deae sepharose ff column, by GE Healthcare - Product details

1

Recombinant Antibody Production in HEK293E

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Example 8

The expression and purification of a recombinant antibody protein were conducted by following methods: HEK293E cells cultured in Freestyle 293 Expression Medium with 10% of Pluronic F-68 (Gibco) at 1×10⁶cell/ml were transfected with equal amount of heavy chain vector and light chain vector DNA at final concentration of 0.5 μg/ml and PEI (Polyethylenimine-linear, Polyscience) at 1.0 μg/ml. DNA to PEI ratio was 1:2. DNA and PEI complexes formed period with Optimal MEM should be 15 minutes at the room temperature. Transfected cells were cultured in the flasks with 5% CO2, 37° C. and 125 rpm shaking speed. 1% Peptone medium was added at 22 to 26 hours post transfection. Conditioned medium was harvested on day 6 and supernatant was centrifuged at 3,000 rpm for 30 minutes. The clarified conditioned medium were then loaded onto nProteinA column (G.E. Healthcare), washed with PBS plus 0.1% triton-X100 and finally the bound IgG was eluted with a solution containing 0.1M glycine at pH 3.5. The eluted antibody protein was dialyzed to PBS and stored at −80*C. To remove endotoxin, the purified protein was further processed by passing through Hitrap DEAE Sepharose F.F. column and the resulting antibody was analyzed to determine the level of purity using size exclusion chromatography (Superdex 200 5/150 GL, G.E. Healthcare).

US10233249B2. Antibodies and methods for treating estrogen receptor-associated diseases (2019-03-19). BEIJING SHENOGEN PHARMA GROUP LTD. [CN]. Inventors: Xueming Qian [CN], Kun Meng [CN], Feng Chen [CN], Xiao Shang [CN], Jing Wang [CN], Lu Li [CN], Congya Zhou [CN].

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2

Purification of Coversin Variants

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Example 1

Genetic constructs encoding Coversin variants 1 (SEQ ID NO: 5) and 2 (SEQ ID NO: 6) were cloned into E. coli expression vectors which were then transformed into an E. coli expression strain. Fermentations of the bacterial cultures were then performed with complex medium using an established fermentation protocol.

The resulting clarified cell culture was concentrated in 20 mM Bis-Tris buffer, pH 6.0, and applied to a DEAE-Sepharose FF column (GE Healthcare) and then eluted with a step gradient of NaCl in the same buffer. Purified fractions were then run on a Phenyl Sepharose HP column using a linear gradient of (NH₄)₂SO₄in 20 mM Bis-Tris buffer. Chromatography fractions were analysed with SDS-PAGE and pooled. For Coversin variant 1, a further purification step was carried out where the pooled protein fractions were applied to a Superdex 75 gel filtration column. For Coversin variant 2, a further purification step was carried out where pooled protein fractions were applied to a Q-sepharose ion exchange column.

Purified fractions of variants 1 and 2 were then run on an SDS-PAGE gel alongside wild type Coversin. The results are shown in FIG. 3 and demonstrate that both Coversin variants express well in E. coli.

US11214602B2. Coversin variants lacking C5 binding (2022-01-04). VOLUTION IMMUNO PHARMACEUTICALS SA [CH]. Inventors: Miles Andrew Nunn [CH].

