Poros mabcapture a, by Thermo Fisher Scientific - Product details

1

Purification of Fc Constructs Using Chromatography

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Example 2

The expressed proteins were purified from the cell culture supernatant by Protein A-based affinity column chromatography, using a Poros MabCapture A (LifeTechnologies) column. Captured Fc constructs were washed with phosphate buffered saline (low-salt wash) and eluted with 100 mM glycine, pH 3. The eluate was quickly neutralized by the addition of 1 M TRIS pH 7.4 and sterile filtered through a 0.2 μm filter.

The proteins were further fractionated by ion exchange chromatography using Poros XS resin (Applied Biosciences). The column was pre-equilibrated with 50 mM MES, pH 6 (buffer A), and the sample was eluted with a step gradient using 50 mM MES, 400 mM sodium chloride, pH 6 (buffer B) as the elution buffer.

After ion-exchange, the target fraction was buffer exchanged into PBS buffer using a 10 kDa cutoff polyether sulfone (PES) membrane cartridge on a tangential flow filtration system. The samples were concentrated to approximately 30 mg/mL and sterile filtered through a 0.2 μm filter.

US11155640B2. Compositions and methods related to engineered Fc constructs (2021-10-26). Momenta Pharmaceuticals, Inc. [US]. Inventors: Carlos J. Bosques [US], Jonathan C. Lansing [US], Daniel Ortiz [US], Laura Rutitzky [US], Nathaniel J. Washburn [US].

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2

Purification of Fc Constructs

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Example 2

The expressed proteins were purified from the cell culture supernatant by Protein A-based affinity column chromatography, using a Poros MabCapture A (LifeTechnologies) column. Captured Fc constructs were washed with phosphate buffered saline (low-salt wash) and eluted with 100 mM glycine, pH 3. The eluate was quickly neutralized by the addition of 1 M TRIS pH 7.4 and sterile filtered through a 0.2 μm filter.

The proteins were further fractionated by ion exchange chromatography using Poros XS resin (Applied Biosciences). The column was pre-equilibrated with 50 mM MES, pH 6 (buffer A), and the sample was eluted with a step gradient using 50 mM MES, 400 mM sodium chloride, pH 6 (buffer B) as the elution buffer.

After ion-exchange, the target fraction was buffer exchanged into PBS buffer using a 10 kDa cutoff polyether sulfone (PES) membrane cartridge on a tangential flow filtration system. The samples were concentrated to approximately 30 mg/mL and sterile filtered through a 0.2 μm filter.

US11827682B2. Engineered Fc constructs (2023-11-28). MOMENTA PHARMACEUTICALS, INC. [US]. Inventors: Carlos J. Bosques [US], Jonathan C. Lansing [US], Daniel Ortiz [US].

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3

Purification of Fc-Fusion Proteins

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Example 2

The expressed proteins were purified from the cell culture supernatant by Protein A-based affinity column chromatography, using a Poros MabCapture A (LifeTechnologies) column. Captured Fc constructs were washed with phosphate buffered saline (low-salt wash) and eluted with 100 mM glycine, pH 3. The eluate was quickly neutralized by the addition of 1 M TRIS pH 7.4 and sterile filtered through a 0.2 μm filter.

The proteins were further fractionated by ion exchange chromatography using Poros XS resin (Applied Biosciences). The column was pre-equilibrated with 50 mM MES, pH 6 (buffer A), and the sample was eluted with a step gradient using 50 mM MES, 400 mM sodium chloride, pH 6 (buffer B) as the elution buffer.

After ion-exchange, the target fraction was buffer exchanged into PBS buffer using a 10 kDa cutoff polyether sulfone (PES) membrane cartridge on a tangential flow filtration system. The samples were concentrated to approximately 30 mg/mL and sterile filtered through a 0.2 μm filter.

US11220531B2. Engineered Fc constructs (2022-01-11). Momenta Pharmaceuticals, Inc. [US]. Inventors: Carlos J. Bosques [US], Jonathan C. Lansing [US], Daniel Ortiz [US].

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4

Antibody Production and Purification

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The sequences obtained from each VH and VL of the antibody clones were codon-optimized for expression in human cells at GeneArt/Invitrogen. The variable regions of these functional variants were subsequently cloned directly by restriction digest for expression in the IgG expression vectors pCP9-kappa and pCP9-gamma. Vectors encoding the IgGs were transfected into PER.C6® cells and the antibodies were purified from cell supernatants using POROS Mabcapture A chromatography (Applied Biosystems). The antibodies were then exchanged into 50 mM sodium acetate, 50 mM sodium chloride, pH 5.5. The quality of each antibody was confirmed by size-exclusion chromatography, SDS-PAGE and isoelectric focusing.

