Polyethylene glycol modified interferon alpha 2b and preparation method and applicatioins thereof

FIELD OF THE INVENTION

0001The present invention relates to interferon-α2b modified with Y-shaped branched polyethylene glycol (PEG) at a single amino acid residue and the preparation thereof, as well as the use of the prepared PEGylated IFN-α2b at a single amino acid residue in pharmaceutical field.

BACKGROUND OF THE INVENTION

0002Interferons (IFNs) are a family of small molecule proteins or glycoproteins produced by eukaryotic cells in response to viral infection and other antigenic stimuli, which display broad-spectrum antiviral, antiproliferative and immunomodulatory effects. IFNs have been widely applied in the treatment of various conditions and diseases, such as viral infections, e.g. hepatitis B, hepatitis C and HIV; inflammatory disorders and diseases, e.g. multiple sclerosis, arthritis, asthma, cystic fibrosis and interstitial lung disease; and tumors, e.g. myelomas, lymphomas, liver cancer, lung cancer, hairy-cell leukemia, and so on (<nplcit id="ncit0001" npl-type="s"><text>Kenji Oritani, Paul W Kincade, et al. Type I interferon and limitin: a comparison of structures, receptors, and functions. Cytokine and Growth Factor Reviews, 12, 337-348, 2001</text></nplcit>; <nplcit id="ncit0002" npl-type="s"><text>Yu-Sen Wang, Stephen Youngster, et al. Structural and biological characterization of PEGylated recombinant interferon alpha-2b and its therapeutic implications. Advance Drug Delivery Reviews, 54, 547-570, 2002</text></nplcit>).

0003IFNs are classified into four types according to their differences in chemical, immunological, and biological properties: interferon- α, β, γ and ε. Interferon-α (IFN-α) is secreted by leukocytes. Human IFNs-α are encoded by a multigene family consisting of about 20 genes, the encoded proteins sharing up to about 90% amino acid sequence homology (<nplcit id="ncit0003" npl-type="s"><text>Henco K., Brosius F.J., et al. J. Mol. Biol., 185, 227-260, 1985</text></nplcit>). Human IFN-α2b is one of the subtypes of the α2 subfamily of human IFN-α family, and is a single chain protein with various biological activities. The single chain protein consists of 165 amino acid residues with 4 Cys, wherein two intrachain disulfide bonds are formed between Cys1-Cys98 and Cys29-Cys138 respectively and the N-terminal amino acid is Cys with one free α-NH₂ group. The residues in positions 31, 49, 70, 83, 112, 121, 131, 133, 134 and 164 of the amino acid sequence are Lys, each of which contains one free ε-NH₂ group. This protein is not glycosylated and sensitive to many proteases. The amino acid sequence of human interferon-α2b is shown in SEQ ID No: 1.

0004IFNs are usually administered parenterally in clinical treatments. The short <i>in vivo</i> half-life (2-4h) and strong immunogenicity of IFNs result in a shorter dosing interval and a higher dosing frequency. As the generated antibodies significantly decrease the therapeutic efficacy, it is difficult to achieve ideal clinical efficacy. The polyethylene glycol (PEG) modification technology developed in recent years has provided a possible solution to the above problems.

0005PEG is an inert, nontoxic and biodegradable organic polymer, and is important in the fields of both biotechnology and pharmaceutics. PEG modification technique is to link PEG to an active protein via covalent bond. After the polyethylene-glycolation (PEGylation), the properties of the protein can be significantly improved, e.g. the prolongation of drug metabolic half-life, the reduction of immunogenicity, the increase of safety, the improvement of therapeutic efficacy, the decrease of dosing frequency, the increase of drug solubility/water solubility, the increase of resistance against proteolysis, the facilitation of drug controlled release and so on. For further details please refer to <nplcit id="ncit0004" npl-type="s"><text>Inada et al. J. Bioact. and Compatible Polymers, 5, 343, 1990</text></nplcit>, <nplcit id="ncit0005" npl-type="s"><text>Delgado et al. Critical Reviews in Therapeutic Drug Carrier Systems, 9, 249, 1992</text></nplcit>, <nplcit id="ncit0006" npl-type="s"><text>Katre. Advanced Drug Delivery Systems, 10, 91, 1993</text></nplcit>, and <patcit id="pcit0001" dnum="USUP4179337A"><text>U.S. Patent publication UP 4179337</text></patcit>.

0006It is disclosed in <patcit id="pcit0002" dnum="US4179337A"><text>U.S. patent No. 4179337</text></patcit>, after linking PEG to an enzyme or insulin, the immunogenicity of the protein was reduced, while simultaneously the activities of the protein were reduced as well. This was also found in G-CSF (<nplcit id="ncit0007" npl-type="s"><text>Satake-Ishikawa et al. Cell Structure and Function, 17, 157-160, 1992</text></nplcit>), IL-2 (<nplcit id="ncit0008" npl-type="s"><text>Katre et al. Proc. Natl. Acad. Sci. USA, 84, 1487, 1987</text></nplcit>), TNF-α (<nplcit id="ncit0009" npl-type="s"><text>Tsutsumi et al. Jpn. J. Cancer Res., 85, 9, 1994</text></nplcit>), IL-6 (<nplcit id="ncit0010" npl-type="s"><text>Inoue et al. J. Lab. Clin. Med., 124, 529, 1994</text></nplcit>) and CD4-IgG (<nplcit id="ncit0011" npl-type="s"><text>Chamow et al. Bioconj. Chem., 5, 133, 1994</text></nplcit>).

0007It was reported that the branched PEG-modified protein exhibited better pH tolerance, thermo-stability and resistance against proteolysis than linear chain PEG-modified proteins (<nplcit id="ncit0012" npl-type="s"><text>Monfardini et al. Bioconjugate Chem., 6, 62, 1995</text></nplcit>). Generally, a PEG molecule modifies a protein by linking itself to the N-terminal α-amino group or ε-amino groups of a Lys residue within the protein molecule. There are normally three types of PEGs for protein modification: a linear chain PEG (<patcit id="pcit0003" dnum="EP0593868A"><text>EP 0593868</text></patcit>), an U-shaped branched PEG (<patcit id="pcit0004" dnum="EP0809996A"><text>EP 0809996</text></patcit>) and an Y-shaped branched PEG (<patcit id="pcit0005" dnum="CN1243779C"><text>CN1243779C</text></patcit>).

0008Currently many kinds of PEGylated proteins have been applied clinically. In 1990, the PEGylated-bovine adenosine deaminase (Adagen) produced by ENZON Inc. was approved by FDA, and used to treat severe combined immunodeficiency disease (pegfamg013102LB, <u>http://www.fda.gov</u>). In 1994, another PEG-modified protein for treating acute lymphoblastic leukemia, the PEGylated asparaginase (pegaspargase, Oncaspar), was also marketed in US (103411s50521b1, <u>http://www.fda.gov</u>). The PEG modified interferon-α2b (PEG IFN-α2b, PEG-Intron) developed by Schering-Plough was approved by FDA for marketing in 2000 and the PEGylated interferon-α (PEG IFN-α2a, Pegasys) produced by Hoffman-la Roche Ltd. was also approved for marketing in 2002, both of which are used to treat hepatitis (103964s50371b1, pegsche011901LB, <u>http://www.fda.gov</u>). In 2002, the PEG modified human granulocyte colony-stimulating factor produced by Amgen Inc. (PEG-filgrastim, Neulasta) was also approved by FDA, which is used to treat metastatic breast cancer (pegfamg013102LB, <u>http://www.fda,gov</u>). The FDA also accepted the application for PEGylated human growth factor antagonist developed by Pharmacia. The PEG combined TNF-α antibody fragment from Celltech and the PEG-TNF receptor from Amgen are tested in the advanced clinical trials. The first PEG-organic molecule conjugate, PEGylated camptothecin, has also entered phase II of clinical trial. In 2004, the PEG modified oligonucleotide (Pegaptanib, Macugen^™) was approved by FDA. The <i>in vivo</i> metabolism of the PEG in the drug (or PEG itself) has already been clearly understood, and PEG has been proven to be a good and safe drug modifier without any adverse effect.

