr to 200 cm/hr after the 1st CV's due to high back pressure from the column storage buffer (50 mM Sodium Phosphate, 20% Ethanol, pH 7.2).
  • the 200L Non-cGMP Protocol column storage parameter will be run at 200 cm/hr for all 3 CV's instead of 300 cm/hr.
  • the pH of column storage effluent acceptable range will be changed from 7.0 + 0.5 to ⁇ 8 as the main goal of the step is for the column storage to be at a neutral pH and doesn't require a tight specification.
  • the eluate pH adjustment buffer will be changed from 2M Tris pH 9.5 to 1 M Tris Base for the 200L Non-cGMP Protocol to standardize this buffer with the post low pH hold neutralization buffer and reduce the volume of buffer required.
  • the pH and conductivity adjustment will be performed on the Capto S Imp Act eluate prior to filtration and start of the hold duration.
  • the 200L Non-cGMP Protocol will have an updated Capto S ImpAct load conductivity acceptance range of ⁇ 16.0 mS/cm.
  • Table 11 describes the Capto Butyl HIC process.
  • Table 12 describes the Capto Butyl HIC process parameters. The properties of a HIC column at this step allow loading of a high salt solution that subsequent elution with a low salt solution.
  • the Pre-Use Sanitization was performed at 200 cm/hr for the 1st CV due to high back pressure while removing the column storage buffer (50 mM Sodium Phosphate, 20% Ethanol, pH 7.2) then the flowrate was increased to the target flowrate of 300 cm/hr.
  • the 200L Non- cGMP Protocol will be performed at 200 cm/hr for the 1st CV then 300 cm/hr for the remaining 2 CV's to match the process performed for the 10L confirmation batch.
  • the Column Storage flow rate was reduced from 300 cm/hr to 200 cm/hr after the 1st CV's due to high back pressure from the column storage buffer (50 mM Sodium Phosphate, 20% Ethanol, H 7.2).
  • the 200L confirmation batch column storage will be run at 200 cm/hr for all 3 CV's instead of 300 cm/hr.
  • the pH of column storage effluent acceptable range will be changed from 7.0 + 0.5 ⁇ 8 as the main goal of the step is for the column storage to be at a neutral pH and this doesn't require a tight specification.
  • Capto Butyl Column was run at 20cm bed height to match the expected 200L Non-cGMP column bed height. All in-process data showed no concern over change in the bed height.
  • Capto Butyl Column asymmetry was run outside of the acceptable range of 0.8 - 1.6 being at 1.67. This was deemed acceptable as the manufacturer resin manual states 0.8 to 1.8 as an acceptable asymmetry range. The column ran as expected and all inprocess data showed no concern over change in asymmetry specification. Additionally, the Capto Butyl plates / m (N/m) specification will be updated to > 2000 N/m for the 200L Non-GMP Protocol. This is based on standard Repligen Opus column specifications and should have no effect on the column operation for this process.
  • Capto Butyl Column was loaded above the acceptance range of ⁇ 8 g rhAC / L being at 8.9 g rhAC / L.
  • the column was loaded at 7.47 g rhAC / L based on initial Capto S ImpAct Elution "STAT" assay concentration data of 1.47 mg/mL. The sample was re-run later and the concentration changed to 1.67 mg/mL. Even though the column was loaded over the acceptable range, there was neither breakthrough during the load nor lower than expected yield.
  • the 8 g rhAC / L will remain as the specification for the 200L Non-cGMP Protocol.
  • the Load pH Adjustment Buffer will change to 1 M Tris Base for the 200L Non-cGMP Protocol to align the base titration buffers throughout the process and adjustment will be performed on day that the Capto S ImpAct Eluate is generated.
  • the Pre-Use Sanitization was performed at 76 cm/hr for the 1st CV due to high back pressure while removing the column storage buffer (50 mM Sodium Phosphate, 20% Ethanol, pH 7.2). After 1 CV the flowrate was increased to 150 cm/hr.
