US20170145068A1 - Novel ifn beta protein analogs - Google Patents

Novel ifn beta protein analogs Download PDF

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US20170145068A1
US20170145068A1 US15/300,288 US201515300288A US2017145068A1 US 20170145068 A1 US20170145068 A1 US 20170145068A1 US 201515300288 A US201515300288 A US 201515300288A US 2017145068 A1 US2017145068 A1 US 2017145068A1
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beta
ifn
protein
composition
deamidated
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Wolf Palinsky
Mara Rossi
Anna R. Pezzotti
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Ares Trading SA
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Ares Trading SA
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/565—IFN-beta
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • IFN-beta Natural interferon-beta
  • IFN-beta is produced by cells mostly in response to viral infections or following an exposure to other biologicals. IFN-beta is implicated in antiviral, anti-proliferative and immuno-modulatory activities.
  • Interferon-beta 1a and Interferon-beta 1b are commercially available for the treatment of relapsing forms of Multiple Sclerosis (M Revel, Pharmacol Ther 2003 October, 100(1): 49-62).
  • IFN-beta 1a is a 166 amino acid glycoprotein with a single N-linked carbohydrate chain on Asn 80 residue.
  • the sequence comprises three cysteines of which two forming a disulphide bond (Cys 31 and Cys 141 ) and one Cys 17 is free and proximal to the surface, but buried.
  • Interferon-beta 1b is non-glycosylated and has a Cys7Ser mutation.
  • Interferon-beta exerts its antiviral function in different ways; one is to elicit antiviral activity from the target cells (inhibition of viral replication) and the other is to induce apoptosis in infected cells (Taniguchi et al., Current Opinion in Immunology, Febuary 2002, 14(1): 111-116).
  • interferon-beta affects cells of the immune system as well as it induces the expression of MHC-class I molecule on cell surface.
  • the virus-infected cells can then be eliminated by cytotoxic T lymphocytes (CTL). This elimination mechanism is based on CTL recognition of viral antigens presented by MHC class I on surface of the infected cells.
  • Immuno-oncology is an evolving approach to cancer care focused at redirecting the patient's immune system to eliminate tumours.
  • tumour cells One of the immuno mechanisms to clear tumour cells by the host system is the killing of tumour cells by CTL. This elimination mechanism is based on CTL recognition of tumour antigens presented by MHC class I on surface of the tumour cells.
  • Viruses and tumours can use immune evasion mechanisms which include the down modulation of MHC-class I expression.
  • Molecules which are able to increase the direct antiviral response and/or the elimination mechanism of infected cells by CTL as well as molecules having a direct anti-proliferative activity and/or the capacity to redirect the host immune system to kill tumour cells have the potentiality to act as a novel and more potent antiviral and cancer therapeutic agent.
  • WO 2006/053134 identifies an IFN-beta 1b protein that shows at most 40% deamidation by storage at 25° C. and 60% relative humidity for 6 month. This protein has an increased anti-viral and anti-proliferative activity. An increased immunomodulatory activity has not been reported.
  • the inventors of the present invention have surprisingly found that deamidation of IFN-beta 1a at an asparagine at amino acid position 25 leads to an increase in immunomodulation, e.g. upregulation of class I MHC and increased antiviral activity.
  • the inventors unexpectedly found that the deamidation-induced increase in immunomodulatory and antiviral activity is dependent on sialylation of IFN-beta 1a.
  • the inventors of the present invention found particular deamidation conditions that lead to an almost complete deamidation of IFN-beta 1a.
  • the deamidated IFN-beta protein of the invention may thus be produced with high biological efficiency and cost-efficiently. Further, the modified IFN-beta protein of the invention may enhance clinical efficacy of, e.g., cancer immunotherapies and antiviral therapies, such as vaccines either alone or in combination with other therapeutic agents or means. In addition, IFN-beta therapies may profit from the use of lower doses of IFN-beta and a reduction of side effects related to IFN-beta treatment.