0009The PEGs that can be linked to a protein drug normally need derivatization, so that one or two terminal groups of the ends of PEGs can be chemically activated to possess a proper functional group which displays activity, and thus can form a stable covalent bond with, at least one functional group of the drug to be linked. For example, PEGs can be linked to ε-NH₂ of Lys residue within the protein peptide chain, or to α-NH₂ of the N-terminal amino acid residue of the protein peptide chain. In the PEGylation of IFN-α described in European patent <patcit id="pcit0006" dnum="EP0809996A"><text>EP0809996</text></patcit>, PEG-NHS is linked through nucleophilic substitution to α-NH₂ of the N-terminal amino acid or ε-NH₂ of Lys in IFN-α. The PEG-NHS mentioned in the above patent is a U-shaped branched PEG derivative (PEG₂-NHS), the molecular formula thereof as below: <chemistry id="chem0001" num="0001"><img file="EP2186830B1_D0001.tif" /></chemistry> wherein, R and R' are independently a low molecular weight alkyl group, n and n' are from 600 to 1500, and the average molecular weight of the PEG is from 26KD to 66KD. The molecular formula of the PEG₂-NHS-modified IFN-α is as below: <chemistry id="chem0002" num="0002"><img file="EP2186830B1_D0002.tif" /></chemistry>

0010Where one or more PEG₂-NHS molecules are linked to α-NH₂ of the N-terminal amino acid or ε-NH₂ of Lys in IFN-α, the obtained products are a mixture of non-PEGylated IFNs-α, PEGylated IFNs-α at a single amino acid residue and PEGylated IFNs-α at multiple amino acid residues. The PEGylated IFN-α at a single amino acid residue can be isolated from the obtained products by any appropriate purification means. IFN-α has one N-terminal amino acid and more than one Lys residues, namely several reactive sites for PEG₂-NHS, so the isolated PEGylated IFNs-α at a single amino acid residue are a mixture of the isomers of the PEGylated IFNs-α at different single amino acid residues.

0011In European patent <patcit id="pcit0007" dnum="EP0593868A"><text>EP 0593868</text></patcit>, linear-chain PEG is used to modify IFN, the molecular formula of the modified product as below: <chemistry id="chem0003" num="0003"><img file="EP2186830B1_D0003.tif" /></chemistry> wherein R is a low molecular weight alkyl group; R₁, R₂, R₃ and R₄ are H or low molecular weight alkyl groups; m is from 1 to the number of possible PEG modification positions in IFN; W is O or NH; x is from 1 to 1000, y and z are from 0 to 1000, x+y+z is from 3 to 1000; and at least one of R₁, R₂, R₃ and R₄ is a low molecular weight alkyl group. Yu-Sen Wang et al (<nplcit id="ncit0013" npl-type="s"><text>Yu-Sen Wang et al, Advanced Drug Delivery Reviews, 54: 547-570, 2002</text></nplcit>. <nplcit id="ncit0014" npl-type="s"><text>Yu-Sen Wang et al, Biochemistry, 39, 10634-10640, 2000</text></nplcit>.) have reported the modification of rIFN-α2b with 12KD linear monomethoxy-PEG (Peg-Intron) and shown that the products analyzed by HPLC-IE are a mixture of more than 14 isomers modified by PEG at different single amino acid residues. The molecular formula of the linear PEGs used by Yu-Sen Wang et al is shown below: <chemistry id="chem0004" num="0004"><img file="EP2186830B1_D0004.tif" /></chemistry> wherein the average molecular weight of the PEG is 12KD.

SUMMARY OF THE INVENTION

0012The PEG derivatives used in the present invention are novel branched, Y-shaped branched PEG derivatives, and their structures are different from those of the U-shaped branched PEGs. The biggest difference between these two kinds of PEGs is that: two-branch PEG chains of the Y-shaped PEG derivatives according to the present invention are connected together through N atom, while the two-branch PEG chains of the U-shaped PEG derivatives in <patcit id="pcit0008" dnum="EP0809996A"><text>EP0809996</text></patcit> are connected together through C atom. The molecular composition of the Y-shaped PEG derivatives according to the present invention is shown as below: <chemistry id="chem0005" num="0005"><img file="EP2186830B1_D0005.tif" /></chemistry> wherein, P_a and P_b are same or different PEGs; j is an integer from 1 to 12; R_i is H, a substituted or unsubstituted C1-C12 alkyl group, a substituted aryl, an aralkyl or a heteroalkyl; X₁ and X₂ are independently a linking group, wherein X₁ is (CH₂)_n, and X₂ is selected from the group consisting of (CH₂)_n, (CH₂)_nOCO, (CH₂)_nNHCO, and (CH₂)_nCO; n is an integer from 1 to 10; and F is a terminal group selected from the group consisting of a hydroxyl group, a carboxyl group, an ester group, acyl chloride, hydrazide, maleimide, pyridine disulfide, capable of reacting with amino, hydroxyl or mercapto group of a therapeutic agent or a substrate to form a covalent bond.

0013In one preferred embodiment of the present invention, the Y-shaped PEG derivative molecule is shown as below: <chemistry id="chem0006" num="0006"><img file="EP2186830B1_D0006.tif" /></chemistry> wherein, R and R' are independently a C1-C4 alkyl group, preferably methyl; m and m' denote the degree of polymerization and can be any integer; m+m' is preferably from 600 to 1500; R_i is H, a substituted or unsubstituted C1-C12 alkyl, a substituted aryl, an aralkyl, or a heteroalkyl group; j is an integer from 1 to 12; and F is a terminal group selected from the group consisting of a hydroxyl group, a carboxyl group, an ester group, carboxylic acid chloride, hydrazide, maleimide, pyridine disulfide, capable of reacting with an amino group, a hydroxyl group or a mercapto group of a therapeutic agent or a substrate to form a covalent bond. Preferably, the average total molecular weight of the PEG is from about 10000 to about 60000 Dalton, most preferably about 40000 Dalton.

0014In one preferred embodiment of the present invention, a possible structural formula of the Y-shaped PEG derivative molecule is shown as formula (I): <chemistry id="chem0007" num="0007"><img file="EP2186830B1_D0007.tif" /></chemistry> wherein R and R' are independently a C1-C4 alkyl group, preferably methyl; m and m' denote the degree of polymerization and can be any integer; m+m' is preferably from 600 to 1500; j is an integer from 1 to 12; and the average total molecular weight of the PEGs is about 40000 Dalton.

0015The present inventors used Y-shaped branched PEG derivatives (YPEG) to modify interferon-α2b (IFN-α2b), and isolated the YPEG-IFNs-α2b, modified by PEG at a single amino acid residue, by Q-Sepharose FF ion-exchange chromatography. Moreover, the isolated YPEG-IFNs-α2b, modified by PEG at a single amino acid residue, were further separated by SP-Sepharose FF chromatography to obtain YPEG-IFN-α2b wherein the YPEG is principally linked to the side chain ε-NH₂ of Lys at position 134 in SEQ ID NO.1, which is called YPEG-IFN-α2b(134). After measurement, it is found that the <i>in vitro</i> activity of the YPEG-IFN-α2b(134) is significantly higher than that of the YPEG-IFN-α2b in which the YPEG is linked to another amino acid residue, and the half-life of the YPEG-IFN-α2b(134) in serum is significantly longer than that of the unmodified IFN-α2b.

