RECOMBINANT HUMAN ACID CERAMIDASE PRODUCTION PROCESS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.

62/562,804, filed September 25, 2017, which is incorporated by reference herein in its entirety.

BACKGROUND

[0002] 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.

[0003] A wide variety of methodologies are known for purification of recombinant proteins for therapeutic use (see, e.g. , Saraswat M et al., BioMed Research International Volume 2013, Article ID 312709 (2013)). Improved processes for protein purification are needed to lower costs, reduce processing times, and increase yields of target proteins. It is also well-known that different purification processes can yield proteins with different functional profiles even though they contain the same amino acid sequence. Moreover, processes for purification of recombinant proteins for human therapy must result in a product that can be administered to a patient with minimal the risk of side effects from the impurities in the recombinant protein composition.

[0004] 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)). Thus, in the case of AC, 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.

[0005] Recently, a composition has been described that comprises a combination of inactive and active rhAC, where the composition does not comprise acid sphingomyelinase (ASM) activity (US 2016/0038574). This application describes use of heat inactivation to remove ASM activity from the preparation. However, 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.

[0006] Described herein is a process for purification of rhAC. In particular, this purification process does not require heat inactivation of a co-purifying protein.

SUMMARY

[0007] 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.

[0008] In some embodiments, the recombinantly produced acid ceramidase is subjected to each of cation exchange chromatography, hydrophobic interaction

chromatography (HIC), and anion exchange chromatography. In some embodiments, cation exchange chromatography comprises CaptoS ImpAct. In some embodiments, hydrophobic interaction chromatography (HIC) comprises Capto Butyl HIC. In some embodiments, anion exchange chromatography comprises Capto Q.

[0009] In some embodiments, the one or more viral inactivation steps comprises contacting the recombinantly produced acid ceramidase with citric acid. In some embodiments, the recombinantly produced acid ceramidase is titrated to pH 3.7 with citric acid.

[0010] In some embodiments, 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.

[0012] In some embodiments, the purified recombinantly produced acid ceramidase has a purity of at least 90%, 93%, 95%, 98%, or 99%, or a purity of 100%.

[0013] In some embodiments, the purified recombinantly produced acid ceramidase has no detectable acid sphingomyelinase activity.

[0014] In some embodiments, the acid sphingomyelinase activity of the

recombinantly produced acid ceramidase is removed without the use of heat.

[0015] Also described herein is a purified recombinantly produced acid ceramidase produced by any of the methods described herein.

[0016] Also described herein is a therapeutic composition comprising the purified recombinantly produced acid ceramidase produced by any of the methods described herein.

[0017] In some embodiments, the therapeutic composition further comprises a pharmaceutically acceptable carrier.

[0018] In some embodiments, the therapeutic composition further comprises one or more pharmaceutically acceptable adjuvants, excipients, or stabilizers. In some embodiments, 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. In some embodiments, the salts are hydrates (e.g., trisodium citrate dihydrate, citric acid monohydrate, sodium phosphate monobasic monohydrate, and/or sodium phosphate dibasic heptahydrate).

[0019] In some embodiments, the therapeutic composition further comprises one or more additional agents that reduce ceramide levels.

[0020] 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. In some embodiments, the therapeutic

composition is administered in vivo to a subject in need thereof. In some embodiments, the therapeutic composition is administered in vitro to a population of cells.

[0021] In some embodiments, 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.

[0022] In some embodiments, the selected cell population comprises mammalian cells.

[0023] In some embodiments, the selected cell population comprises bone marrow cells, stem cells, and/or fibroblasts. In some embodiments, the stem cells are mesenchymal stem cells.

[0024] In some embodiments, 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.

[0025] In some embodiments, the subject has a joint disease or disorder. In some embodiments, the joint disease or disorder comprises osteoarthritis, rheumatoid arthritis, mucopolysaccharidosis, degenerative joint disease, joint injury, or Farber lipogranulomatosis.

[0026] In some embodiments, the subject has a neurodegenerative disease or disorder.

In some embodiments, 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. In some embodiments, motor neuron disease is spinal muscular atrophy.

[0027] In some embodiments, the subject has a cardiac disease or disorder. In some embodiments, the cardiac disease or disorder comprises heart disease, cardiac injury, atherosclerosis, thrombosis, or cardiomyocyte apoptosis.

[0028] In some embodiments, the subject has diabetes.

[0029] In some embodiments, the subject has a pathogenic infection in a subject having cystic fibrosis, COPD, and/or an open wound. In some embodiments, the pathogenic infection comprises a viral, fungal, prionic, or bacterial infection.

[0030] In some embodiments, subject has a ceramide accumulation infection.

[0031] In some embodiments, the subject has Farber disease. [0032] In some embodiments, the method of treatment further comprises

administering to the subject an antipyretic, an antihistamine, a corticosteroid or any combination thereof prior to, concurrently with, or after administration of the composition.

[0033] In some embodiments, 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.

[0034] In some embodiments, the method of treatment further comprises one or more repeat administrations of the therapeutic composition.

[0035] In some embodiments, the method of treatment further comprises

administering one or more additional agents which reduce ceramide levels.

[0036] Additional objects and advantages will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0037] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.

[0038] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description, serve to explain the principles described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] For a more complete understanding of the principles disclosed herein, and the advantages thereof, reference is made to the following descriptions taken in conjunction with the accompanying drawings, in which:

[0040] FIG. 1 shows a flow diagram of one embodiment of the purification process.

UF/DF = ultrafiltration/diafiltration.

[0041] 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.

[0042] FIG. 3 shows the Capto Butyl elution process chromatogram.

[0043] FIG. 4 shows the Capto Q flow through process chromatogram. [0044] FIG. 5 shows AC activity ("act") in the process steps of the third 10L run. Post

VI = post viral inactivation; BDS = bulk drug substance.

[0045] 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.

[0046] 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 Boost™ Feeds 7a and 7b, with 5%: 0.5% bolus additions; Condition 2: Efficient Feed B™ 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.

[0047] FIG. 8 shows ammonia concentrations for the different conditions described for FIGs. 7 A and 7B.

[0048] FIGs. 9A and 9B show viable cell density (FIG. 9A) and product titer (FIG.

