Immune globulin main including Man7GlcNAc2, Man8GlcNAc sugar type
A technology of immunoglobulin and glycan, applied in peptides, chemical instruments and methods, antibodies, etc.
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Embodiment 1
[0145] Cloning of DX-IgG1 for expression in P. pastoris
[0146]The light (L) and heavy (H) chains of DX-IgG1 (anti-CD20IgG1) consist of mouse variable regions and human constant regions. The light chain is disclosed as SEQ ID NO:1 and the heavy chain as SEQ ID NO:2. Heavy and light chain sequences were synthesized using overlapping oligonucleotides purchased from Integrated DNA Technologies (IDT). For the light chain variable region, 15 overlapping oligonucleotides (SEQ ID NOs: 5-19) were purchased and annealed in a PCR reaction using Extaq (Takada) to generate a light chain variable region fragment with a 5' MlyI site . This light chain variable region fragment was then subjected to overlapping PCR using 5'MlyI primer CD20L / up (SEQ ID NO:20), 3'variable / 5'constant region primer LfusionRTVAAPS / up (SEQ ID NO:21), The 3' constant region primers LfusionRTVAAPS / lp (SEQ ID NO: 22) and 3' CD20L / lp (SEQ ID NO: 23) were ligated to the light chain constant region (SEQ ID NO: 3) (...
Embodiment 2
[0147] is an anti-CD20 mouse / human chimeric IgG1 purchased from Biogen-IDEC / Genentech, San Francisco, CA.
[0148] PCR amplification. Eppendorf Mastercycler was used for all PCR reactions. PCR reactions containing template DNA, 125 μM dNTPs, 0.2 μM each forward and reverse primers, ExTaq polymerase buffer (Takara Bio Inc.) and EX Taq polymerase or pFU Turbo polymerase buffer (Stratagene) and pFU Turbo polymerase enzymes. DNA fragments were amplified using 30 cycles of 97°C for 15 seconds, 55°C for 15 seconds, 72°C for 90 seconds, an initial denaturation step at 97°C for two minutes and a final extension step at 72°C for seven minutes.
[0149] PCR samples were separated by agarose gel electrophoresis, and DNA bands were extracted and purified using a gel extraction kit from Qiagen. All DNA purifications were eluted in 10 mM Tris, pH 8.0, except for the final PCR (overlapping all three fragments), which was eluted in deionized H2O.
[0150] Example 2
[0151] Transf...
Embodiment 3
[0157] Purification of IgG1
[0158] Monoclonal antibodies were captured from culture supernatants using STREAMLINE protein A columns. Antibodies were eluted in Tris-Glycine pH 3.5 and neutralized using 1M Tris pH 8.0. Further purification was performed using hydrophobic interaction chromatography (HIC). The specific type of HIC column depends on the antibody. For JC-IgG and DX-IgG, with 20 mM Tris (7.0), 1 M (NH 4 ) 2 SO 4 Use a phenyl sepharose column with the buffer (octyl sepharose can also be used), use 1M to 0M (NH 4 ) 2 SO 4 eluted with a linear gradient buffer. Antibody fractions from the phenyl sepharose column were pooled and exchanged into 50 mM NaOAc / Tris pH 5.2 buffer for final purification by cation exchange (SPSEPHAROSE Fast Flow) (GE Healthcare) column. Antibodies were eluted with a linear gradient using 50 mM Tris, 1 M NaCl (pH 7.0).
[0159] Treatment of Ig-high mannose with alpha-1,2 mannosidase
[0160] For alpha-1,2 mannosidase treatment, 5 ...
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