0016Therefore, the present invention provides PEGylated IFNs-α2b at a single amino acid residue, the structure of which is as below: <chemistry id="chem0008" num="0008"><img file="EP2186830B1_D0008.tif" /></chemistry> wherein P_a and P_b are same or different PEGs; j is an integer from 1 to 12; R_i is H, a substituted or unsubstituted C1-C12 alkyl group, a substituted aryl, an aralkyl, or a heteroalkyl group; X₁ and X₂ are independently a linking group, wherein X₁ is (CH₂)_n, and X₂ is selected from the group consisting of (CH₂)_n, (CH₂)_nOCO, (CH₂)_nNHCO and (CH₂)_nCO, wherein n is an integer from 1 to 10.

0017In one preferred embodiment of the present invention, the PEGylated IFNs-α2b of the present invention is of the structural formula (II) below: <chemistry id="chem0009" num="0009"><img file="EP2186830B1_D0009.tif" /></chemistry> wherein R and R' are independently a C1-C4 alkyl group, preferably methyl; j is an integer from 1 to 12; m and m' denote the degree of polymerization and can be any same or different integers. In this structure, a Y-shaped branched PEG molecule is linked to an IFN-α2b molecule via one single amino acid residue. The average molecular weight of the YPEG-IFN-α2b in formula (II) depends principally on the degree of polymerization, m and m'. Where m+m' is preferably from 600 to 1500, the corresponding average molecular weight of the YPEG is from about 26000 to about 66000 Dalton. Where m+m' is preferably from 795 to 1030, the corresponding average molecular weight of the YPEG is from about 35000 to about 45000 Dalton. Where m+m' is preferably from 885 to 1030, the corresponding average molecular weight of the YPEG is from about 39000 to about 45000 Dalton. Where m+m' is most preferably 910, the corresponding average molecular weight of the YPEG is 40000 Dalton. The ratio of m and m' can be in a range from 0.5 to 1.5, preferably from 0.8 to 1.2.

0018In one preferred embodiment, in the PEGylated IFN-α2b of the present invention, a PEG molecule is linked to IFN-α2b via an amido bond formed by α-amino group of the N-terminal amino acid or the side chain ε-amino group of Lys residue of IFN-α2b corresponding to position 31, 49, 70, 83, 112, 121, 131, 133, 134, or 164 as shown in SEQ ID No.1.

0019In a further preferred embodiment, in the PEGylated IFN-α2b of the present invention, a PEG molecule is linked to IFN-α2b via an amido bond principally formed by the side chain ε-amino group of Lys residue of IFN-α2b corresponding to position 134 as shown in SEQ ID No.1.

0020Optionally, the IFN-α2b of the present invention can be extracted from natural sources or obtained by the recombinant biotechnology. Preferably, the IFN-α2b is human IFN-α2b (hIFN-α2b) having the amino acid sequence of SEQ ID No.1, which is extracted from natural sources or obtained by the recombinant biotechnology. More preferably, the human IFN-α2b is recombinant human IFN-α2b (rhIFN-α2b). The rhIFN-α2b can be artificially synthesized, or be expressed from prokaryotic expression systems such as <i>E</i>. <i>coli,</i> or be expressed from eukaryotic yeast expression systems such as <i>Pichia,</i> or be expressed from insect cell expression systems or mammalian cell expression systems such as CHO. The preparation methods of the natural or recombinant IFN-α2b and the activity tests of IFN-α2b and YPEG modified IFN-α2b are known in prior art.

0021Similar to IFN-α2b, the YPEG-IFN-α2b of the present invention can also be used clinically to treat tumors and viral infections, such as hepatitis, hairy-cell leukemia, cell-mediated lympholysis, Kaposi's sarcoma and so on. In clinical, the YPEG-IFN-α2b of the present invention is clearly improved, as compared to IFN-α2b, in stability, solubility, half-life in serum and clinical therapeutic efficacy. For the mode of administration, the YPEG-IFN-α2b of the present invention can be administered to the patients in the form of a composition comprising a pharmaceutically effective amount of the YPEG-IFN-α2b and a pharmaceutically acceptable carrier or excipient. Hence, the present invention, in another aspect, also provides a composition comprising a pharmaceutically effective amount of the PEGylated IFN-α2b of the present invention and a pharmaceutically acceptable carrier or excipient. Preferably, the composition comprises mannitol, amino acids, sodium chloride and sodium acetate, wherein the amino acids are preferably selected from the group consisting of aspartic acid, asparagine and glycine.

0022In another aspect, the present invention also provides the use of the PEGylated IFN-α2b of the invention or the composition comprising the PEGylated IFN-α2b of the invention in the preparation of a medicament for treating a disease in need of IFN-α2b treatment. Preferably, the disease in need of IFN-α2b treatment is selected from the group consisting of viral infections e.g. hepatitis B, hepatitis C, hepatitis D and condyloma acuminatum, tumors e.g. hairy-cell leukemia, chronic myeloid leukemia, low-grade malignant non Hodgkin's leukemia, cell-mediated lympholysis, Kaposi's sarcoma, multiple myeloma, malignant melanoma, cutaneous T-cell lymphoma, laryngeal papilloma, recurrent or metastatic renal cell carcinoma, inflammatory disorders and diseases e.g. multiple sclerosis, arthritis, asthma, cystic fibrosis and interstitial lung disease, and myeloproliferative diseases related thrombocythemia.

0023In order to obtain the YPEG modified IFN-α2b, in one embodiment of the present invention, initially the PEG moiety of activated YPEG derivatives such as PEG N-hydroxyl succinimidyl ester (YPEG-NHS) is covalently linked to an amino (-NH₂) group of the protein through nucleophilic substitution, wherein the amino group includes N-terminal α-amino group of the protein and an ε-amino group of Lys residue. The reaction equation for the generation of YPEG-IFN-α2b from IFN-α2b and YPEG is as below: <chemistry id="chem0010" num="0010"><img file="EP2186830B1_D0010.tif" /></chemistry>

0024The reaction conditions are mild, the pH is in a range from 4.5 to 9.5, the temperature is between 0-25°C, and stirring or other blending measures are necessary. For detailed conditions please refer to the Examples in <b>DETAILED DESCRIPTION OF THE INVENTION</b>. All YPEGs with different molecular weights can be linked to IFN-α2b using the above method. The products include the modified by PEG at a single amino acid residue (YPEG-IFN-α2b), the modified by PEG at two amino acid residues (YPEG₂-IFN-α2b) and the modified by PEG at multiple amino acid residues (YPEG_n-IFN-α2b), wherein the products modified by PEG at a single amino acid residue can be the predominant products by adjusting the reaction condition.

0025Subsequently, the YPEG-IFN-α2b, modified by PEG at a single amino acid residue, can be isolated from the mixture of all kinds of the YPEG modified IFNs-α2b using a method such as cation exchange chromatography, anion exchange chromatography, or exclusion chromatography, and then the IFNs-α2b modified by PEG at different single amino acid residues can be further resolved to obtain the YPEG-IFN-α2b in which the YPEG is linked at a specific position. Conventional purification methods include cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography and exclusion chromatography. Characteristic analysis can be performed by a known method in the art, e.g. the mass spectroscopy, the polyacrylamide gel electrophoresis and the high-performance liquid exclusion chromatography can be used to analyze the molecular weight of the products, so as to distinguish the products modified by PEG at a single amino acid residue from those modified by PEG at two or multiple amino acid residues and unmodified IFN-α2b. The above mentioned purification methods can also be used to further resolve the products modified by PEG at a single amino acid residue to obtain different isomers with the PEG modification at different single positions. The <i>in vitro</i> biological activities of all kinds of the PEG modified products can be measured according to any known assay for IFN-activity, e.g. cytopathic effect inhibition. For IFNs modified by PEG at a single amino acid residue, the PEG moieties in the different isomers have different effects on maintaining the active domains of IFNs, resulting in the great differences in the biological activities of different isomers. Generally speaking, the <i>in vitro</i> activities of IFNs are remarkably decreased after PEG modification. However, according to the present invention, the <i>in vitro</i> specific activity of the isolates of three peaks obtained by ion exchange chromatography have been measured, and the results indicate that the isolate of peak 3 (SP2) has significantly higher specific activity than the isolates of other peaks and PEGASYS (Hoffmann-La Roche, Basel, Switzerland), and has significantly longer half-life in serum than unmodified IFN-α2b.