Gilman M.S., Furmanova-Hollenstein P., Pascual G., B. van ‘t Wout A., Langedijk J.P, & McLellan J.S. (2019). Transient opening of trimeric prefusion RSV F proteins. Nature Communications, 10, 2105.

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5

Recombinant IgG1 Antibodies Targeting RSV F

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Fully human IgG1 anti-RSV F protein antibodies CR9501, CR9503, CR9515 and CR13039 were constructed by cloning the heavy (VH) and light (VL) chain variable regions into a single IgG1 expression vector. PER.C6 cells (Crucell) were transfected with the IgG1 expression constructs and the expressed antibodies were purified from culture supernatants using POROS Mabcapture A chromatography (Applied Biosystems) and then buffer exchanged to 50 mM NaAc, 50 mM NaCl, pH 5.5. Antibody concentration was measured by optical absorption at 280 nm. Antibody quality was also confirmed by size-exclusion chromatography (SEC), SDS–PAGE and isoelectric focusing. CR9501 comprises VH and VL regions of 58C5, which binds specifically to RSV F protein in its prefusion conformation and not to the postfusion conformation21 . CR9503 comprises VH and VL regions of motavizumab26 (link). CR9515 comprises VH and VL regions of D25 antibody27 (link). CR13039 comprises VH and VL regions of MPE8 antibody28 (link).

Krarup A., Truan D., Furmanova-Hollenstein P., Bogaert L., Bouchier P., Bisschop I.J., Widjojoatmodjo M.N., Zahn R., Schuitemaker H., McLellan J.S, & Langedijk J.P. (2015). A highly stable prefusion RSV F vaccine derived from structural analysis of the fusion mechanism. Nature Communications, 6, 8143.

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6

Bispecific Antibody Production and Purification

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All bsAbs (C, D, and F) used in this study were expressed in CHO cells and produced at Regeneron Pharmaceuticals. Commercially available chromatographic resins used were obtained from their manufacturers: MabSelect SuRe and MabSelect SuRe pcc (GE Healthcare), and POROS MabCapture A (Thermo Scientific). All chemicals used were supplied by J.T. Baker. Developmental resins were supplied by GE Healthcare Custom Design Resin Group.

Tustian A.D., Laurin L., Ihre H., Tran T., Stairs R, & Bak H. (2018). Development of a novel affinity chromatography resin for platform purification of bispecific antibodies with modified protein a binding avidity. Biotechnology Progress, 34(3), 650-658.

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7

Continuous Capture and Chromatography

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A harvested fed-batch CHO cell culture fluid at 2.3 g/L titer was prepared at Merck & Co., Inc., Kenilworth, NJ. The feedstock was stored at -80°C and then thawed and sterile filtered (Asepti-Cap ® , Advanced Microdevices, India) before use. Hollow fiber membranes from Spectrum Labs (49-157 cm 2 membrane area, 0.65 µm pore size, 0.75 mm ID, and 41.5 cm length) and static mixers from Koflo Corporation (L = 28.6 cm, ID = 1.0 cm) were used to construct each stage in the CCTC system. Two different protein A capture resins were used: POROS MabCapture A from Thermo Fisher Scientific (Waltham, MA) and custom made prototype Praesto D5980 resin from Purolite Corporation (Bala Cynwyd, PA). The POROS MabCapture A has an average diameter of approximately 45 µm and is made of poly (styrene divinyl benzene). The Praesto D5980 resin is smaller (~25 µm) with an agarose backbone. MabCapture A resin will be referred to as Resin 1 while the Purolite resin will be referred to as Resin 2. All buffers were filtered through AseptiCap KS Polyethersulfone Capsule Filters with 0.45/0.2 µm pore size (Advanced Microdevices, India).

Fedorenko D., Dutta A.K., Tan J., Walko J., Brower M., Pinto N.D.S., Zydney A.L, & Shinkazh O. (2020). Improved protein A resin for antibody capture in a continuous countercurrent tangential chromatography system. Biotechnology and bioengineering, 117(3).

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Poros mabcapture a

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7 protocols using poros mabcapture a

Purification of Fc Constructs Using Chromatography

Purification of Fc Constructs

Purification of Fc-Fusion Proteins

Antibody Production and Purification

Recombinant IgG1 Antibodies Targeting RSV F

Bispecific Antibody Production and Purification

Continuous Capture and Chromatography

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