9B) for runs with different sparging and agitation as described in Table 30.

[0049] 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.

[0050] FIG. 11 shows effect of excipients on AC function. PBS = rhAC in PBS; PBS

& TW80 = rhAC in PBS + 0.001% Tween ^® 80; PBS & sucrose = rhAC in PBS + 1% sucrose; and PBS, TW80 & sucrose = rhAC in PBS + 1% sucrose, 0.001% Tween 80.

[0051] 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.

Table 1: Description of the Sequences

DETAILED DESCRIPTION

[0053] The titles, headings and subheadings provided herein should not be interpreted as limiting the various aspects of the disclosure. Accordingly, the terms defined below are more fully defined by reference to the specification in its entirety. All references cited herein are incorporated by reference in their entirety.

[0054] Unless otherwise defined, scientific and technical terms used herein shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0055] In this application, the use of "or" means "and/or" unless stated otherwise. In the context of a multiple dependent claim, the use of "or" refers back to more than one preceding independent or dependent claim in the alternative only. It is further noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the," and any singular use of any word, include plural referents unless expressly and unequivocally limited to one referent. As used herein, the term "include" and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items. [0056] As described 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.

[0057] Units, prefixes, and symbols are denoted in their Systeme International de

Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Measured values are understood to be approximate, taking into account significant digits and the error associated with the measurement.

[0058] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0059] As used herein, "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.

[0060] There are three types of AC described to date (Nikolova-Karakashian et al.,

Methods Enzymol. 311 : 194-201 (2000)). These are classified as acid, neutral, and alkaline ceramidases according to their pH optimum of enzymatic activity. ACs have optimal enzymatic activity at a pH<5. The human AC was the first ceramidase to be cloned (Koch et al., /. Biol. Chem. 271:33110-33115 (1996)). It is localized in the lysosome and is mainly responsible for the catabolism of ceramide. Dysfunction of this enzyme because of a genetic defect leads to a sphingolipidosis disease called Lipogranulomatosis or Farber disease (Koch et al., /. Biol. Chem. 271:33110-33115 (1996), Young et al., /. Lipid. Res. 54:5-19 (2013)).

[0061] 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. 276(38):35352-60 (2001)), which is the product of the Asahl gene (NCBI UniGene GenelD No. 427). AC protein [Homo sapiens] (NCBI Accession No. AAC50907) is shown in SEQ ID NO: 1.

[0062] 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).

[0063] As used herein, "active acid ceramidase" or "active AC" refers to AC precursor proteins that has undergone autoproteolytic cleavage into the active form

(composed of a- and β-subunits). The mechanism of human AC cleavage and activation is reported in Shtraizent et al., /. Biol. Chem. 283(17): 11253-11259 (2008)). Activation is promoted by the intracellular environment, and, based on highly conserved sequences at the cleavage site of ceramidase precursor proteins across species, is expected to occur in most, if not all, cell types.

[0064] 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.

[0065] 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 β-subunits). The mechanism of human AC cleavage and activation is reported in Shtraizent et al., /. Biol. Chem. 283(17): 11253-11259 (2008)). This is promoted by the intracellular environment, and, based on highly conserved sequences at the cleavage site of ceramidase precursor proteins across species, is expected to occur in most, if not all, cell types. Thus, 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. Embodiments in which 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.

[0066] 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.

[0067] The recombinant acid ceramidase of the therapeutic composition may, in some embodiments, contain a greater amount of the inactive AC precursor than active AC.

Alternatively, the purified recombinant acid ceramidase of the therapeutic composition may, in some instances, contain a lesser amount of inactive AC precursor than active AC. [0068] In some embodiments, 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 inactive AC precursor and 20 to 10 wt % of the active AC. In some embodiments, 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. In yet a further embodiment, the purified recombinant acid ceramidase may contain 60 wt % inactive ceramidase precursor and 40 wt % active ceramidase. In some aspects, 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.

[0069] As used herein, "recombinant human acid ceramidase" or "rhAC" refers to protein encoded by the human ASAH1 gene and produced by the process described herein. The amino acid sequence of rhAC (AC preprotein) is SEQ ID NO: 1.

[0070] As used herein, "acid sphingomyelinase activity" or "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)).

[0071] As used herein, "production," "production process," or "processing" refers to the production and purification of a biotherapeutic. Production can encompass

bioengineering, equipment design, molecular biology, cell genetics, cell culture technology, and analytical chemistry.

[0072] As used herein, "chromatography" refers to any laboratory technique for separation of mixtures.

[0073] As used herein, "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.

[0074] As used herein, "cation exchange chromatography" or "CIEX" refers to a process that separates substances based on their charges using an ion-exchange resin containing negatively charged groups. Capto S Imp Act is an exemplary CIEX media. [0075] As used herein, "hydrophobic interaction chromatography" or "HIC" refers to a process that separates substances based on their hydrophobicity. Capto Butyl HIC is an exemplary HIC media.

[0076] As used herein, "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)).

[0077] 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. Such 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. In some embodiments, the salts are hydrates (e.g., trisodium citrate dihydrate, citric acid

monohydrate, sodium phosphate monobasic monohydrate, and/or sodium phosphate dibasic heptahydrate).

[0078] 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.

[0079] Treatment according to this aspect of the present invention is carried out using methods that will be apparent to the skilled artisan. 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)).

[0080] The 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. [0081] In some embodiments, treatment is carried out by introducing a ceramidase protein into the cells. An approach for delivery of proteins or polypeptide agents (e.g., active ceramidase, inactive ceramidase precursor proteins) involves the 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.

[0082] Yet another approach for delivery of proteins or polypeptide agents involves preparation of chimeric proteins according to U.S. Pat. No. 5,817,789 to Heartlein et al. 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. Thus, when the chimeric protein is delivered to the cell, the chimeric protein will adsorb to the targeted cell, and the targeted cell will internalize the chimeric protein.

[0083] 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.

[0084] In one embodiment, 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

lipogranulomatosis.

[0085] In another embodiment, 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. Exemplary types of 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.

[0086] In another embodiment, 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. Exemplary types of 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.

[0087] In another embodiment, 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.

[0088] In another embodiment, 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. Exemplary types of pathogenic infections include, without limitation, viral, fungal, prionic, and bacterial.