0026In a further embodiment, the Y-branched PEG linked peptide isolated from the SP2 is sequenced using Edman degradation, and the results showed that the primary component of SP2 was YPEG-IFN-α2b(134).

0027Hence, in another aspect, the present invention also provides the preparation and purification methods for YPEG-IFN-α2b(134), comprising: <ol id="ol0001" compact="compact"><li>(a) under an alkaline condition, preferably at pH 9.0, allowing Y-shaped branched PEG as shown in formula (I) below to react with IFN-α2b, and obtaining PEGylated IFN-α2b.; <chemistry id="chem0011" num="0011"><img file="EP2186830B1_D0011.tif" /></chemistry> wherein R and R' are independently a C1-C4 alkyl group, preferably methyl; j is an integer from 1 to 12; m and m' denote the degree of polymerization and can be any integer; and m+m' is preferably from 600 to 1500;</li><li>(b) capturing the reaction products in step (a) with an anion exchange resin, preferably Q Sepharose FF, and eluting the products in an anion gradient, preferably in a chloride ion gradient, to obtain modified products;</li><li>(c) eluting the reaction products captured in step (b) with a cation exchange resin, preferably SP Sepharose FF, in a cation gradient, preferably in a sodium ion gradient, and collecting each peak separately:</li><li>(d) determining the activity of the product from each peak, and selecting the peak corresponding to the reaction product with highest activity.</li></ol>

BRIEF DESCRIPTION OF THE DRAWINGS

0028<ul id="ul0001" list-style="none" compact="compact"><li><figref idref="f0001">FIG.1</figref>: SDS-PAGE of 2 batches of IFN-α2b modified with YPEG (40KD). The concentration of the separation gel was 12%, and Coomassie brilliant blue R-250 was used as staining dye. It can be seen from the SDS-PAGE result of <figref idref="f0001">FIG.1</figref>, under the condition, the PEG modification rate of rHuIFN-α2b was between 30-50%, and was stable. The primary modified products were IFN-α2b modified by PEG at a single amino acid residue (YPEG-IFN-α2b), and there were also some IFN-α2b modified by PEG at multiple amino acid residues (YPEGn-IFN-α2b). Lane 1: IFN-α2b, NHS-YPEG (40KD) modification reaction at 0h; Lane 2: Batch 060604-1 of IFN-α2b, NHS-YPEG (40KD) modification reaction at 2h; Lane 3: Batch 060604-2 of IFN-α2b, NHS-YPEG (40KD) modification reaction at 2h; Lane 4: marker (GE Lifescience).</li><li><figref idref="f0002">FIG.2</figref>: The resolving profile of YPEG-IFN-α2b modification isomers by SP-Sepharose FF.</li><li><figref idref="f0003">FIG.3</figref>: Silver-stained SDS-PAGE (12%) of the YPEG-IFN-α2b samples purified by SP-Sepharose FF. Lane 1: molecular weight marker (GE Lifescience); Lane 2: SP-Sepharose FF purification peak 1 of YPEG-IFN-α2b; Lane 3: SP-Sepharose FF purification peak 2 of YPEG-IFN-α2b, Lane 4: SP-Sepharose FF purification peak 3 of YPEG-IFN-α2b; Lane 5: SP-Sepharose FF purification peak 4 of YPEG-IFN-α2b.</li><li><figref idref="f0004">FIG.4</figref>: Apparent molecular weight of the SP-Sepharose FF purified YPEG-rHuIFN-α2b sample determined by 7.5% reducing SDS-PAGE with silver staining. Lane 1: molecular weight marker (GE Lifesciences); Lane 2: YPEG-HuIFN-α2b SP1, 2µg; Lane 3: YPEG-rHuIFN-α2b SP2, 2µg; Lane 4: YPEG-rHuIFN-α2b SP3, 2µg.</li><li><figref idref="f0005">FIG.5</figref>: The molecular weights of the SP-Sepharose FF purified YPEG-IFN-α2b samples determined by MALDI-TOF MS.</li><li><figref idref="f0006">FIG.6</figref>: The molecular weight of YPEG-NHS (40KD) determined by MALDI-TOF MS.</li><li><figref idref="f0007">FIG.7</figref>: The serum drug concentration and activity after a single s.c. injection of 30 µg·kg^-1 YPEG-rhIFN-α2b into Crab-eating Macaque (<i>Macaca fascicularis</i>).</li><li><figref idref="f0007">FIG.8</figref>: The blank control of Trypsinase Peptide Mapping of the trypsin digested YPEG-rHuIFN-α2b SP2. Two small peaks were detected respectively at 71.674min and 17.589min; and the trypsin peak was detected between 2-3 min.</li><li><figref idref="f0008">FIG.9</figref>: The analysis of Trypsinase Peptide Mapping of the trypsin digested (0h) YPEG-IFN-α2b SP2 sample by HPLC-RP C₁₈. The retention time of YPEG-IFN-α2b SP2 was 62.114min; and the elution peak at 71.908min was the background, 2-3min.</li><li><figref idref="f0008">FIG.10</figref>: The analysis of Trypsinase Peptide Mapping of the trypsin digested (48h) YPEG-IFN-α2b SP2 sample by HPLC-RP C₁₈. No substrate peak (62.114min) was detected between 60.5min-63.2min, demonstrating that the sample was completely digested.</li><li><figref idref="f0009">FIG.11</figref>: Sephacryl S-100 HR separation profile of the YPEG modified peptides from the trypsin completely digested YPEG-IFN-α2b SP2 sample. The samples were collected according to the peaks, S100-1 was YPEG modified peptides, namely the target sample.</li></ul>

DETAILED DESCRIPTION OF THE INVENTION

0029The present invention will be further described by the following examples, but any example or the combination thereof should not be understood as limiting the scope or embodiment of the invention. The scope of the invention is limited only by the appended claims. In combination with the description and prior art, skilled persons in the art would clearly understand the scope limited by the claims.

Example 1

Preparation of Y-shaped branched PEG modified recombinant human IFN-α2b

(1) Small-scale preparation of Y-shaped branched PEG modified recombinant human IFN-α2b

0030166.1mg of YPEG (formula (I), average molecular weight 40KD, equal-arm, lot number RD010P041, Beijing JenKem Technology Co., Ltd.) was weighted and dissolved in 1ml of 2mM HCl (Guangdong Guanghua Chemical Factory Co., Ltd.). And 40mg IFN-α2b (Xiamen Amoytop Biotech Co., Ltd.) and 50mM boric acid-borax buffer (pH 9.0, Sinopharm Shanghai Chemical Reagent Co., Ltd.) were added to a final reaction volume of 10ml. In this reaction system, the final concentration of IFN-α2b was 4mg/ml, and the reaction molar ratio of IFN-α2b and YPEG was 1:2. The reaction system was kept under 0-20°C for 2h with stirring. The PEGylated IFNs-α2b were then generated, and the reaction was stopped by adding glacial acetic acid (Shantou Xilong Chemical Co., Ltd.) to make pH&lt;4.0. A sample was subjected for SDS-PAGE. The reaction system was diluted 50 times with water and then 0.2µm filtered before stored at 4°C for further use.