  • the 200L NoncGMP Protocol will be performed at 200 cm/hr for the 1st CV then 300 cm/hr.
  • Capto Q process was run at 150 cm/hr throughout the process, with exceptions for the Pre-Use Sanitization and Storage steps, instead of 300 cm/hr due to system back pressure limiting the flowrate.
  • the 200L Non-cGMP Protocol will be run at the target 300 cm/hr throughout the process with continued exceptions for the Pre-Use Sanitization and Storage steps.
  • c Prior to the 10L run it was discovered that the specified Capto Q equilibration and wash buffer, 10 mM Sodium Phosphate, 130 mM Sodium Chloride, pH 6.8, did not match the desired conductivity specification of ⁇ 14mS/cm.
  • the Capto Q column load was run with a residence time of 6 min, due to the lower operating flowrate of 150 cm/hr and the increase of the bed height from 10 cm to 15.4 cm.
  • the 200L Non-cGMP Protocol will operate with a target residence time of 4 min based on the Capto Q Optimization Study.
  • the Column Storage flow rate was reduced from 150 cm/hr to 76 cm/hr after the 1st CV due to high back pressure from the column storage buffer (50 mM Sodium Phosphate, 20% Ethanol, pH 7.2).
  • the 200L Non-cGMP Protocol column storage flowrate parameter will be set to 200 cm/hr for all CV's.
  • the pH of the product will be the close to the viral filtration filtrate sample to support the hold time and temperature.
  • the conductivity of the product will be slightly increased from the viral filtration filtrate sample and bracketed by the Adjusted Capto S ImpAct elution. Based on this assessment, 18-25 °C for ⁇ 7 days for the Capto Q Flow will be used through Hold duration and temperature.
  • Capto Q asymmetry and plates / meter (N/m) specification will be updated to 0.8 - 1.8 and > 1700 N/m respectively for the 200L Non-GMP Protocol. These numbers are based on standard GE Ready to Process column specifications and should have no effect on the column operation for this process.
  • the Capto Q Load pH and conductivity target and acceptable range will be updated for the 200L Non-cGMP Protocol to pH 7.4 ⁇ 0.1 and 20 mS/cm ⁇ 1 mS/cm to optimize the Capto Q process step.
  • the column equilibration effluent pH parameter will be updated to a target and acceptable range of 7.4 ⁇ 0.2 for the 200L Non-cGMP Protocol to match the change in equilibration buffer.
  • the equilibration effluent conductivity parameter will remain as "Same as equilibration buffer ⁇ 10%".
  • Table 15 describes the virus filtration process.
  • Table 16 describes the filtration parameters.
  • Viral filtration loading capacity of the Planova 20N for the 200L will remain at 20 - 200 g/rhAC/m 2 .
  • Viral Filtration Flow through Hold duration and temperature will be changed to 18-25 °C for ⁇ 7 days.
  • Table 17 describes the ultrafiltration/diafiltration (DF/UF) process.
  • Table 18 describes the DF/UF n parameters.
  • UF/DF Sanitization buffer was 0.2 M NaOH as stated in the buffer section, not 0.5 M NaOH.
  • the 200L Non-cGMP Protocol will use 0.2 M NaOH for sanitization.
  • a ⁇ 48 hr hold in caustic will be incorporated in the 200L Non-cGMP protocol to allow setup of the system to occur prior to the day of use.
  • the membranes are stable in 0.2 M NaOH as the manufacturer uses this buffer for long term storage of the membranes.
  • UF/DF flush buffer was USP Water not water for injection (WFI) water as WFI was not required for the 10L confirmation run.
  • WFI Water not water for injection
  • the 200L Non-cGMP protocol will use WFI Quality water.
  • the 10L confirmation run had higher than anticipated permeate flux rates.
  • the flux rates were between 120 L/m 2 *h for sanitization, 96 L/m 2 *h for flush, 50 - 65 L/m 2 *h for initial concentration.