[0089] 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,

Clostridium tetani, Corynebacterium dipththeriae, Escherichia coli, enterohemorrhagic E. coli, enterotoxigenic E. coli, Haemophilus influenzae type B and non-typable, Helicobacter pylori, Legionella pneumophila, Listeria monocytogenes, Mycobacterium spp.,

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

Streptococcus, Streptococcus mutans, Treponema pallidum, Vibrio cholerae, and Yersinia pestis. In some embodiments, the pathogenic infection is a Pseudomonas infection.

[0090] 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, mobillivirus such as measles virus, rubulavirus (such as mumps virus), pneumonoviridae (e.g., pneumovirus, human respiratory syncytial virus), metapneumovirus (e.g., avian pneumovirus and human metapneumo virus), picornaviridae (e.g., enterovirus, rhinovirus, hepatovirus such as human hepatitis A virus, cardiovirus, and apthovirus), reoviridae (e.g., orthoreo virus, orbivirus, rotavirus, cypo virus, fijivirus, phytoreo virus, and oryzavirus), retroviridae (e.g., mammalian type B retroviruses, mammalian type C retroviruses, avian type C retroviruses, type D retrovirus group, BLV-HTLV retroviruses, lenti virus (such as human

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

(mononucleosis), Dengue virus (dengue fever, shock syndrome), Epstein-Barr virus

(mononucleosis, Burkitt's lymphoma), Human T-cell lymphotropic virus type 1 (T-cell leukemia), Influenza A, B, and C (respiratory disease), Japanese encephalitis virus

(pneumonia, encephalopathy), Poliovirus (paralysis), Rhinovirus (common cold), Rubella virus (fetal malformations), Vaccinia virus (generalized infection), Yellow fever virus (jaundice, renal and hepatic failure), and Varicella zoster virus (chickenpox).

[0091] Pathogenic fungi include, without limitation, the genera Aspergillus (e.g.,

Aspergillus fumigates), Blastomyces, Candida (e.g., Candida albicans), Coccidiodes, Cryptococcus, Histoplasma, Phycomyces, Tinea corporis, Tinea unguis, Sporothrix schenckii, and Pneumocystis carinii. Pathogenic protozoan include, without limitation, Trypanosome spp., Leishmania spp., Plasmodium spp., Entamoeba spp., and Giardia spp., such as Giardia lamblia.

[0092] As described herein, an "open wound" refers to a type of injury in which an epithelial layer, i.e., skin, is torn, cut, and/or punctured. In some embodiments, 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. [0093] In another embodiment, 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

accumulation infection.

[0094] 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.

[0095] Mammalian subjects according to these aspects of the present invention include, for example, human subjects, rodent subjects, equine subjects, porcine subjects, feline subjects, and canine subjects.

[0096] In all embodiments that involve administering the purified recombinant AC to a subject, 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.

Additionally, and/or alternatively, 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.

[0097] Typically, the purified recombinant AC will be administered to a subject in a vehicle that delivers the ceramidase to the target cell, tissue, or organ. Exemplary 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.

[0098] In some embodiments, 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.

[0099] 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. For oral therapeutic administration, 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.

[00100] 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. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as fatty oil.

[00101] 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.

[00102] 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. In all cases, 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.

[00103] The purified recombinant AC may also be administered directly to the airways in the form of an aerosol. For use as aerosols, 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. The purified recombinant AC may also be administered in a non-pressurized form.

[00104] 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. 5,059,421 to Loughrey et al.; Wolff et al., Biochim. Biophys. Acta 802:259-73 (1984)), transdermal patches, implants, implantable or injectable protein depot compositions, and syringes. Other delivery systems which are known to those of skill in the art can also be employed to achieve the desired delivery of ceramidase to the desired organ, tissue, or cells.

[00105] Administration can be carried out as frequently as required and for a duration that is suitable to provide effective treatment. For example, administration can be carried out with a single sustained-release dosage formulation or with multiple daily doses.

[00106] 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. In such embodiments, 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.

[00107] In one embodiment, one or more additional agents which reduce ceramide levels may be administered with the purified recombinant AC. This includes, without limitation, inhibitors of acid sphingomyelinase (e.g., amitryptiline (Becker et al., Am. J. Respir. Cell. Mol. Biol. 42:716-724 (2010)) and inhibitors of ceramide synthases (e.g., Shiffmann et al., Biochimie 94:558-565 (2012)).

[00108] 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.

[00109] In one embodiment of the present invention, 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. 57:495-9 (1998)), chondrocytes (MacRae et al., /. Endocrinol. 191(2):369-77 (2006)), lung epithelium (Chan & Goldkorn, Am. J. Respir. Cell Mol. Biol. 22(4):460-8 (2000)), erythrocytes (Lang et al., Cell. Physiol. Biochem. 15: 195-202 (2005)), cardiomyocytes (Parra, V. et al., Cardiovasc. Res. 77(2): 387-97 (2007)), and lymphocytes (Gombos et al., Immunol. Lett. 104(l-2):59-69 (2006)), eggs, embryos, neurons, sperm, synovial fibroblasts, and embryonic stem cells. In dome embodiments, the cell types are eggs (fertilized or

unfertilized), embryos, primary cells (e.g., neurons), sperm, synovial fibroblasts, and embryonic stem cells. Moreover, the 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)). Therefore, it is expected that, for each of the cell types recited above, 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.

[00110] Cells according to this aspect of the present invention can be provided by methods that will be apparent to the skilled artisan. By way of example, 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).

[00111] 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. In this and all aspects of the present invention (including the in vivo methods discussed below), 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.

[00112] 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.

[00113] 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)).

[00114] Chondrocytes according to this aspect of the present invention 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.

[00115] Suitable cells according to this and all other aspects of the present invention include mammalian cells, e.g., human cells, equine cells, porcine cells, feline cells, and/or canine cells.

[00116] In this and all aspects of the present invention involving cell populations, embodiments in which the cells are all of one type, as well as embodiments in which the population is a mixture of two or more cell types, are both contemplated.

[00117] The purified recombinant AC and methods of treating the populations of cells with purified recombinant AC include all those set forth supra.