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3

Purification of Glycosylated Horseradish Peroxidase

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In the next stage, 500 mL of the filtrated final cultivation supernatant were ultra- and diafiltrated with a mini-TFF membrane (Pall Corporation Minimate™ TFF Capsule, Omega 10K) with buffer A (50 mM Tris-HCl, pH 8.0) to obtain a final volume of ~8 mL. Afterwards a negative anion exchange chromatography (negative-AIEX) step with a DEAE-Sepharose FF column (GE Healthcare, Vienna, Austria) was performed on an Äkta pure (GE Healthcare, Vienna, Austria). The column was equilibrated with 10 CV buffer A, then the sample was loaded at 156 cm·h⁻¹. For elution, 100% buffer B (50 mM Tris-HCl, 2M NaCl, pH 8) was reached through a linear gradient in 20 min at 156 cm·h⁻¹. Absorbance was monitored at 214 nm, 280 nm and 404 nm. This yielded highly glycosylated HRP in the flowthrough and bound host cell impurities on the column [59 ]. The flow through was collected, concentrated and rebuffered with a centrifuge filter (5 kDa cut off) with storage buffer (50 mM Tris-HCl; 150 mM NaCl; pH 8). Samples were sterile filtrated with regenerated cellulose (RC) filters and stored at 4 °C.

Pekarsky A., Mihalyi S., Weiss M., Limbeck A, & Spadiut O. (2020). Depletion of Boric Acid and Cobalt from Cultivation Media: Impact on Recombinant Protein Production with Komagataella phaffii. Bioengineering, 7(4), 161.

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4

Recombinant Chimeric Antibody Purification

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Example 8

The expression and purification of the recombinant chimeric antibody protein produced above were conducted by following methods: HEK293E cells cultured in Freestyle 293 Expression Medium with 10% of Pluronic F-68 at 1×10⁶cell/ml were transfected with equal amount of heavy chain vector and light chain vector DNA with final concentration of 0.5 μg/ml and PEI (Polyethylenimine-linear, Polyscience) of 1.0 μg/ml. DNA to PEI ratio was 1:2. DNA and PEI complexes formed period with Optimal MEM should be 15 minutes at the room temperature. Transfected cells were cultured in the flasks with 5% CO₂, at 37° C. and at 125 rpm shaking speed. 1% Peptone medium was added at 22 to 26 hours post transfection. Conditioned medium was harvested on day 6 and supernatant was centrifuged at 3,000 rpm for 30 minutes. The clarified conditioned medium was then loaded onto ProteinA column (G.E. Healthcare), washed with PBS plus 0.1% triton-X100 and finally the bound IgG was eluted with a solution containing 0.1M glycine at pH 3.5. The eluted antibody protein was dialyzed to PBS and stored at −80° C. To remove endotoxin, the purified protein was further processed by passing through Hitrap DEAE Sepharose F.F. column and the resulting antibody was analyzed to determine the level of purity using size exclusion chromatography (Superdex 200 5/150 GL, G.E. Healthcare).

US10822416B2. Anti-PD-L1 antibodies (2020-11-03). MABSPACE BIOSCIENCES (SUZHOU) CO., LTD [CN]. Inventors: Xueming Qian [US], Teng Fei [CN], Zhen Li [CN].

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5

Recombinant Chimeric Antibody Production

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Example 8

The expression and purification of the recombinant chimeric antibody protein produced above were conducted by following methods: HEK293E cells cultured in Freestyle 293 Expression Medium with 10% of Pluronic F-68 at 1×10⁶cell/ml were transfected with equal amount of heavy chain vector and light chain vector DNA with final concentration of 0.5 μg/ml and PEI (Polyethylenimine-linear, Polyscience) of 1.0 μg/ml. DNA to PEI ratio was 1:2. DNA and PEI complexes formed period with Optimal MEM should be 15 minutes at the room temperature. Transfected cells were cultured in the flasks with 5% CO₂, at 37° C. and at 125 rpm shaking speed. 1% Peptone medium was added at 22 to 26 hours post transfection. Conditioned medium was harvested on day 6 and supernatant was centrifuged at 3,000 rpm for 30 minutes. The clarified conditioned medium was then loaded onto ProteinA column (G.E. Healthcare), washed with PBS plus 0.1% triton-X100 and finally the bound IgG was eluted with a solution containing 0.1M glycine at pH 3.5. The eluted antibody protein was dialyzed to PBS and stored at −80° C. To remove endotoxin, the purified protein was further processed by passing through Hitrap DEAE Sepharose F.F. column and the resulting antibody was analyzed to determine the level of purity using size exclusion chromatography (Superdex 200 5/150 GL, G.E. Healthcare).