  • the UF/DF Cross flow flux was out of specified acceptance range of 4 - 8 L/min m 2 at 8.9 - 9.0 L/min/m 2 due to a reduction in the UF/DF membrane area without adjusting the feed flowrate to correspond to the new membrane area.
  • the 200L Non-cGMP Protocol will change the specification from cross flow flux to feed flow with a specification of 4-8 L/min/m 2 as it can be controlled easier at the larger process scale.
  • the UF/DF Hold duration and temperature will be changed to 18-25 °C for ⁇ 2 days.
  • the rhAC concentration after initial concentration step has a target specification of > 11 g/L but a range of 11 - 15 g/L.
  • the 200L Non-cGMP Protocol will change this to a target of > 11 g/L.
  • Table 19 describes the process parameters formulation with buffer.
  • Table 20 describes the drug substance filtration process parameters.
  • Ethyl Vinyl Acetate (Sartorius Film S71) will be the Product Final Container for the 200L Non-cGMP Protocol and will be stored at -20°C.
  • the temperature of the Bulk Drug Substance was initially stored at 2 - 8 °C and -20°C. All material was eventually stored -20°C.
  • the 200L Non-cGMP Protocol will store and ship the Bulk Drug Substance at -20°C.
  • the Filter Loading (g/m 2 ) will be increased from ⁇ 1000 g/m 2 to ⁇ 10000 g/m 2 for the 200L Non-cGMP Protocol. All process steps are filtered so there is little concern over increasing the filter loading capacity.
  • the final filter is sized by the bag manufacturer for the appropriate L/m 2 capacity.
  • Example 2 Analytical data on 10L purification batch [00157] The 10L confirmation batch process and analytical data was evaluated for technical transfer to the 200L Non-cGMP Engineering / Toxicology run.
  • Table 21 lists 10L confirmation batch step and process yield data.
  • the sample titer/concentration data is based on the RP-HPLC concentration assay for all samples besides the BDS sample which used the A280 concentration assay.
  • FIGs. 2-4 present the Capto S ImpAct process (FIG. 2), Capto Butyl process (FIG. 3), and Capto Q process chromatograms (FIG. 4). These chromatograms show generation of AC of high purity.
  • Table 22 presents data on the rhAC concentrations by reversed phase high- performance liquid chromatography (RP HPLC) and A280nm measurement.
  • RP HPLC reversed phase high- performance liquid chromatography
  • Table 23 presents data on the purity of rhAC from the 10L process.
  • HMW high molecular weight
  • LMW low molecular weig
  • Table 24 lists a summary of data using reduced SDS-PAGE analysis in relation to a reference standard (Ref Std) of rhAC.
  • Table 25 lists a summary of data using non-reducing SDS-PAGE analysis. In both cases, the profile of the 10L process batch aligned with that of the reference standard of rhAC.
  • Table 26 lists isoelectric point (pi) data on the five isoforms of rhAC generated by imaged capillary isoelectric focusing. These isoforms may likely indicate glycosylation heterogeneity, which would be expected for AC. Table 26: icIEF, pis of the Five Isoforms, BDS Data Summary
  • Table 27 presents data on residual host cell (HC) DNA measured by qPCR, as described previously (see, e.g., WO2014118619).
  • Table 27 Residual Host Cell (HC) DNA by qPCR Data Summary
  • AC activity was measured using an ultra performance liquid chromatography (UPLC) method with the pure enzyme.
  • a substrate stock solution was prepared comprising 200 ⁇ C12-NBD Ceramide (#10007958, Cayman Chemical), 0.2% Igepal CA-630 (#1- 3021, Sigma), 0.2 M Citrate/Phosphate (C/P) buffer, pH 5.0, 0.3 M NaCl (#S271-1, Fisher Scientific), and 10% Fetal Bovine Serum (FBS, #35-010-CV, Corning Cellgro).
  • a 0.2 M C/P buffer solution containing 0.3 M NaCl was prepared and kept at room temperature in amber vials for up to 6 months.