[00118] Another embodiment of the present aspect of AC treatment relates to a method of promoting chondrogenesis with the purified recombinant AC. In one embodiment, 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

differentiation of mesenchymal stem cells into chondrocytes.

[00119] Suitable cells populations according to this aspect of the present invention 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.

[00120] Suitable stem cells according to this and all other aspects of the present invention include, but are not limited to, bone marrow cells, adipocytes, and skin cells.

Additional stem cells according to this aspect of the present invention 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.

[00121] 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.

[00122] In at least one embodiment, 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.

[00123] The purified recombinant AC and methods of treating the populations of cells with purified recombinant AC include all those set forth supra.

[00124] 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. Alternatively, proteins of the present invention may be prepared using recombinant expression systems.

[00125] In some aspects of the methods of the invention, the human recombinant AC is produced in CHO cells using the methods described in WO2014/118619, incorporated herein by reference in its entirety.

[00126] Generally, 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. When multiple nucleic acid molecules are inserted, 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.

[00127] The preparation of the 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.

4,237,224 to Cohen and Boyer, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation into a suitable host cell.

[00128] A variety of genetic signals and processing events that control many levels of gene expression (e.g., DNA transcription and messenger RNA (mRNA) translation) can be incorporated into the nucleic acid construct to maximize peptide production. For the purposes of expressing a cloned nucleic acid sequence encoding a desired recombinant protein, it is advantageous to use strong promoters to obtain a high level of transcription. Depending upon the host system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E. coli, its bacteriophages, or plasmids, 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. 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.

[00129] 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. Depending on the vector system and host utilized, any number of 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. For a review on maximizing gene expression see Roberts and Lauer, Methods in Enzymology 68:473-82 (1979).

[00130] 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.

[00131] 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).

[00132] A variety of suitable host-vector systems may be utilized to express the recombinant protein or polypeptide. Primarily, 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.

[00133] 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. In some embodiments, 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. 6,596,509 to Bauer et al.), 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. In one embodiment of the present invention, 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.

[00134] In one embodiment of the present invention, 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. Alternatively, 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.

[00135] In some embodiments, 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. In other embodiments, 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 Alternatively, 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.

[00136] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present invention are described in various levels of detail in order to provide a substantial understanding of the present technology. The definitions of certain terms as used in this specification are also provided. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

EXAMPLES

[00137] The following examples are provided to illustrate certain disclosed embodiments and are not to be construed as limiting the scope of this disclosure in any way.

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.

[00139] For example, the original downstream process described in He, X. et al., JBC; 35(29):32978-32986 (2003), for purification of rhAC comprises 20X concentration;

Concanavalin A-sepharose (Con A) chromatography; Blue sepharose chromatography;

Superose 12 gel filtration (GF), and ultrafiltration/diafiltration (UF/DF). 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.

[00140] 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

2016/0038574. The process outlined in 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).

[00141] General guidelines were followed for the purification process. Product storage was room temperature if < 24 hours or 2-8°C if > 24 hours. Water used throughout the process was USP Purified Water. Chemicals and reagents used were USP Multicompendial grade, except Tris-HCl which there is no USP compendia. The J.T. Baker Tris-HCl used is cGMP produced, using Tromethamine, USP as a raw material source.

[00142] Single-use sterile containers for rhAC product were used throughout. The materials of construction for wetted parts were polypropylene, PETG, EVA, polystyrene, polycarbonate, and polyethylene. No known incompatibilities were detected.

[00143] Single-use tubing for buffers and rhAC product were used throughout. The materials of construction were C-Flex, Silicone, and Pure Weld XL. Animal-derived content free per EMEA/410/01. No known incompatibilities were detected. The density of the product was assumed to be l.Og/mL for all process steps.

[00144] CHO clones expressing rhAC were developed using technologies to enhance expression of AC (see, e.g., WO2014118619). The 10L process used Clone 47. [00145] 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.

Table 2: Buffer Characteristics

Ethanol, pH 7.2 a. 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.

b. During manufacture of the Packing Buffer (100 mM Sodium Phosphate, 500 mM Sodium Chloride, pH 7.4), 0.3 g/L NaOH Pellets were not used but instead were titrated into the correct pH range with liquid 10N NaOH. The addition of either form of NaOH is to titrate the buffer so using NaOH pellets or 10N NaOH will not impact the buffer. The 200L Non-cGMP Protocol will include the use of NaOH pellets to match the original makeup of the Capto Butyl Chromatography Equilibration/Wash 1 Buffer. However, in this case the buffer will not be required for the 200L Non-cGMP Protocol as columns will come prepacked and qualified. c. 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.

d. 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.

e. The 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.

f. Capto Butyl Chromatography Wash 2 Buffer (225 mM Acetate, 50 mM Sodium Chloride, pH 4.0) conductivity specification was set incorrectly. The conductivity of the 10L

Confirmation Batch buffer lot was 8.77 mS/cm and was 8.74 mS/cm for the small scale Process Development buffer. Based on these two buffer makeups, the conductivity specification will be changed to 8.7 + 2.0 mS/cm for the 200L Non-cGMP Protocol. g. Glacial Acetic Acid USP Multicompendial grade was used for all occasions where Acetic Acid was referenced in the 10L protocol.

h. Capto Butyl Chromatography Elution Buffer (5 mM Tris, pH 9.0) density is 0.99 g/mL. In some embodiments, 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.

i. Different Capto Q pH Load Adjustment and Capto Q Load Conductivity Adjustment Buffers were used for the 10L Confirmation run then those originally proposed in the 10L Protocol. Replacement of the Capto Q pH Load Adjustment buffer from lOmM Sodium Phosphate, pH 6.8 to lOOmM Sodium Phosphate increased the buffering capacity. The Capto Q Load Conductivity Adjustment Buffer will be changed to 10 mM Sodium

Phosphate, 2 M Sodium Chloride, pH 7.4 so pH is already at the set point and decrease the amount of titration buffer required as the Capto Q Load conductivity target has increased from 13 mS/cm to 20 mS/cm. The new Capto Q Load pH and conductivity target and acceptable range will be 7.4 + 0.1 and 150 + 20 mS/cm for the 200L Non-cGMP Protocol.

j. The Capto Q Chromatography Equilibration buffer will be changed for the 200L

Non-cGMP protocol to 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.

k. All buffers targeting a specific pH target had the option of titration with NaOH or HC1 or Acetic Acid (if used previously in the buffer) as needed even if not stated explicitly in the 10L Protocol. The 200L Non-cGMP Protocol will state the option of titration for each buffer with "As needed" for clarity.