US11753473B2. Anti-PD-L1 antibodies (2023-09-12). SUZHOU TRANSCENTA THERAPEUTICS CO., LTD. [CN]. Inventors: Xueming Qian [US], Teng Fei [CN], Zhen Li [CN].

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6

Purification of Protein by Chromatography

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Filtrated cultivation supernatant was ultra- and diafiltrated with a mini-TFF membrane (Pall Corporation Minimate™ TFF Capsule, Omega 10K) into AIEX buffer A (25 mM sodium citrate pH 6.0). Then, a proprietary purification step-chain was used. Briefly, an AIEX step with a DEAE-Sepharose FF column (GE Healthcare, Vienna, Austria) was performed on an Äkta pure (GE Healthcare, Vienna, Austria) and elution was done with AIEX buffer B (25 mM sodium citrate pH 6.0; 1 M NaCl). The elution fraction was used for a subsequent precipitation step with saturated (NH₄)₂SO₄ and centrifugation to apply the supernatant on a Capto-Phenyl hydrophobic interaction chromatography (HIC) column (GE Healthcare, Vienna, Austria) on an Äkta pure (GE Healthcare, Vienna, Austria). Loading of the HIC column was done with HIC buffer A (25 mM sodium citrate pH 6.0; 20% saturated (NH₄)₂SO₄) and elution with AIEX buffer A. Again, the elution fractions were pooled. Absorbance was monitored for both purification steps at 280 nm, 370 nm and 404 nm. The HIC Pool was rebuffered into 1 mM phosphate buffer pH 7.4 with a mini-TFF membrane (Pall Corporation MinimateTM TFF Capsule, Omega 10K) and concentrated with a centrifuge filter (5 kDa cut off). Samples were sterile filtrated with regenerated cellulose (RC) filters and stored at 4 °C.

Pekarsky A., Mihalyi S., Weiss M., Limbeck A, & Spadiut O. (2020). Depletion of Boric Acid and Cobalt from Cultivation Media: Impact on Recombinant Protein Production with Komagataella phaffii. Bioengineering, 7(4), 161.

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7

Laccase Purification from T. trogii

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The supernatant from a 7-day submerged culture of T. trogii YDHSD was filtered and concentrated 20-fold using a stirred ultrafiltration system with a 10 kDa cutoff membrane (Amicon 8200; Millipore, USA). The concentrated supernatant was fractionated by ammonium sulfate precipitation. The 40-60% fraction was then dialyzed against 20 mM sodium acetate buffer (pH 4.8). The dialyzed sample was applied onto a DEAE-Sepharose FF column (1.6 × 12.5 cm GE Healthcare), which was equilibrated with the same buffer. Laccase was eluted with a linear 0 to 0.5 M NaCl gradient in 200 ml of the equilibration buffer at a rate of 1 ml/min. Active fractions were combined and concentrated. The concentrated sample was applied to a Sephacryl S-200 column (1.6 × 46 cm GE Healthcare) equilibrated with 50 mM phosphate buffer (pH 6.5) containing 0.15 M NaCl and eluted at 0.25 ml/min. Fractions with high laccase activity were pooled, concentrated, dialyzed against 50 mM phosphate buffer (pH 6.5), and stored at 4°C.

Ai M.Q., Wang F.F, & Huang F. (2015). Purification and Characterization of a Thermostable Laccase from Trametes trogii and Its Ability in Modification of Kraft Lignin. Journal of microbiology and biotechnology, 25(8).

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Deae sepharose ff column

Lab products found in correlation

7 protocols using deae sepharose ff column

Recombinant Antibody Production in HEK293E

Purification of Coversin Variants

Purification of Glycosylated Horseradish Peroxidase

Recombinant Chimeric Antibody Purification

Recombinant Chimeric Antibody Production

Purification of Protein by Chromatography

Laccase Purification from T. trogii

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