  • Substrate buffer was prepared fresh depending on the number of assays to be run on a given day. For example, to prepare 100 ⁇ of substrate buffer, 10 ⁇ of substrate stock solution was dried by airflow, followed by addition of 10 ⁇ of FBS and 90 ⁇ of C/P buffer solution. The solution was suspended by gentle vortex.
  • the UPLC system configuration was as follows: Waters Acquity H-Class UPLC system consists of Quaternary Solvent Manager, Sample Manager, and Fluorescence Detector. The column was ACQUITY BEH C18 1.7 ⁇ , 2.1x30mm (#186002349). The guard column was ACQUITY BEH C18 VanGuard Pre-column, 1.7 um, 2.1 x 5 mm
  • the mobile phase comprised A: 0.1% ammonium acetate buffer (pH 7.2) and B: Acetonitrile (Fisher, #A998-4).
  • the Quaternary Solvent Manager reference for instrument method setup was Time (min)/flow rate (ml/min)/Channel A%/B%/C%/D%/curve at 0.0/1.2/68/32/00/00; 0.1/1.2/00/100/00/00/6; and 0.4/1.2/68/32/00/00/11.
  • the total running time was 0.8 minutes.
  • FIG. 5 presents data on AC activity in different process steps of the third 10L run and shows acceptable activity.
  • Table 28 summarizes BDS analytic results for the engineering lot versus the 10L process run.
  • Table 29 summarizes process parameter modifications made to the 10L process outlined in Example 1 for the 200L Non-cGMP protocol. All of the modifications listed in Table 29 were made during the rhAC preparation using the 200L Non-cGMP protocol.
  • Clarificati and Duration 18-25 °C ⁇ 24 and Duration: 18-25 °C ⁇ 6 on hrs., or 2-8°C > 24 hrs. hrs.
  • ImpAct Load Adjustment ImpAct Load Adjustment ImpAct Load Adjustment
  • Inactivatio and Duration 18-25 °C ⁇ 24 and Duration: 18-25 °C ⁇ 24 n hrs., or 2-8°C > 24 hrs. hrs.
  • Adjustment Buffer Not Water as needed to titrate ImpAct
  • Capto S will be performed only on either post Capto S ImpAct
  • FIG. 6A shows the cell viability data for different conditions.
  • FIG. 6B shows that the highest product titer (i.e., rhAC concentration) was found with Condition 3.
  • the feed optimization study was comprised of 6 separate fed-batch conditions with three 125mL shake flasks per condition.
  • the Cell Boost feeds all contain glucose, and in the case of Cell Boost Feed A, glutamine as well.
  • the conditions tested were as follows: Condition 1: Cell BoostTM Feeds 7a and 7b, with 5%: 0.5% bolus additions
  • a sampling schedule was followed to collect viable cell density, cell viability, and titer data to support the decision as to which fed-batch condition would be optimal.
  • FIG. 7A Cell viability over time for the different conditions is shown in FIG. 7A.
  • Product titer at Day 12 is shown in FIG. 7B, showing the highest titers for Condition 5.
  • An outlying variable that separates Condition 5 from the other conditions is the temperature reduction on Day 4. Due to a slower metabolic rate, Condition 5 consumed less glutamine than the other conditions, and therefore had a lesser ammonia concentration. The lower concentration of ammonia may have contributed to the prolonged cell viability of Condition 5.
  • Condition 5 being identical to Condition 1 with the exception of the temperature shift to 33 °C, experienced enhanced protein production with 50% more product.
  • An outlying variable that separates Condition 5 from the other conditions is the temperature reduction on Day 4. Due to a slower metabolic rate, Condition 5 consumed less glutamine than the other conditions, and therefore had a lesser ammonia concentration, as shown in FIG. 8. The lower concentration of ammonia may have contributed to the prolonged cell viability of Condition 5.
  • Condition 5 being identical to Condition 1 with the exception of the temperature shift to 33°C, experienced enhanced protein production with 50% more product.
  • Condition 5 had >1.5x rhAC concentration compared with all other conditions.