1. 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.

[00146] 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

Table 4: Packed Chromatography Columns

a. The GE Capto Butyl resin minimum quantity required was 2000mL as the Capto Butyl column was packed to 20cm requiring more resin.

b. 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.

c. 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. d. 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.

[00147] Clarification process media used in the purification protocol are described in Table 5. The clarification process is described in Table 6.

Table 5: Clarification Process Media

Table 6: Clarification Process Description

Each 0.102m filter was flushed individually at the specified L/m *h flow rate to sufficient flushing of filters.

⁽²⁾ Volume L/m ² calculation is based on Stage 1 filter surface area of 0.306m ² .

a. 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. b. The 0.2μηι filter used was a Sartopore 2 300 0.45/0.2μηι 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 . There were no issues of clogging during the 10L confirmation batch as the 0.03m ² Sartopore 2 filter passed an integrity test post run. The 200L Non-cGMP Protocol will use a Sartopore 2 0.8m ² 0.8/0.2μιη 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.

c. The 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. In some embodiments, 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.

d. 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.

[00148] Virus inactivation used in the purification protocol is described in Table 7. Virus inactivation process parameters are described in Table 8.

Table 7: Virus Inactivation Step Description

10L Confirmation Batch

Step Buffer/ Feed Volume Required

Data

1 M Citric Acid:

CIEX Load 1 M Citric

Not Available 0.046mL/mL of

Adjustment Acid Clarified Harvest

Table 8: Virus Inactivation Process Parameters

a. The amount of 2 M Tris, pH 9.5 required to get the post low pH VI hold product to pH 7.0 was 4.4% of harvest instead of the estimated 0.775% of harvest material. 1 M Tris Base will be used for the 200L Non-cGMP Protocol to reduce the volume of buffer required for titrations. The estimated volumes are pH 4.0: 0.001 L/L of Clarified Filtered Harvest and pH 7.0: 0.019 L/L of Clarified Filtered Harvest.

b. Post VI Filter used was a Sartopore 2 0.05m ² 0.45/0.2μηι PES dual layer filter instead of a single layer 0.2μιη 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 0.45/0.2μιη 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 0.8/0.2μιη 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 .

c. All estimated titration volumes will be updated in the 200L Non-cGMP Protocol with the actual L/L volumes required to reflect the actual process used unless the titration buffer is changing.

d. The post viral inactivation product Hold duration and temperature will be changed to 18-25 °C for≤ 24 hrs.

[00149] 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.

Table 9: Capto S ImpAct CIEX Chromatography Process Description

CIEX Eluate

Table 10: Capto S ImpAct CIEX Process Parameters

a. To meet specifications the Capto S ImpAct Load was diluted with USP water to a conductivity of 13.98 mS/cm from 16.56 mS/cm. This required 0.24 mL of USP / mL of Post VI material.

b. 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.

c. 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.

d. 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%.

e. 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. In addition, 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.

f. 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.

g. The Capto S ImpAct Eluate Hold duration and temperature will be updated for the 200L Non-cGMP Protocol to 18-25 °C for < 7 days.

h. The Capto S ImpAct asymmetry and plates / m (N/m) specification will be updated to 0.8 - 1.8 and > 2000 N/m respectively for the 200L Non-cGMP Protocol. These numbers are based on standard Repligen Opus column specifications and should have no effect on the column operation for this process.

i. The 200L Non-cGMP Protocol will have an updated Capto S ImpAct load conductivity acceptance range of < 16.0 mS/cm.

[00150] 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.

Table 11: Capto Butyl HIC Process Description

Table 12: Capto Butyl HIC Process Parameters

a. 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.

b. The Capto Butyl Column Load was run at 4 minute residence time instead of the specified 2 minutes. The Capto Butyl Column was run at 300 cm/hr and 20cm bed height which equates to a 4-minute residence time. The process step yield of 77.27% was equivalent to the process development experiment 7530-080 yield of 76%. All in-process data showed no concern over change in the residence time.

c. 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. In addition 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.

d. Cap to 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. e. 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.

f. 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. g. 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.

h. During elution the Capto Butyl Elution peak UV absorbance didn't reach the end collection specification of main elution peak. Instead collection ended at the specified 3.5CVs. A small percentage of product was lost due to not collecting to the UV end point of the main elution peak. The Elution step will be extended to 5 CV's from 3.5 CV's to allow the elution UV trace to return to baseline.

i. The Capto Butyl Eluate Hold duration and temperature will be changed to 18-25 °C for < 7 days.

j. The 200L Non-cGMP Protocol will be updated to state "> 5" as going over 5 CV's for equilibration is not an issue. [00151] Table 13 describes the Capto Q AIEX process. Table 14 describes the Capto Q AIEX process parameters. These data confirm the ability of an AIEX step to remove residual DNA from the solution.

Table 13: Capto Q AIEX Chromatography Process Step Description

Table 14: Capto Q AIEX Process Parameters

a. 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.

b. The 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. A new buffer, 10 mM Sodium Phosphate, 115 mM Sodium Chloride, pH 6.8, was developed which matched the conductivity specification and this was what was used for the 10L run. However, due to a low yield the Capto Q step as a whole was further studied and new improved conditions were developed. The 200L Non-cGMP Protocol will now use 10 mM Sodium Phosphate, 185 mM Sodium Chloride, pH 7.4 based on this Capto Q Optimization Study.

d. 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.

e. 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.

f. Column Storage Volume required to get the column effluent pH to the acceptable range was 5 CV's instead of the 3 CV's per the 10L protocol. The parameter for the 200L Non- cGMP Protocol will be increased to 5 CV's of 50 mM Sodium Phosphate, 20% Ethanol, pH 7.2 Storage Buffer. 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. g. Instead of 10 mM Sodium Phosphate, pH 6.0 the Capto Q Load pH Adjustment buffer used was 100 mM Sodium Phosphate Acid. This was thought to have been a better buffer for titrating the load down to pH 6.8. The 100mm Sodium Phosphate Acid Buffer worked well and will be used for the 200L Non-cGMP Protocol.