  • the invention provides a composition comprising interferon-beta (IFN-beta) protein, preferably IFN-beta 1a, at least 80% of which is deamidated at an amino acid asparagine located at an amino acid position corresponding to amino acid position 25 of an interferon-beta 1a protein according to SEQ ID NO:1.
  • IFN-beta interferon-beta
  • the invention further provides a modified interferon-beta (IFN-beta) 1a protein wherein the amino acid asparagine at an amino acid position corresponding to amino acid position 25 of an interferon-beta 1a protein according to SEQ ID NO:1 is deamidated.
  • IFN-beta interferon-beta
  • the invention further provides the composition or the modified IFN-beta 1a protein for use in therapy, for use as immunomodulating agent, for use as a vaccine or in cancer immunotherapy.
  • the modified IFN-beta 1a protein according to the invention has a sequence as defined by SEQ ID NO:2.
  • the invention further provides the composition or the modified IFN-beta 1a protein for use in the treatment of a condition selected from the group consisting of viral infections, cancer, and neuronal disorder.
  • the invention further provides a method of deamidating a protein, preferably IFN-beta, more preferably IFN-beta 1a, comprising:
  • FIGS. 1A and 1B show the primary structure (SEQ ID NO:1) and 3D model structure of human IFN- ⁇ -1a.
  • IFN- ⁇ -1a is a 166 amino acid glycoprotein with a single N-linked carbohydrate chain on Asn 80 residue.
  • the sequence comprises three cysteines of which two forming a disulphide bond (Cys31 and Cys 141 ) and one Cys 17 is free and proximal to the surface but buried.
  • FIG. 2A RP-UPLC Profiles or artificial degraded IFN-beta 1a
  • FIG. 2B is a partial enlargement of FIG. 2A
  • FIG. 3A Deamidation Level of IFN-beta 1a untreated;
  • FIG. 3B is a partial image enlargement of FIG. 3A
  • FIG. 4A Deamidation Level of IFN-beta 1a artificially deamidated
  • FIG. 4B is a partial image enlargement of FIG. 4A
  • FIG. 5 Deamidation Level of IFN-beta 1a untreated and artificially deamidated IFN-beta 1a in an Overlay
  • FIG. 6A Deamidation Level of IFN-beta 1a artificially de-sialylated
  • FIG. 6B is a partial image enlargement of FIG. 6A
  • FIG. 7A Deamidation Level of IFN-beta 1a artificially deamidated and de-sialylated
  • FIG. 7B is a partial image enlargement of FIG. 7A
  • FIGS. 8 A and B Deamidation Level of IFN-beta 1a artificially de-sialylated and artificially deamidated and de-sialylated IFN-beta 1a in an Overlay
  • FIG. 9 MHC class I Immunomodulatory Bioassay: Dose Respones Curves
  • FIG. 10 Immunomodulatory Biological Activity of IFN-beta 1a
  • FIG. 11 Antiviral Activity by A549/EMCV System: Dose Response Curves
  • FIG. 12 Antiviral Activity of IFN-beta 1a
  • the present invention provides a composition comprising interferon-beta (IFN-beta), preferably IFN-beta 1a, protein at least 80% of which is deamidated at an amino acid asparagine located at an amino acid position corresponding to amino acid position 25 of an interferon-beta 1a protein according to SEQ ID NO:1 (cf. FIG. 1 ).
  • IFN-beta interferon-beta
  • protein at least 80% of which is deamidated at an amino acid asparagine located at an amino acid position corresponding to amino acid position 25 of an interferon-beta 1a protein according to SEQ ID NO:1 (cf. FIG. 1 ).
  • the invention provides a modified IFN-beta 1a protein wherein the amino acid asparagine at an amino acid position corresponding to amino acid position 25 of an interferon-beta 1a protein according to SEQ ID NO:1 is deamidated.