0031Q-Sepharose FF Chromatography was used to separate the remaining PEG and PEG hydrolates, IFNs-α2b modified by YPEG at multiple amino acid residues, IFNs-α2b modified by YPEG at a single amino acid residue and the unmodified IFN-α2b. Q-Sepharose FF (GE Healthcare) column (Φ12mm×90mm, 1CV=10ml) was regenerated with 3CV of 20mM boric acid/borax buffer (pH9.0)-1M NaCl (BBI), and then equilibrated with 5CV of 20mM boric acid/borax buffer (pH9.0). UV detection wavelength was set at 280nm. The entire sample stored at 4°C was loaded. After loading, the column was equilibrated with 3CV of boric acid/borax buffer (pH9.0), and then 20mM boric acid/borax buffer (pH9.0)-12mM NaCl was used for elution until the first peak was completely eluted, which peak was the remaining PEG 20mM boric acid/borax buffer (pH9.0)-60mM NaCl was then used for elution, and the sample collected in this elution peak was primarily the YPEG-IFNs-α2b, modified by PEG at a single amino acid residue. And then 20mM boric acid/borax buffer (pH9.0)-500mM NaCl was used for elution and the elution peak was the unmodified IFN-α2b.

0032The target products were primarily the YPEG-IFNs-α2b modified by PEG at a single amino acid residue, with a yield rate of 20-40%.

2) Large-scale preparation of Y-shaped branched PEG modified recombinant human IFN-α2b

00334982.4mg of YPEG (formula (I), average molecular weight 40KD, equal-arm, lot number RD010P041, Beijing JenKem Technology Co., Ltd.) was weighted and dissolved in 25ml of 2mM HCl. And 1200mg IFN-α2b and 50mM boric acid/borax buffer (pH 9.0) were added to a final reaction volume of 200ml. In this reaction system, the final reaction concentration of IFN-α2b was 6mg/ml, and the reaction molar ratio of IFN-α2b and YPEG was 1:2. The reaction system was kept under 0-25°C for 2h with stirring. The reaction was stopped by adding glacial acetic acid to make pH&lt;4.0. A sample was subjected for SDS-PAGE. The reaction system was diluted 50 times with water and then 0.2µm filtered before stored at 4°C for further use.

0034Q-Sepharose FF Chromatography was used to separate the remaining PEG and PEG hydrolates, IFNs-α2b modified by YPEG at multiple amino acid residues, IFNs-α2b modified by YPEG at a single amino acid residue and the unmodified IFN-α2b. Q-Sepharose FF (GE Healthcare) column (Φ38mm×265mm, 1CV=300ml) was regenerated with 3 CV of 20mM boric acid/borax buffer (pH9.0)-1M NaCl, and then equilibrated with 5CV of 20mM boric acid/borax buffer (pH9.0). UV detection wavelength was set at 280nm. The entire sample stored at 4°C was loaded. After loading, the column was equilibrated with 3CV of boric acid/borax buffer (pH9.0), and then 20mM boric acid/borax buffer (pH9.0)-12mM NaCl was used for elution until the first peak was completely eluted, which peak was the remaining PEG 20mM boric acid/borax buffer (pH9.0)-60mM NaCl was then used for elution, and the sample collected in this elution peak was primarily the YPEG-IFNs-α2b modified by PEG at a single amino acid residue. And then 20mM boric acid/borax buffer (pH9.0)-500mM NaCl was used for elution and the elution peak was the unmodified IFN-α2b.

0035The target products were primarily the YPEG-IFNs-α2b modified by PEG at a single amino acid residue, with a yield rate of 35-50%.

0036<figref idref="f0001">FIG.1</figref> shows SDS-PAGE results for 2 batches of IFNs-α2b modified with YPEG (40KD). It can be seen from <figref idref="f0001">figure 1</figref> that under the condition, the PEG modification rate of rhIFN-α2b was between 35-50% and remained stable. The primary modified products were modified by PEG at a single amino acid residue (YPEG-IFN-α2b), and there were also some products modified by PEG at multiple amino acid residues (YPEG_n-IFN-α2b).

Example 2

Resolving YPEG-IFNs-α2b by SP-Sepharose FF

0037The Q-Sepharose FF captured YPEG-IFNs-α2b sample was adjusted to pH 5.0 with 20% acetic acid, then diluted 15 times with 5mM NaAc/HAc (pH5.0, Shantou Xilong Chemical Co., Ltd.). The sample was loaded at 0.5mg/ml loading capacity to SP-Sepharose FF 100ml (GE Healthcare) column (Φ18mm×394mm). The column was equilibrated with 3CV of 5mM NaAc/HAc (pH5.0), and then eluted with 2.5CV of the gradient of 0%-30% 5mM NaAc/HAc-70mM NaCl (pH5.0), following with 50CV of the gradient of 30%-100% 5mM NaAc/HAc-70mM NaCl (pH5.0). YPEG-IFN-α2b was resolved as 4 elution peaks by SP-Sepharose FF 100ml. The samples were collected according to these peaks and then measured by SDS-PAGE with silver staining respectively. According to the SDS-PAGE results, it can be seen that peak 1 resolved by SP-Sepharose FF was primarily the products modified by YPEG at multiple amino acid residues (YPEG_n-IFN-α2b). Peak 2 by SP-Sepharose FF was primarily the products modified by PEG at a single amino acid residue (YPEG-IFN-α2b), and also contained some products modified by PEG at multiple amino acid residues. Peak 3 and peak 4 by SP-Sepharose FF were both the products modified by PEG at a single amino acid residue. Peaks 2-4 resolved by SP-Sepharose FF were isomers with YPEG-modification at different single positions, and were named respectively as YPEG-IFN-α2b SP1, YPEG-IFN-α2b SP2 and YPEG-IFN-α2b SP3. The resolution profile and silver-stained SAD-PAGE results were shown in <figref idref="f0002">FIG. 2</figref> and <figref idref="f0003">FIG.3</figref> respectively.

0038Every sample of YPEG-IFN-α2b SP1-3 was supplemented with sodium chloride, sodium acetate, mannitol, aspartic acid and was sterilized with 0.22µm filter before stored at 4°C for further use.

Example 3

Characteristic analysis of YPEGIFN-α2b modification isomers

(1) Protein concentration

0039The concentrations of YPEG-IFN-α2b modification isomers were determined by Kjeldahl method.

(2) Protein apparent molecular weight

0040The apparent molecular weights of YPEG-IFN-α2b modification isomers were determined by SDS-PAGE. The method was according to <nplcit id="ncit0015" npl-type="s"><text>Laemmli et al (Nature 227: 680, 1970</text></nplcit>). The concentration of the gel was 7.5%, and the gel was visualized by silver staining. The apparent molecular weights of YPEG-IFN-α2b modification isomers were almost the same, about 123KD (<figref idref="f0004">FIG.4</figref>).

(3) Molecular weight determined by MALDI-TOF MS

0041MALDI-TOF MS (Autoflex TOF/TOF system, Bruker Daltonics, Germany) was used to determine the molecular weights of YPEG-rHuIFN-α2b modification isomers. Sinapinic acid (SA, C₁₁H₁₂O₅, M.W. 224.22, lot number: 2006 236870 002, Bruker Daltonics, Germany) was used as matrix. Protein Calibration Standard II (Part No.207234, Bruker Daltonics, Germany) was used as protein molecular weight standard, and the software for data analysis was FlexAnalysis Ver.3.0.54.0. The MS molecular weights of YPEG-IFN-α2b modification isomers were almost the same, about 59000 Dalton (<figref idref="f0005">FIG.5</figref>).