  • Parameters measured included viable cell density, reactor cell viability, reactor glucose concentration, ammonia concentration, PCO2 concentration, glutamine concentration, reactor pH, and titer concentration.
  • Table 30 presents the bioreactor process conditions for Runs 1, 2, and 3.
  • FIG. 9A Cell viability (FIG. 9A) and product titer (FIG. 9B) are shown for the different runs. Data show that the 20 ⁇ sparge configuration was best due to higher kLa (volumetric oxygen transfer coefficient of liquid film). Because of the low sensitivity of cells to shear forces, agitation can be increased at the end of the run to increase titers.
  • kLa volumemetric oxygen transfer coefficient of liquid film
  • BalanCD ® CHO Media in XDR-10, XDR 200 and cGMP XDR 200 bioreactors were compared.
  • the purpose/scope of this reactor study was to demonstrate equivalence in scale- up from 10L to 200L.
  • Figure 11 shows that AC activity was similar for each excipient. Further, storage of the solutions found that all excipients had similar profiles at 4°C and room temperature over 25 weeks ( Figure 12). Thus, activity and stability of rhAC produced by these methods was acceptable in a number of different excipients.

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Abstract

La présente invention concerne le domaine de la purification de la céramidase acide humaine recombinante.
PCT/US2018/052463 2017-09-25 2018-09-24 Procédé de production de céramidase acide humaine recombinante WO2019060837A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019150192A1 (fr) * 2018-02-02 2019-08-08 Enzyvant Therapeutics Gmbh Méthodes de traitement de la maladie de farber
EP3568154A4 (fr) * 2017-01-13 2020-11-18 Icahn School of Medicine at Mount Sinai Composés et méthodes pour traiter la maladie de farber

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150183821A1 (en) * 2013-03-08 2015-07-02 Genzyme Corporation Integrated Continuous Manufacturing of Therapeutic Protein Drug Substances

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150183821A1 (en) * 2013-03-08 2015-07-02 Genzyme Corporation Integrated Continuous Manufacturing of Therapeutic Protein Drug Substances

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "Capto(TM) S ImpAct", GE HEALTHCARE BIO-SCIENCES AB, DATA FILE 29-0670-18 AB, September 2014 (2014-09-01), pages 1 - 8, XP055583986, Retrieved from the Internet <URL:https://www.gelifesciences.co.jp/catalog/pdf/29067018ABDF.pdf> [retrieved on 20181030] *
ANONYMOUS: "Hydrophobic interaction chromatography. Capto(TM) Phenyl (high sub), Capto Butyl, and Capto Octyl", GE HEALTHCARE BIO-SCIENCES AB , DATA FILE 28-9558-57 AC- KA1384080119DF, September 2011 (2011-09-01), pages 1 - 4, XP055583995, Retrieved from the Internet <URL:https://cdn.gelifesciences.com/dmm3bwsv3/AssetStream.aspx?mediaformatid=10061&destinationid=10016&assetid=14385> [retrieved on 20181030] *
ANONYMOUS: "Ion exchange chromatography- Capto(TM) S, Capto Q, and Capto DEAE", GE HEALTHCARE BIO-SCIENCES AB, DATA FILE 11- 0025-76 AG-KA138518121DF, May 2012 (2012-05-01), pages 1 - 8, XP055584001, Retrieved from the Internet <URL:https://cdn.gelifesciences.com/dmm3bwsv3/AssetStream.aspx?mediaformatid=10061&destinationid=10016&assetid=11114> [retrieved on 20181030] *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3568154A4 (fr) * 2017-01-13 2020-11-18 Icahn School of Medicine at Mount Sinai Composés et méthodes pour traiter la maladie de farber
EP4295903A3 (fr) * 2017-01-13 2024-03-27 Icahn School of Medicine at Mount Sinai Composés et méthodes pour traiter la maladie de farber
WO2019150192A1 (fr) * 2018-02-02 2019-08-08 Enzyvant Therapeutics Gmbh Méthodes de traitement de la maladie de farber

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