  • the modified IFN-beta 1a protein according to the invention has a sequence as defined by SEQ ID NO:2.
  • At least 85%, 90%, 95% or at least 96% of the IFN-beta protein, e.g. IFN-beta 1a, in the composition is deamidated, most preferably 96%.
  • Deamidation is one of the possible modification routes of proteins. This type of modification occurs mainly at asparagine and glutamine residues. For each asparagine undergoing deamidation, three possible modified products are formed:
  • the last two modified products yield a change in the net charge of the protein, since the neutral amide side chain of the asparagine has been converted into the more acidic carboxyl group of aspartic acid.
  • composition as described herein comprises deamidated IFN-beta protein, e.g. IFN-beta 1a, in which the asparagine residue of the amino acid asparagine to be deamidated is replaced by an aspartate residue, an iso-aspartate residue or cyclic imidid residue such as succinimide.
  • IFN-beta protein e.g. IFN-beta 1a
  • the asparagine residue of the amino acid asparagine to be deamidated is replaced by an aspartate residue, an iso-aspartate residue or cyclic imidid residue such as succinimide.
  • 65% to about 70%, more preferably about 68% of the asparagine residue is replaced by asparate, about 15% to about 20%, more preferably about 18%, is replaced by isoasparate and about 8% to about 13%, preferably about 11%, is replaced by succinimide.
  • the asparagine residue of the amino acid asparagine to be deamidated is replaced by an aspartate residue, an iso-aspartate residue or cyclic imidid residue such as succinimide.
  • compositions as described herein further exhibit an increased immunomodulatory activity, preferably an increased upregulation of class I MHC complexes compared to an IFN-beta protein, preferably of SEQ ID NO:1, produced in CHO cells.
  • an increased immunomodulatory activity preferably an increased upregulation of class I MHC complexes compared to an IFN-beta protein, preferably of SEQ ID NO:1, produced in CHO cells.
  • the deamidated or modified INF-beta proteins both as described herein may play a role in cancer immunotherapy or antiviral vaccination by restoring antigen presentation on cell surfaces of tumour or infected cells which in turn would result in increased number of targets on cell surfaces for CTLs activity with a consequent increased efficacy in terms of cell killing activity by CTLs.
  • This immunomodulatory activity might be exploited in IFN-beta monotherapies or in combination with conventional therapies, such as antiviral or cancer (immuno-)therapies.
  • compositions as described further exhibit an increased antiviral activity (cf. Example 3.2).
  • the modified IFN-beta 1a protein or composition both as described herein might comprise an IFN-beta protein that is glycosylated, particularly sialylated.
  • the glycosylation might be an N-linked glycosylation attached to a nitrogen of asparagine or arginine residues; 0-linked glycosylation attached to hydroxy oxgen of a serine, threonine, tyrosine, hydroxylysine or hydroxyproline residues; phospho-serine-linked glycosylation attached to serine residues or C-linked glycosylation attached to an carbon on a tryptophan residue.
  • the glycosylation is an N-linked glycosylation.
  • the glycosylation is attached to an asparagine corresponding to asparagine 80 of SEQ ID NO:1.
  • the modified IFN-beta 1a protein or the composition both as described herein might comprise an IFN-beta protein that has the glycosylation pattern of an IFN-beta protein produced in any eukaryotic cell, such as Chinese hamster ovary (CHO) cells, human embryonic kidney cells (293T), human hepatocellular carcinoma cells (HepG2), human T cell leukemia cells (Jurkat), human T cell acute lymphoblastic leukemia cells (Molt4), human EBV-immortalized B-cell line (Dakiki), human rhabdomyosarcoma cells (RD) and human fibrosarcoma cells (HT1080), preferably CHO cells.
  • CHO Chinese hamster ovary
  • HepG2 human hepatocellular carcinoma cells
  • HepG2 human T cell leukemia cells
  • Jurkat human T cell acute lymphoblastic leukemia cells
  • Molt4 human EBV-immortalized B-cell line
  • RD human
  • the glycan chains of the modified IFN-beta 1a protein might be a tri or tetra-antennary organization, preferably as performed in CHO cells.