h. During collection of the Capto Q Wash the UV absorbance never dropped below the specified end collection point prior to the end of the 3.5CV elution block. There was a minimal amount of product lost due to not collecting to the end point of the main elution peak. The wash buffer column volume will be extended to 5 CV's to allow the flow through to return to baseline prior to ending the wash.

i. The Capto Q flow through Acid/Base adjustment was not used in the 10L Confirmation Batch and will not be needed for the 200L Non-cGMP Protocol as the load and wash will already be at pH 7.4. j. The Capto Q Flow through Hold duration and temperature will be changed to 18-25°C for < 7 days for the 200L Non-cGMP Protocol. Note that the product will be in a new buffer matrix due to the change in pH and conductivity but will remain in a lOmM Sodium

Phosphate/Sodium Chloride buffer system. 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.

k. The 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.

1. 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.

m. 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%".

n. The Equilibration volume used was 5.1 CV's which exceeded the specified 5 CV's. Going over the 5 CV's is not an issue since equilibration is based upon pH and Conductivity testing. The specification will be updated in the 200L Non-cGMP protocol to state "> 5".

[00152] Table 15 describes the virus filtration process. Table 16 describes the filtration parameters.

Table 15: Virus Filtration Process Description

Planova 20N 10L Confirmation

Buffer/ Feed Volume (L/m ² )

Process Steps Batch Data

10 mM Sodium

Phosphate, 150 mM

Equilibration > 6

Sodium Chloride, pH 6.5 L/m ² / 0.778 L 7.4

Viral Filtration Not Volume

AIEX Flow through 5.19 L Sample based

10 mM Sodium

Post-flush Phosphate, 150 mM ≥4

4.4 L/m ² / 0.53 L Sodium Chloride, pH 7.4

Table 16: Virus Filtration Process Parameters

a. Pre-Filtration of sample was not performed as it looked visually clean and no titration of the Capto Q flow through was performed. The 200L Non-cGMP Protocol will have a 0.2μιη filtration step prior to viral filtration for increased protection of the viral filters.

b. Viral filtration loading capacity of the Planova 20N for the 200L will remain at 20 - 200 g/rhAC/m ² .

c. The Viral Filtration Flow through Hold duration and temperature will be changed to 18-25 °C for≤ 7 days.

[00153] Table 17 describes the ultrafiltration/diafiltration (DF/UF) process. Table 18 describes the DF/UF n parameters.

Table 17: UF/DF Process Description

Table 18: UF/DF Process Parameters 10 kD UF/DF Acceptance 10L Confirmation

Target /Set Point

Process Parameters Range Batch Data

Recirculation time

prior to product 5 > 5 6 minutes recovery (min)

10 mM Sodium

lx Holdup Volume /

System Flush and Phosphate, 150 mM

2x old-up 135mL

Product Recovery Sodium Chloride, volumes See footnote d

pH 7.4

Concentration rhAC

13.23 mg/ml in final UF Product 10 + 1.0 See footnote f (g/L)

18-25 °C≤24 hrs.,

Product HOLD Temp or 2-8°C > 24 hrs. 18-25 °C / 2-8 °C < 4 hrs at 18-25 °C post UF/DF See footnote g

18-25 °C≤24 hrs.,

Product HOLD

or 2-8°C > 24 hrs. 18-25 °C / 2-8 °C < 4 hrs at 18-25 °C Duration UF/DF

See footnote g

a. 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.

b. UF/DF flush buffer was USP Water not water for injection (WFI) water as WFI was not required for the 10L confirmation run. The 200L Non-cGMP protocol will use WFI Quality water.

c. 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.

d. In order not to dilute the product below the acceptable range a lx Hold-up Volume was used at the end for the system flush and product recovery instead of a 2x Hold-up Volume. This may have resulted in a small percentage loss of product but overall step recovery was still >92%. The 200L Non-cGMP Protocol will continue to have a system flush specification of 2x Hold-up volume.

e. 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.

f. Concentration was 13.23 mg/mL outside of the 10 +/- 1 mg/mL. The acceptable range wasn't critical at this step since dilution below 10 mg/mL + 1 mg/mL is possible.

Dilution with PBS buffer to a concentration within specifications was performed for the 10L confirmation run. The 200L Non-cGMP Protocol will have a new specification of > 9.0 g/L and allow for dilution.

g. The UF/DF Hold duration and temperature will be changed to 18-25 °C for < 2 days. h. 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.

[00154] Table 19 describes the process parameters formulation with buffer. Table 20 describes the drug substance filtration process parameters.

Table 19: Process Parameters Formulation with Buffer

Table 20: Drug Substance Filtration Process Parameters

a. 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.

b. 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.

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.

[00155] Thus, the 10L process was used to assess the yield and analytics of the purification process. During the 10L process, numerous modifications and improvements to the protocol were noted, as described.

[00156] Any observations or discrepancies observed were addressed and the path forward for the Non-cGMP 200L Protocol based on upon knowledge gained from the batch, additional development studies performed, industry standards, and historical experience of the group.

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.

[00158] Table 21 lists 10L confirmation batch step and process yield data.

Table 21: 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.

[00159] 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.

[00160] Table 22 presents data on the rhAC concentrations by reversed phase high- performance liquid chromatography (RP HPLC) and A280nm measurement. Once the rhAC was eluted from the Capto S ImpAct column the 200L Non-cGMP Protocol and batch records will specify the use of the A280nm assay for in-process concentration determination and not the RP-HPLC Concentration assay. After Capto S ImpAct the size-exclusion chromatography HPLC (SEC-HPLC) assay shows that the purity is high enough to allow for the use of the A280 and a comparison between the RP-HPLC and A280nm concentration data show that they match very well. The RP-HPLC concentration assay will only be used for Cell Culture samples, Clarified Harvest and the Capto S ImpAct Load samples. The A280 Assay will remain the release assay for protein concentration of the Drug Substance.

Table 22: Concentration rhAC (In-Process and Bulk Drug Substance) by RP HPLC & A280nm Data Summary

[00161] Table 23 presents data on the purity of rhAC from the 10L process.