(4) Endotoxin content test

0042Based on limulus assay (<nplcit id="ncit0016" npl-type="s"><text>Pharmacopoeia of the People's Republic of China, 2005, Volume 3, Appendix X C</text></nplcit>), the endotoxin content of every YPEG-IFN-α2b sample was less than 5.0EU/mg.

(5) In vivo activity and Pharmacokinetics of YPEG-IFN-α2b SP2 in animal.

□ In vivo activity of YPEG-IFN-α2b SP2 in animal.

0043The action mechanism of IFN is partially to induce the production of 2',5'-AS (2',5'-oligoadenylate synthetase), which in turn exerts its antiviral effects. Using ¹²⁵I as a tracer, the pharmacodynamic parameters of IFN are reflected by the <i>in vivo</i> 2',5'-AS activity. 2',5'-AS catalyzes the synthesis of 2',5'-A (2',5'-oligoadenylate) from ATP in the presence of Poly(I)-Poly(C) agar (The activity of 2',5'-AS can be represented by the concentration of the synthesized 2',5'-A). First, 2',5'-AS in the samples are absorbed and activated by Poly(I)·Poly(C) agarose, then catalyzes the substrate ATP to generate 2',5'-A. A mixture of ¹²⁵I labeled 2',5'-A, anti-2',5'-A serum and secondary antibody is added into the sample which then is incubated and centrifugated to separate the mixture. The supernatant is discarded and a Gamma Counter was used to measure the radioactivity of the sediment. The binding rate of the initially added ¹²⁵I labeled 2',5'-A is calculated. Four-parameter Logistic regression is used to generate standard curve, and then the concentration of the 2',5'-AS-induced 2',5'-A products in an unknown sample could be estimated.

0044Using the above mentioned 2', 5'-A method, the results in Tablel and <figref idref="f0007">FIG.7</figref> showed the serum 2', 5'-A concentration after a single s.c. injection of 30µg·kg^-1 YPEG-IFN-α2b SP2 into Crab-eating Macaque (<i>Macaca fascicularis</i>) (18 Crab-eating Macaques, Guangxi Weimei Biotechnology Co., Ltd., Certification No. S.CXK GUI 2002-0001, body weight 2.5-5.5kg, 6 female and 12 male, raised in separate cages, fed with standard monkey feed, drink freely). It can be seen from <figref idref="f0007">FIG.8</figref> that the average time-to-peak was 64±27.71h, and the concentration to peak was 292.30±148.08 Pmol·dL^-1. After administration, the activity of 2', 5'-AS in serum was clearly increased, and the time-to-peak of 2', 5'-A in serum was delayed than that of YPEG-rhIFNα2b SP2. <tables id="tabl0001" num="0001"><table frame="topbot"><title>Table1. The serum 2',5'-A concentrations over time, after a single s.c. injection of 30 µg·kg^-1 YPEG-rhIFN-α2b SP2 into Crab-eating Macaque. ( Pmol·dL^-1 )</title><tgroup cols="7"><colspec colnum="1" colname="col1" colwidth="25mm" /><colspec colnum="2" colname="col2" colwidth="23mm" colsep="0" /><colspec colnum="3" colname="col3" colwidth="23mm" colsep="0" /><colspec colnum="4" colname="col4" colwidth="23mm" colsep="0" /><colspec colnum="5" colname="col5" colwidth="23mm" colsep="0" /><colspec colnum="6" colname="col6" colwidth="24mm" colsep="0" /><colspec colnum="7" colname="col7" colwidth="25mm" colsep="0" /><thead><row><entry morerows="1" align="center" valign="middle">time(h)</entry><entry namest="col2" nameend="col4" align="center" valign="middle">No. of crab-eating Macaque</entry><entry morerows="1" align="center" valign="middle">Mean</entry><entry namest="col6" nameend="col7" morerows="1" align="center" valign="middle">SD</entry></row><row><entry align="center" valign="middle">1</entry><entry align="center" valign="middle">2</entry><entry align="center" valign="middle">3</entry></row></thead><tbody><row rowsep="0"><entry align="center" valign="middle">0</entry><entry align="center" valign="middle">19.32</entry><entry align="center" valign="middle">13.63</entry><entry align="center" valign="middle">20.74</entry><entry align="right" valign="middle">17.90</entry><entry align="center" valign="middle">±</entry><entry valign="middle">3.76</entry></row><row rowsep="0"><entry align="center" valign="middle">1</entry><entry align="center" valign="middle">40.17</entry><entry align="center" valign="middle">218.67</entry><entry align="center" valign="middle">--</entry><entry align="right" valign="middle">129.42</entry><entry align="center" valign="middle">±</entry><entry valign="middle">126.22</entry></row><row rowsep="0"><entry align="center" valign="middle">2</entry><entry align="center" valign="middle">45.74</entry><entry align="center" valign="middle">14.30</entry><entry align="center" valign="middle">80.23</entry><entry align="right" valign="middle">46.76</entry><entry align="center" valign="middle">±</entry><entry valign="middle">32.98</entry></row><row rowsep="0"><entry align="center" valign="middle">4</entry><entry align="center" valign="middle">14.89</entry><entry align="center" valign="middle">69.41</entry><entry align="center" valign="middle">138.23</entry><entry align="right" valign="middle">74.18</entry><entry align="center" valign="middle">±</entry><entry valign="middle">61.81</entry></row><row rowsep="0"><entry align="center" valign="middle">8</entry><entry align="center" valign="middle">49.12</entry><entry align="center" valign="middle">243.43</entry><entry align="center" valign="middle">141.66</entry><entry align="right" valign="middle">144.74</entry><entry align="center" valign="middle">±</entry><entry valign="middle">97.19</entry></row><row rowsep="0"><entry align="center" valign="middle">10</entry><entry align="center" valign="middle">119.51</entry><entry align="center" valign="middle">274.99</entry><entry align="center" valign="middle">109.89</entry><entry align="right" valign="middle">168.13</entry><entry align="center" valign="middle">±</entry><entry valign="middle">92.67</entry></row><row rowsep="0"><entry align="center" valign="middle">12</entry><entry align="center" valign="middle">72.75</entry><entry align="center" valign="middle">152.81</entry><entry align="center" valign="middle">112.87</entry><entry align="right" valign="middle">112.81</entry><entry align="center" valign="middle">±</entry><entry valign="middle">40.03</entry></row><row rowsep="0"><entry align="center" valign="middle">24</entry><entry align="center" valign="middle">10.05</entry><entry align="center" valign="middle">321.23</entry><entry align="center" valign="middle">159.12</entry><entry align="right" valign="middle">163.47</entry><entry align="center" valign="middle">±</entry><entry valign="middle">155.63</entry></row><row rowsep="0"><entry align="center" valign="middle">48</entry><entry align="center" valign="middle">45.60</entry><entry align="center" valign="middle">622.42</entry><entry align="center" valign="middle">164.49</entry><entry align="right" valign="middle">277.50</entry><entry align="center" valign="middle">±</entry><entry valign="middle">304.56</entry></row><row rowsep="0"><entry align="center" valign="middle">96</entry><entry align="center" valign="middle">400.67</entry><entry align="center" valign="middle">352.65</entry><entry align="center" valign="middle">123.58</entry><entry align="right" valign="middle">292.30</entry><entry align="center" valign="middle">±</entry><entry valign="middle">148.08</entry></row><row rowsep="0"><entry align="center" valign="middle">168</entry><entry align="center" valign="middle">10.87</entry><entry align="center" valign="middle">286.38</entry><entry align="center" valign="middle">4.17</entry><entry align="right" valign="middle">100.47</entry><entry align="center" valign="middle">±</entry><entry valign="middle">161.03</entry></row><row rowsep="0"><entry align="center" valign="middle">240</entry><entry align="center" valign="middle">2.74</entry><entry align="center" valign="middle">323.83</entry><entry align="center" valign="middle">10.48</entry><entry align="right" valign="middle">112.35</entry><entry align="center" valign="middle">±</entry><entry valign="middle">183.19</entry></row><row><entry align="center" valign="middle">312</entry><entry align="center" valign="middle">20.65</entry><entry align="center" valign="middle">238.65</entry><entry align="center" valign="middle">1.54</entry><entry align="right" valign="middle">86.94</entry><entry align="center" valign="middle">±</entry><entry valign="middle">131.72</entry></row></tbody></tgroup></table></tables>