  • the modified IFN-beta 1a protein or the composition both as described herein might comprise an IFN-beta protein that is sialylated, preferably with N-acetylneuraminic acid, at the end of glycan chains attached to said modified IFN-beta 1a protein.
  • sialylation of the glycan chains is preferably the sialylation as performed by CHO.
  • all glycan side chains are sialylated, e.g. as performed by CHO cells.
  • composition of the invention might comprise as active agent an IFN-beta protein alone or an IFN-beta protein in combination with other therapeutic agents as described herein, optionally together with a pharmaceutically acceptable carrier, adjuvant or additive as described herein, e.g. as an aqueous solution optionally with further carriers, adjuvants or additives as described herein.
  • modified IFN-beta 1a protein as described herein might be used alone or in combination with other therapeutic agents or pharmaceutically acceptable carriers both as described herein.
  • compositions as described herein might comprise a pharmaceutically acceptable carrier, adjuvant or additive.
  • Such carriers, adjuvants or additives are known in the art and depend on the indication and localization of application in the body. The choice of them might also consider the releasing rate of the therapeutic agent, e.g. immediate release or retard.
  • the compositions or modified IFN-beta 1a protein both as described herein are preferably for systemic administration.
  • the composition or modified IFN-beta 1a protein might be administered orally, locally, e.g., on the skin, or parentally, preferably via injection of infusion.
  • Carriers, adjuvants or additives for parenteral administration are aqua sterilisata, reagents influencing the pH, such as organic and anorganic acids and salts thereof, buffers for adjusting the pH, isotonicity agents, such as natrium chloride, natrium hydrogen carbonate, glucose, fructose, detergents and emulgators, such as Tween®, Cremophor®, oils, such as peanut oil, soja bean oil, ricinus oil, synthetic fatty acid ester, polymeric carriers, complexing agents, preservatives and stabilizers.
  • reagents influencing the pH such as organic and anorganic acids and salts thereof
  • buffers for adjusting the pH isotonicity agents, such as natrium chloride, natrium hydrogen carbonate, glucose, fructose, detergents and emulgators, such as Tween®, Cremophor®
  • oils such as peanut oil, soja bean oil, ricinus oil, synthetic fatty
  • composition as described herein might further comprise an anti-oxidant, such as natrium sulfit or methionine, preferably methionine.
  • an anti-oxidant such as natrium sulfit or methionine, preferably methionine.
  • modified IFN-beta 1a protein as described herein might be in combination with an anti-oxidant as described herein.
  • composition or modified IFN-beta 1a protein both as described herein is for use in therapy, as immunomodulating agent, as a vaccine or in cancer immunotherapy.
  • composition or modified IFN-beta 1a protein both as described herein might be either alone or in combination with other therapeutic agents, such as antiviral agents, e.g. ribavirin, vaccines, immunomodulators such as tumor immunotherapeutics, cytokines, interleukins or chemokines (L-BLP-25 or other tumour-associated antigens).
  • antiviral agents e.g. ribavirin
  • vaccines e.g. ribavirin
  • immunomodulators such as tumor immunotherapeutics, cytokines, interleukins or chemokines (L-BLP-25 or other tumour-associated antigens).
  • An immunomodulating therapy might be designed to elicit or amplify an immune response.
  • the immunomodulating therapy might reduce or suppress the immune response.
  • the immunomodulating therapy elicits or amplifies the immune response.
  • inventive composition or modified IFN-beta 1a protein both as described herein as immunomodulating agent might be used alone or in combination with other immunomodulators as described herein.
  • the vaccine as described herein might be a prophylactic, a therapeutic vaccine or a cancer vaccine.
  • the vaccine might further comprise pharmaceutically acceptable carriers for injection, nano-patch or oral delivery of the vaccine.