Table 23: Purity of rhAC by SEC-HPLC Data Summary

HMW = high molecular weight; LMW = low molecular weig

[00162] 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 24: SDS-PAGE Reduced Data Summary

Table 25: SDS-PAGE Non Reduced Data Summary

[00163] 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

RSD = relative standard deviation.

[00164] 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

[00165] AC activity was measured using an ultra performance liquid chromatography (UPLC) method with the pure enzyme. A substrate stock solution was prepared comprising 200 μΜ 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. Then, 1 mg of C12-NBD ceramide (supplied in chloroform) was dried by nitrogen flow and 742.5 μΐ of 100% ethanol, and 15.2 μΐ of stock Igepal CA-630 (Sigma #1-3021) was added to the dried substrate vial. The substrate stock solution was kept at -20°C and was stable for up to one year.

[00166] Substrate buffer was prepared fresh depending on the number of assays to be run on a given day. For example, to prepare 100 μΐ of substrate buffer, 10 μΐ of substrate stock solution was dried by airflow, followed by addition of 10 μΐ of FBS and 90 μΐ of C/P buffer solution. The solution was suspended by gentle vortex.

[00167] The AC reaction mixture was then prepared. For each assay tube

(#NC9281766, Fisher Scientific), 3 μΐ of substrate buffer was added to 3 μΐ of pure acid ceramidase (50 ng/μΐ), mixed by vortex and then incubated at 37°C for one hour. The reaction was stopped by adding 100% ethanol (1:10; 54 μΐ) followed by centrifugation at 13000g for 5 min. Then, 35 μΐ of supernatant was removed and applied onto a UPLC system with fluorescent detector for analysis.

[00168] For sample analysis, 5 μΐ of supernatant is injected into the UPLC system. The concentration of fatty acid product was calculated by comparing the peak area against the standard curve (see below). The acid ceramidase activity in the sample was reported as micromoles C12-NBD fatty acid produced/liter/hour after multiplying by 20 (dilution factor; i.e., 3 μΐ of pure enzyme was diluted to 60 μΐ prior to UPLC analysis).

[00169] 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 1.7μιη, 2.1x30mm (#186002349). The guard column was ACQUITY BEH C18 VanGuard Pre-column, 1.7 um, 2.1 x 5 mm

(#186003975). The mobile phase comprised A: 0.1% ammonium acetate buffer (pH 7.2) and B: Acetonitrile (Fisher, #A998-4).

[00170] 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.

[00171] The Fluorescence Detector (Waters Acquity system) reference for instrument method setup was 435/525/single/data rate 10/gain 1. The reference for instrument method setup was column temperature: 50°C. [00172] For chromatography analysis, there were two peaks in the UPLC chromatogram. The first at a retention time of 0.4 min was the product of acid ceramidase, C12-NBD fatty acid. The second at a retention time of 0.6 min was the non-hydrolyzed substrate, C12-NBD ceramide.

[00173] For calculation of the standard curve, serial dilutions (0.001, 0.01, 0.1, 1.0, 10.0 uM) were made in ethanol from the C12-NBD fatty acid stock solution (#790440, Avanti Polar Lipids). The standard curve was created with Empower 3 Software (Waters Corp.) by applying the peak area (at retention time 0.4 min) with the given concentration of C12-NBD fatty acid.

[00174] FIG. 5 presents data on AC activity in different process steps of the third 10L run and shows acceptable activity. [

[00175] Table 28 summarizes BDS analytic results for the engineering lot versus the 10L process run.

Table 28: BDS Analytical Results Data Summary

Analytical Method Engineering Lot Specification 10L Run Results

Criteria

Residual Host Cell

<10 pg/mg of Protein < 0.6 pg/mg

DNA

Bioburden <10 CFU/mL <10 CFU/mL

Endotoxin (LAL) < 0.5 EU/mg of protein 0.1 EU/mg

[00176] All final bulk drug substance (BDS) Engineering Run Specifications were met for the 10L confirmation batch, which demonstrates that the process is performing as intended.

Example 3: Modifications made for the 200L Non-cGMP protocol

[00177] As noted in Example 1, numerous modifications for optimizing future protocols were made during the 10L process.

[00178] 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.

Table 29: Process Parameter Modifications for 200L Non-cGMP Protocol

Product Hold Temperature Product Hold Temperature

Clarificati and Duration: 18-25 °C≤ 24 and Duration: 18-25 °C≤ 6 on hrs., or 2-8°C > 24 hrs. hrs.

Neutralization / Capto S Neutralization / Capto S

Viral

ImpAct Load Adjustment ImpAct Load Adjustment

Inactivatio

Buffer: Buffer:

n

2 M Tris, pH 9.5 1 M Tris Base

Viral

Inactivatio Filter Loading: < 1000 g/m ² Filter Loading: < 260 L/m ² n

Product Hold Temperature Product Hold Temperature

Viral

Inactivatio and Duration: 18-25 °C≤ 24 and Duration: 18-25 °C≤ 24 n hrs., or 2-8°C > 24 hrs. hrs.

Load Conductivity

Load Conductivity Adjustment Buffer: USP

Capto S

Adjustment Buffer: Not Water as needed to titrate ImpAct

Specified Capto S Impact Load

conductivity to < 16 mS/cm.

