The purity of the isolated charge variants and starting material was determined by weak cation-exchange chromatography using a ProPac WCX-10 analytical column (4×250 mm, Dionex). Mobile phase A was 20 mM MES, 50 mM NaCl, pH 5.6, and mobile phase B was 20 mM MES, 240 mM NaCl, pH5.6. The initial concentration of phase B was 30%, and a linear gradient was run from 30% to 70% over 35 min followed by a linear increase from 70% to 100% over 0.1 min and a constant 100% flow from 35.1 to 40 min. The reference and charge variant samples were diluted with mobile phase A to approximately 0.25 mg/mL. The column was flushed and equilibrated with the initial running buffer until the baseline was stable. Peak areas were quantified relative to monobody, and protein was detected at 214 nm.
Propac wcx 10
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The ProPac WCX-10 is a weak cation-exchange chromatography column designed for the separation and purification of biomolecules, such as proteins and peptides. It features a silica-based stationary phase with carboxylic acid functional groups for the retention and separation of positively charged analytes.
Automatically generated - may contain errors
Lab products found in correlation
Sodium phosphate, by Merck Group (1 mentions)
Amicon ultra 15 centrifugal filter unit, by Merck Group (1 mentions)
Unicorn 7, by GE Healthcare (1 mentions)
Kta avant 25, by GE Healthcare (1 mentions)
Agilent chemstation software, by Agilent Technologies (1 mentions)
Hplc system, by Shimadzu (1 mentions)
14 protocols using propac wcx 10
1
Charge Variant Purity Analysis
2
Charge Variant Analysis by HPLC
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Determination of charge variants was performed using a ProPac WCX-10 (4 × 250 mm) analytical column (Dionex, United States) connected to HPLC system (Agilent Technologies 1100/1200 Series, United States) with UV detection at 220 nm.
3
Purification of Romosozumab Variants
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Example 2
Purification or enrichment for different romosozumab species from a composition comprising wild-type romosozumab and the romosozumab PARG (SEQ ID NO: 8) C-terminal variant is achieved by Cation Exchange Chromatography (CEX) fractionation. CEX separates proteins based on differences in their surface charges. At a set pH, positively charged variants of wild-type romosozumab are separated on a cation-exchange column (e.g., Dionex Pro Pac WCX-10 analytical column, 2.0 mm×250 mm) and eluted using a salt gradient (e.g., Mobile Phase A: 10:90 (v/v) ACN, 19 mM MES pH 6.2; Mobile Phase B: 10:90 (v/v) ACN, 19 mM MES, 250 mM NaCl, pH 6.2). The different C-terminal variants of romosozumab are charged differently and the more positively charged variant elutes later in CEX. Thus, the elution order is: PG (wild-type), P-amide (amidated proline of wild-type), PARG (SEQ ID NO: 8) variant, and PAR-amide. The fraction collector can be programmed to collect CEX eluents containing different variants at different elution times.
4
Rituximab Variant Separation by CEX
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Cation exchange chromatography (CEX) separates protein variants based on differences in charge, separate rituximab main compound from acidic and basic variants. Samples were treated with carboxypeptidase B to remove C-terminal lysine residues (CPB; Roche, Mannheim, Germany), and buffer exchanged into 25 mM sodium phosphate pH 7.0. CEX separation was performed using a weak CEX resin (ProPac WCX-10, 4 × 250 mm, Dionex, Germering, Germany), using a linear sodium chloride gradient. UV detection was carried out at 280 nm.
5
Cation-exchange Chromatography for Protein Variant Separation
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Cation-exchange chromatography (CEX) was used to separate protein variants based on charge. CEX separation was performed using a Dionex ProPac WCX-10 cation-exchange column (4 × 250 mm, 10-micron). UV detection was performed at 214 nm.