  • Such carriers are known by those skilled in the art and might comprise adjuvants, such as albumin, preservatives, such as benzyl alcohol, phenol, m-cresol, formaldehyde or thimerosal, antibiotics and/or stabilizers, such as MSG or 2 phenoxyethanol.
  • composition or modified IFN-beta 1a protein both as described herein might be used in the treatment of a condition selected from the group consisting of viral infections, cancer, and neuronal disorder in a subject.
  • composition or modified IFN-beta 1a protein both as described herein might be used in the treatment of viral infections caused e.g. by hepatitis C virus (HCV), Influenza, Dengue, etc.
  • HCV hepatitis C virus
  • Influenza Influenza
  • Dengue Dengue
  • HCV might cause hepatitis C or chronic liver-related diseases such as cirrhosis, liver failure, and hepatocellular carcinoma which are also treated by the inventive composition or modified IFN-beta 1a protein, both as described herein.
  • Cancers that might be treated by the composition or modified IFN-beta 1a protein both as described herein might be selected from the group consisting of breast cancer, lung cancer, such as non-small cell lung cancer, liver cancer, colorectal cancer, prostate cancer, ovary cancer, brain cancer, biliary cancer, pancreas cancer, etc.
  • composition or modified IFN-beta 1a protein both as described herein might also be used for the treatment of neuronal disorders, such as nerve traumata.
  • neuronal disorders such as nerve traumata.
  • they may be used for nerve regeneration, such as regeneration of the visual nerve, e.g. wherein said treatment comprises regulation of synaptic plasticity or enhancement of axonal.
  • composition or modified IFN-beta 1a protein both as described herein might also be used for the treatment of neuronal disorders, such as multiple sclerosis.
  • a preferred subject as described herein might be a dialysis patient with a HCV infection.
  • the treatments as described herein might comprise immunomodulation, preferably upregulation of class I MHC complexes as described herein.
  • composition and modified IFN-beta 1a protein both as described herein might further be used as described herein in combination with chemoimmunotherapy, chemotherapy, radiotherapy, a vaccine and/or a further therapeutic agent, such as an antiviral agent such as ribavirin, a cytokine such as IL-2 or IFN-alpha or a chemotherapeutic antibody such as rituximab.
  • an antiviral agent such as ribavirin
  • a cytokine such as IL-2 or IFN-alpha
  • chemotherapeutic antibody such as rituximab.
  • inventive composition or modified IFN-beta 1a protein both as described herein might be used in combination with chemoimmunotherapy, such immunotherapies being based on tumor-associated antigens, for example in combination with L-BLP25.
  • the inventive or modified IFN-beta 1a protein both as described herein might be used as described herein in combination with chemotherapy and/or radiotherapy.
  • the modified protein or composition might be used to enhance antitumor immune response through T cell cytotoxicity during metronomic chemotherapy, as well as to increase efficacy of combined chemo- (or radio-)therapy.
  • the invention further provides a method of deamidating a protein, comprising:
  • the protein to be deamidated is IFN-beta, particularly IFN-beta 1a.
  • the incubation as described herein might be conducted at a pH of about 8.9 to about 9.5., preferably at about pH 9.2.
  • the incubation as described herein might be conducted in any suitable buffer for deamidation.
  • buffers are known by those skilled in the art.
  • the incubation is conducted in ammonium hydrogen carbonate, more preferably in a final concentration of 0.2 M.
  • the incubation as described herein further is at a temperature of about 20° C. to about 25° C., such as about 21° C. to about 24° C., preferably at about 23° C.
  • the purification as described herein may comprise any purification methods known in the art.
  • the purification comprises ultra-filtration, more preferably ultra-filtration with ammonium acetate pH 3.8.
  • IFN- ⁇ -1a degraded forms were prepared by chemical (basic pH for deamidated), and enzymatic (sialidase for de-sialylated) treatment of IFN- ⁇ -1a DS (drug substance) material.