Capto S Pre-Use Sanitization Flow Pre-Use Sanitization Flow rate: 1 ^st CV: 200 cm/hr, ImpAct rate: 300 cm/hr Remaining CV's: 300 cm/hr

Capto S Equilibration Volume: > 5 ImpAct Equilibration Volume: 5 CV CV

Capto S

ImpAct Load Residence Time: 2 min Load Residence Time: 4 min

Capto S Load Conductivity: < 16 Load Conductivity: <16 ImpAct mS/cm mS/cm

Column Storage Flow Rate:

Column Storage Flow Rate: 200 cm/hr

Capto S 300 cm/hr Column effluent Column effluent pH target ImpAct pH target and acceptable and acceptable range: 7.2 and range: 7.0 + 0.5 ≤8.0

Capto S Eluate pH Adjustment Eluate pH Adjustment Buffer: ImpAct Buffer: 2 M Tris, pH 9.5 1 M Tris Base

Capto Butyl Load titration

Capto Butyl Load titration was able to be performed

Capto S will be performed only on either post Capto S ImpAct

ImpAct Capto S ImpAct Elution prior

Elution or prior to Capto

to filtration

Butyl Load

Product Hold Temperature Product Hold Temperature

Capto S and Duration: 18-25 °C≤ 24 and Duration: 18-25 °C / < 7 ImpAct hrs., or 2-8°C > 24 hrs. days

Packed column asymmetry Packed column asymmetry

Capto S

factor: 0.8 to 1.6 factor: 0.8 to 1.8 ImpAct

Plates / m: > 1500 Plates / m: > 2000

Capto Column Bed Height: 15 cm + Column Bed Height: 20 + 1 Butyl 1 cm cm

Packed column asymmetry Packed column asymmetry

Capto

factor: 0.8 - 1.6 factor: 0.8 - 1.8 Butyl

Plates / m: > 1500 Plates / m: > 2000

Sodium Chloride, pH 6.8 Sodium Chloride, pH 7.4

Example 4: Clone selection for scale-up

[00179] To prepare for scale-up of the production process, the optimum clone was determined.

[00180] Three clones were tested to determine which would be the highest-producing for the rhAC molecule using BalancCD ^® CHO Grow A as a base medium and HyClone™ Cell Boost™ Feeds A and B for a feed strategy. For these experiments, Cell Boost 7a was always used at 10X that of Cell Boost 7B. [00181] The 3 clones tested under (4) conditions for this study are as follows:

- Condition 1 : Clone 77 - Ratio of feeds 5%, 0.5% Cell Boost 7a & b

- Condition 2: Clone 09 - Ratio of feeds 5%, 0.5% Cell Boost 7a & b

- Condition 3: Clone 47 - Ratio of feeds 5%, 0.5% Cell Boost 7a & b

- Condition 4: Clone 47 - Repeat (1) with different feed ratio 4%, 0.4% Cell Boost 7a & b

Viable cell density, cell viability, and titer data were collected at Day 12 to determine the highest-performing clone. 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.

Example 5: Feed optimization

[00182] Shake flask studies were performed to determine the optimal feed strategy for clone 47 in BalanCD ^® CHO Growth A medium.

[00183] 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 Boost™ Feeds 7a and 7b, with 5%: 0.5% bolus additions

Condition 2: Efficient Feed B™ 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

Condition 6: Cell Boost Feeds 7a and 7b, with 4%:0.4% bolus additions

[00184] 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.

[00185] 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.

[00186] 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. [00187] 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.

[00188] Thus, regardless of temperature, all Cell Boost feed 7a & 7b conditions outperformed the Efficient Feed B™ and Cell Boost 5 & 6 conditions, except in the case of cell viability. Condition 5 experienced prolonged cell viability whereas all other conditions shared similar profiles. In terms of viable cell density, all Cell Boost conditions peaked above 3E7vc/mL while all other conditions peaked below 2.5E7vc/mL. In the case of titer,

Condition 5 had >1.5x rhAC concentration compared with all other conditions.

[00189] As such the Cell Boost feed 7a and 7b with a Day 4 temperature shift were chosen for future experiments.

Example 6: Sparging and agitation optimization

[00190] Experiments were performed to optimize sparging and agitation in the production process. Clone 47 with BalanCD ^® CHO Media in Xcellerex™ XDR-10 bioreactors was used.

[00191] The purpose/scope of this reactor study was to determine the control parameters in which to operate the XDR-10 reactors for the optimal growth of cells and titer production. Initially two reactors were set up, seeded, and operated comparing two different sparge configurations. The parameters from the highest performing reactor were applied to the next reactor.

[00192] Parameters measured included viable cell density, reactor cell viability, reactor glucose concentration, ammonia concentration, PCO2 concentration, glutamine concentration, reactor pH, and titer concentration.

[00193] Table 30 presents the bioreactor process conditions for Runs 1, 2, and 3.

Table 30: Bioreactor rocess conditions

High Limit

[00194] Cell viability (FIG. 9A) and product titer (FIG. 9B) are shown for the different runs. Data show that the 20μιη 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.

[00195] Three clones were tested to determine which would be the highest-producing for the rhAC molecule using BalancCD ^® CHO Grow A as a base medium and HyClone™ Cell Boost™ Feeds A and B for a feed strategy.

Example 7: Scale-up analysis

[00196] Next, scale-up of the production process was assessed. Clone 47 with

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.

[00197] Data collected from the highest performing XDR 10 conditions described in Examples 1-6 are applied to XDR 200 cell culture runs.

-Engineering run XDR 10 (10L)

-Engineering run XDR 200 (200L)

-cGMP XDR 200 - Toxicology (Tox) Batch (cGMP 200L)

-cGMP XDR 200 - Second Tox Batch (cGMP 200L2)

[00198] A variety of parameters were assessed for the different scale-ups, including viable cell density, reactor cell viability, reactor glucose concentration, ammonia

concentration, PCO2 concentration, glutamine concentration, reactor pH, and titer concentration. [00199] Cell viability data (Figure 10A) and product titer (Figure 10B) shows that the cGMP 200L rung (cGMP 200L and cGMP 200L2) performed the best and were within typical process variability.

[00200] These data indicate that the protocols outlined in these examples have an acceptable yield of rhAC.

Example 8: Formulation screening

[00201] Various excipients were examined to determine which one was best based on rhAC activity.

[00202] The following excipients were tested at room temperature and 4°C:

-rhAC in PBS

-rhAC in PBS + 0.001% Tween® 80

-rhAC in PBS + 1% sucrose

-rhAC in PBS + 1% sucrose, 0.001% Tween 80

[00203] 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.

[00204] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and Examples detail certain embodiments and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the embodiment may be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof. This description and exemplary embodiments should not be taken as limiting.

[00205] For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities, percentages, or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term "about," to the extent they are not already so modified. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[00206] When terms, such as "less than or equal to" or "greater than or equal to," precede a list of numerical values or ranges, the terms modify all of the values or ranges provided in the list. In some embodiments, the numerical values are rounded to the nearest whole number or significant figure.