② Pharmacokinetics of YPEG-IFNs-α2b and rhIFN-α2b in Crab-eating Macaque

0045A single s.c. injection of 10, 30 or 100 µg·kg^-1 YPEG-IFN-α2b was given to Crab-eating Macaque. For the administration group, 1ml of venous blood was taken from the hind leg opposite to the injected side at the time before, 1h, 2h, 4h, 8h, 10h, 12h, 24h, 48h, 72h, 96h, 168h, 240h, and 312h after administration. For the group with a single s.c. injection of IFN-α2b (30µg·kg^-1), 1ml of blood was taken at the time before, 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, 8h, and 24h after administration. After kept at 4°C for 30 min, the blood samples were centrifugated at 2000rpm for 10min under low temperature, then the serum was separated immediately and stored at -20°C for further analysis.

0046The quantitative double sandwich immunoassay was used. A monoclonal antibody specific to the recombinant human IFN-α was pre-coated on microtiter plate. The standard and the samples were pipetted into the microtiter wells, wherein the IFN-α2b or YPEG-IFN-α2b SP2 would bind to the immobilized antibody. The plate was washed to remove unbound substances, and then anti-human IFN-α IgG (secondary antibody) was added into the wells. After the reaction was complete, the plate was washed and the horseradish peroxidase (HRP) was added into the wells. After washing away the unbound enzyme and reagents, the color generated by adding HRP substrate solution into each well was proportional to the amount of the bound IFN-α2b or YPEG-rhIFN-α2b SP2 in the first step. The reaction was stopped and the color intensity was measured. The higher the OD value of absorbance, the higher the concentration of IFN-α2b or YPEG-IFN-α2b SP2 in the sample. The standard curves were plotted for IFN-α2b and YPEG-IFN-α2b SP2 respectively so as to measure the serum drug concentration in the blood samples.

0047According to the protocol in the description of the kit (American Biomedical Co., lot number 3271), 100µl standard or blood sample was added into each well, and mixed with plate mixer gently. According to the anticipated concentration of an unknown sample, the sample was diluted with the dilute solution to the concentration ranges of the standard curve. The IFN-α2b or YPEG-IFN-α2b SP2 standard curve for each plate was plotted so as to calculate the concentration of the unknown sample in that plate. The plate was incubated at room temperature for 1h, and washed once with plate washing solution. 100µl secondary antibody was added to each well, and kept under room temperature for 1h. The plate was washed 3 times, and 100µl HRP conjugate was added to each well. The plate was incubated under room temperature for 1h and washed 4 times. 100µl TMB substrate was added into each well, and kept under room temperature in the dark for 15min. 100µl stop solution was added to each well, and mixed gently to stop the reaction. The absorbance OD value at 450nm was measured with a microplate reader within 5min to determine the concentration of each sample.

0048After a single subcutaneous injection of low-dose, medium-dose, or high-dose (10, 30 and 100 µg·kg^-1, respectively) of YPEG-IFN-α2b into Crab-eating Macaque, the half-lives were 48.87±11.67, 51.94±3.52 and 49.60±2.97h, respectively. After a single subcutaneous injection of 30 µg·kg^-1 IFN-α2b into Crab-eating Macaque, the half-life was 3.22±0.10h. The half-life of IFN-α2b was prolonged at least ten times after YPEG modification.

(6) The

in vitro

biological activity of each YPEG-IFN-α2b modification isomers was estimated using cytopathic effect inhibition assay.

0049According to the method described in <nplcit id="ncit0017" npl-type="s"><text>Determination Method of Interferon Activity (Pharmacopoeia of the People's Republic of China, 2005, Volume 3, Appendix X C</text></nplcit>), interferon protects human amniotic cells (WISH) from the damage caused by vesicular stomatitis virus (VSV). Crystal violet was used to stain survived WISH cells, and the absorbance OD value was measured at 570nm. The interferon protection effect curve was plotted for WISH cells, so as to determine the <i>in vitro</i> biological activity of interferons. The results of <i>in vitro</i> biological activity of each samples are shown in Table 2, and 3 parallel tests were carried out for each sample. After YPEG modification, in all the modification isomers of the products modified by PEG at a single amino acid residue, the SP2 sample showed the highest <i>in vitro</i> specific activity, which was 1-2 times higher than SP1, SP3 and PEGASYS (manufactured by Hoffmann-La Roche, Basel, Switzerland; packaged separately by Shanghai Roche Pharmaceuticals Ltd., product lot number B1016, package lot number SH0020), and was also 1-2 times higher than the unresolved sample. <tables id="tabl0002" num="0002"><table frame="all"><title>Table2. In vitro biological activity results for each modification isomer of YPEG-IFN-α2b (3 parallel tests)</title><tgroup cols="5"><colspec colnum="1" colname="col1" colwidth="37mm" /><colspec colnum="2" colname="col2" colwidth="28mm" /><colspec colnum="3" colname="col3" colwidth="28mm" /><colspec colnum="4" colname="col4" colwidth="37mm" /><colspec colnum="5" colname="col5" colwidth="37mm" /><thead><row><entry align="center" valign="middle">Sample</entry><entry align="center" valign="middle">PEG Type</entry><entry align="center" valign="middle">PEG M.W. (KD)</entry><entry align="center" valign="middle">No. of Modification Positions</entry><entry align="center" valign="middle">Average Specific Activity (×10⁶IU/mg)</entry></row></thead><tbody><row><entry valign="middle">YPEG-IFN-α2b SP1</entry><entry align="center" valign="middle">Y-branched</entry><entry align="center" valign="middle">40</entry><entry align="center" valign="middle">1</entry><entry align="center" valign="middle">1.07±0.172</entry></row><row><entry valign="middle">YPEG-IFN-α2b SP2</entry><entry align="center" valign="middle">Y-branched</entry><entry align="center" valign="middle">40</entry><entry align="center" valign="middle">1</entry><entry align="center" valign="middle">2.65±0.185</entry></row><row><entry valign="middle">YPEG-IFN-α2b SP3</entry><entry align="center" valign="middle">Y-branched</entry><entry align="center" valign="middle">40</entry><entry align="center" valign="middle">1</entry><entry align="center" valign="middle">1.13±0.215</entry></row><row><entry valign="middle">YPEG-IFN-α2b unresolved sample</entry><entry align="center" valign="middle">Y-branched</entry><entry align="center" valign="middle">40</entry><entry align="center" valign="middle">1</entry><entry align="center" valign="middle">1.68±0.217</entry></row><row><entry valign="middle">PEGASYS</entry><entry align="center" valign="middle">U-branched</entry><entry align="center" valign="middle">40</entry><entry align="center" valign="middle">1</entry><entry align="center" valign="middle">0.934±0.042</entry></row></tbody></tgroup><tgroup cols="5" rowsep="0"><colspec colnum="1" colname="col1" colwidth="37mm" /><colspec colnum="2" colname="col2" colwidth="28mm" /><colspec colnum="3" colname="col3" colwidth="28mm" /><colspec colnum="4" colname="col4" colwidth="37mm" /><colspec colnum="5" colname="col5" colwidth="37mm" /><tbody><row><entry namest="col1" nameend="col5" align="justify">Note: unresolved sample refers to the sample before resolving YPEG-rhIFN-α2b by SP-Sepharose FF</entry></row></tbody></tgroup></table></tables>