  • the experimental design foresaw physic-chemical and biological characterization of untreated and artificially deamidated IFN- ⁇ -1a aimed at evaluating the impact of deamidation on IFN- ⁇ -1a immunomodulatory and antiviral activity.
  • the design included the testing of the untreated and deamidated samples after sialic acid removal as well in order to evaluate the role, if any, of sialylation in regulating these specific biological activities.
  • IFN-beta-1a DS buffer 50 mM Ammonium Acetate pH 3.8
  • Sample corresponding to untreated IFN-beta 1a shows protein content aligned to the value expected for an untreated IFN-beta 1a ( ⁇ 300 ⁇ g/ml). Protein content detected both in IFN-beta 1a artificially deamidated and IFN-beta 1a artificially deamidated and desialylated is higher than the untreated IFN-beta 1a because of sample ultrafiltration and concentration in Amicon Ultra.
  • deamidation treatment applied ensures almost complete IFN- ⁇ -1a deamidation with a level of nearly 97%.
  • de-sialylation treatment does not alter the deamidation level with respect to the untreated sample.
  • the immunomodulatory assay is based on the capability of IFN- ⁇ -1a to up regulate the MHC class I expression in A549 cells in a dose related manner.
  • the expression of MHC class I is detected by flow cytometry using a specific fluorescent labelled antibody.
  • A549 cells (32.000 cells/well) were incubated with 12 different concentrations of IFN- ⁇ -1a ranging from 0.000381 ng/mL up to 1600 ng/mL for 48 hours at 37° C., 5% CO 2 .
  • untreated cells were run as well.
  • cells were harvested and the expression of MHC class I was evaluated by FACS analysis using a FITC-conjugated anti-hMHC class I antibody. FACS analysis was performed according to the internal procedure.
  • the dose response curve of the reference and samples are fitted by 4PL algorithm and the concentration able to lead the 50% of the maximum possible expression (EC 50 ) is automatically calculated.
  • Results are expressed as activity percentage with respect to the reference material on the basis of the EC 50 values. For each sample, results are the average of three independent assays performed over three different weeks and each of which is composed by two independent runs (a total of 6 analytical runs).
  • IFN-beta-1a The antiviral activity of IFN-beta-1a was evaluated measuring the protection exerted by the IFN-beta-1a on A549 cells against the cytopathic effect of Encephalomyocarditis virus (EMCV). A brief description of the method is shown below.
  • A549 cells were plated (40.000 cells/well) in a 96-well microtiter plate containing IFN-beta-1a in a concentration range from 0.016 ng/ml up to 2 ng/ml and then incubated for 20 hours at 37° C., 5% CO 2 . At the end of the incubation time, EMCV suspension was added in each well. After about 24 hours of incubation at 37° C., 5% CO 2 , ATPLite 1 Step was added and the cps were measured in each well by a luminometer microplate reader to assess the proliferation and the vitality of the cells.
  • the dose response curve of the reference and samples is fitted by 4PL algorithm and the concentration able to lead the 50% of the maximum possible expression (EC 50 ) is automatically calculated.
  • Results are expressed as activity percentage with respect to the reference material on the basis of the EC 50 values. For each sample, results are the average of three independent assays performed over three different days.
  • the deamidation process lead to an IFN- ⁇ -1a with a higher capability acquired to increase the biological response revealed. Contrarily, this deamidated feature cannot be seen in IFN- ⁇ -1a deamidated/de-sialylated sample when compared to the IFN- ⁇ -1a de-sialylated, the deamidation process in this case is not able to rescue the effect of the de-sialylated process on the biological activity.

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US20070292391A1 (en) * 2004-06-01 2007-12-20 Ares Trading S.A. Stabilized Interferon Liquid Formulations
US20120082647A1 (en) * 2010-10-01 2012-04-05 Baker Darren P Interferon-beta for use as monotherapy or in combination with other cancer therapies

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