(7) The resolution of the modification position in YPEG-IFN-α2b SP2

0050The solvent system of YPEG-IFN-α2b SP2 was changed to 50mM NH₄HCO₃ (pH8.0) by ultrafiltration with 5K ultrafilter (Millipore, polyethersulfone material), and the protein concentration was determined to be 3.82 mg/ml using UV spectroscopy. TPCK Trypsin (Promega) was dissolved (0.5µg/µl) in the solution provided by the manufacturer. Samples were added according to Table 3: <tables id="tabl0003" num="0003"><table frame="all"><title>Table3. Reaction composition of YPEG-1FN-α2b SP2 trypsin digestion</title><tgroup cols="2"><colspec colnum="1" colname="col1" colwidth="55mm" /><colspec colnum="2" colname="col2" colwidth="47mm" /><thead><row><entry valign="top"><b>Reaction Composition</b></entry><entry align="center" valign="top"><b>Volume</b></entry></row></thead><tbody><row><entry>50mM NH₄HCO3, pH8.0</entry><entry align="center">7.08ml</entry></row><row><entry>PEG-IFN-α2b SP2 (3.82mg/ml)</entry><entry align="center">1.32ml</entry></row><row><entry>Trypsin (0.5µg/µl)</entry><entry align="center">0.2ml</entry></row><row><entry>Total reaction volume</entry><entry align="center">8.6ml</entry></row></tbody></tgroup></table></tables>

0051The reaction system was kept in a water bath at 37°C for 48h, then 1.52ml of 20% acetic acid was added to stop the reaction. A small amount of sample was taken for HPLC-RP C18 peptide mapping. The instrument for analysis was Waters HPLC system, with a controller of type 600, 2487 double wavelength detector, and the software for data processing was Empower 2. The HPLC analytical column was Jupiter C 18 (particle diameter 5µm, pore diameter 300Å, Φ4.6×150min, produced by Phenomenex, USA). Mobile phase A was 0.1% TFNH₂O, Mobile phase B was 0.1 % TFA/90% ACN/H₂O, the flow rate was 1ml/min, and the detection wavelength was set at 214nm. Please refer to Table 4 for the elution gradients, and the results were shown in <figref idref="f0007 f0008">FIG8-10</figref>. <tables id="tabl0004" num="0004"><table frame="all"><title>Table4. The elution gradients for HPLC-RP C18 peptide mapping of the trypsin digested YPEG-IFN-α2b SP 2</title><tgroup cols="5"><colspec colnum="1" colname="col1" colwidth="29mm" /><colspec colnum="2" colname="col2" colwidth="32mm" /><colspec colnum="3" colname="col3" colwidth="29mm" /><colspec colnum="4" colname="col4" colwidth="29mm" /><colspec colnum="5" colname="col5" colwidth="34mm" /><thead><row><entry align="center" valign="top" /><entry align="center" valign="top">Time (min )</entry><entry align="center" valign="top">A%</entry><entry align="center" valign="top">B%</entry><entry align="center" valign="top">ACN%</entry></row></thead><tbody><row><entry align="center">1</entry><entry align="center">0</entry><entry align="center">100</entry><entry align="center">0</entry><entry align="center">0</entry></row><row><entry align="center">2</entry><entry align="center">8</entry><entry align="center">100</entry><entry align="center">0</entry><entry align="center">0</entry></row><row><entry align="center">3</entry><entry align="center">68</entry><entry align="center">40</entry><entry align="center">60</entry><entry align="center">54</entry></row><row><entry align="center">4</entry><entry align="center">72</entry><entry align="center">40</entry><entry align="center">60</entry><entry align="center">54</entry></row><row><entry align="center">5</entry><entry align="center">75</entry><entry align="center">100</entry><entry align="center">0</entry><entry align="center">0</entry></row><row><entry align="center">6</entry><entry align="center">80</entry><entry align="center">100</entry><entry align="center">0</entry><entry align="center">0</entry></row></tbody></tgroup></table></tables>

0052Based on the detection result, it can be determined that the sample was almost completely digested. The products were treated with DTT reduction after the reaction was stopped. The Sephacryl S-100HR column (Φ18×255mm, 1CV=64ml; GE Healthcare) was pre-equilibrated with 3CV of 20mM PBNa-400mM NaCl (pH7.0), and 3% CV of the YPEG-IFN-α2b SP2 sample by TPCK trypsin digested completely was loaded by hydrostatic pressure. 20mM PBNa-400mM NaCl (pH7.0) was used for elution, and the detection wavelength was set at 280nm. The sample from the first elution peak was collected (sample number: YPEG-IFN-α2b S100-1, <figref idref="f0009">FIG.11</figref>), and the solvent system was changed to 5mM PBNa (pH 7.0) with 5K ultrafilter. Vacuum freeze-drying was done. The N-terminal amino acids of the freeze-dried sample were determined using Edman degradation, and the sequence of the 7 amino acids at the N-terminus of the sample was XYSPXAW (Table 5), wherein X denotes α-amino acid cysteine (Cys), a non-α-amino acid or another modified amino acid that cannot be detected using Edman degradation. According to the sequence shown in SEQ ID NO: 1, it can be determined that the YPEG-IFN-α2b SP2 was primarily the products modified with YPEG at Lys134. <tables id="tabl0005" num="0005"><table frame="all"><title>Table5. Sequencing result for the N-terminal amino acids of YPEG-IFN-α2b S100-1</title><tgroup cols="3"><colspec colnum="1" colname="col1" colwidth="39mm" /><colspec colnum="2" colname="col2" colwidth="49mm" /><colspec colnum="3" colname="col3" colwidth="69mm" /><thead><row><entry align="center" valign="middle">Sample</entry><entry align="center" valign="middle">Detected N-terminal Sequence</entry><entry align="center" valign="middle">The corresponding PEG modification position.</entry></row></thead><tbody><row><entry align="center" valign="middle">YPEG-IFN-α2b S100-1</entry><entry align="center" valign="middle">XYSPXAW</entry><entry align="center" valign="middle">Lys134</entry></row></tbody></tgroup><tgroup cols="3" rowsep="0"><colspec colnum="1" colname="col1" colwidth="39mm" /><colspec colnum="2" colname="col2" colwidth="49mm" /><colspec colnum="3" colname="col3" colwidth="69mm" /><tbody><row><entry namest="col1" nameend="col3" align="justify">Note: X denotes α-amino acid cysteine, a non-α-amino acid or another modified amino acid that cannot be detected using Edman degradation.</entry></row></tbody></tgroup></table></tables>

Sequence listings

0053<ul id="ul0002" list-style="none"><li><110> BIOSTEED GENE EXPRESSION TECH. CO., LTD.</li><li><120> INTERFERON ALPHA 2B MODIFIED BY POLYETHYLENE GLYCOL, THE PREPARATION AND USE THEREOF</li><li><130> P2007474C</li><li><160> 1</li><li><170> PatentIn version 3.3</li><li><210> 1 <211> 165 <212> PRT <213> Homo sapiens</li><li><400> 1 <img file="EP2186830B1_D0012.tif" /></li></ul>