6
Cation-Exchange Purification of Proteins
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CEX separation was performed using a weak CEX column (ProPac WCX-10, 4 × 250 mm, Dionex, Germany) connected to an Agilent 1260 HPLC system. Mobile phase A consisted of 10 mM sodium phosphate (pH 7.5), and mobile phase B consisted of 10 mM sodium phosphate and 100 mM sodium chloride (pH 7.5). Samples were diluted using equilibration buffer consisting of mobile phases A and B at a ratio of 85 : 15 (v/v) to a final concentration of 2 mg/mL. For C-terminal lysine cleavage, 15 μL of 1 mg/mL carboxypeptidase B (CPB; Roche, Germany) was added to 1 mL of the sample solution, and the mixture was incubated at 37°C for 30 min. The separation gradient was set as follows: 0 to 3 min, holding at 15% B, 3 to 6 min, 15% B–30% B, 6 to 36 min, 30%–70% B, and 36 to 38 min, 70%–100% B. The column was then washed with 100% B for 5 min, followed by equilibration using 15% B for 15 min. The column temperature was set at 30°C and the absorbance of the eluent was monitored at 214 nm.
7
Ion Exchange Chromatography-HPLC Separation
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Ion exchange chromatography-high performance liquid chromatography (IEC-HPLC) separation was performed using a weak cation exchange resin (ProPac WCX-10, 2.0 × 250 mm, 10 μm particle size, Dionex). Mobile Phase A (20 mM Sodium Phosphate, pH 7.10) and Mobile Phase B (200 mM Sodium Chloride in 20 mM Sodium Phosphate, pH 7.10) were used at a gradient of 0 to 20% B at a flow rate of 0.5 mL/minute to elute the samples with the column temperature of 50 °C and UV detection wavelength of 214 nm.
8
Purification of Romosozumab Variants
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Example 2
Purification or enrichment for different romosozumab species from a composition comprising wild-type romosozumab and the romosozumab PARG LSEQ ID NO: 8) C-terminal variant is achieved by Cation Exchange Chromatography (CEX) fractionation. CEX separates proteins based on differences in their surface charges. At a set pH, positively charged variants of wild-type romosozumab are separated on a cation-exchange column (e.g., Dionex Pro Pac WCX-10 analytical column, 2.0 mm×250 mm) and eluted using a salt gradient (e.g., Mobile Phase A: 10:90 (v/v) ACN, 19 mM MES pH 6.2; Mobile Phase B: 10:90 (v/v) ACN, 19 mM MES, 250 mM NaCl, pH 6.2). The different C-terminal variants of romosozumab are charged differently and the more positively charged variant elutes later in CEX. Thus, the elution order is: PG (wild-type), P-amide (amidated proline of wild-type), PARG (SEQ ID NO: 8) variant, and PAR-amide. The fraction collector can be programmed to collect CEX eluents containing different variants at different elution times.
9
Ion Exchange Chromatography of Biomolecules
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The IEX chromatography was performed on a liquid chromatograph (KNAUER,) Detection was performed at 280 nm. Flow rate was 0.8 mL/min, the injection volume was 80μL and the column compartment temperature was set at 25°C. Instrument control and data acquisition were performed using ChromGate software. The monolithic IEX column used was ProPac WCX-10, 4 mm × 250 mm (Thermo Scientific, USA). The mobile phases used were mobile phase A (0.01 M Sodium phosphate (Merck, Darmstadt, Germany) buffer pH 6.6) and mobile phase B (0.01 M Sodium phosphate buffer + 0.1M Sodium chloride pH 6.6). The elution was performed by an ascending gradient from 30% to 100% eluent B followed by isocratic elution for 2 min before returning the eluent composition to the starting condition (100% eluent A).
10
Fractionation and Characterization of mAb Charge Variants
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Preparative separation of the mAb charge variants was carried out on a 9 × 250 mm ProPac™ WCX‐10 (Thermo Fisher Scientific Inc.) using an ÄKTA avant 25 and UNICORN 7.0 (GE Healthcare GmbH). mAb1 fractions were eluted using a linear NaCl gradient and pooled to obtain the charge variants as shown in Figure S1. As a control, all fractions of one run were pooled to obtain a sample of all charge variants exposed to the same conditions as the fractions. The charge variants and control sample were concentrated to around 2–5 g/L and rebuffered to 10 mM sodium phosphate pH 6.8 using 10 kDa molecular weight cut‐off Amicon® Ultra‐15 centrifugal filter units (Sigma‐Aldrich Chemie GmbH). In total, the fractionation experiment was carried out twice, and all resulting samples were checked by weak cation exchange chromatography (WCX) and capillary electrophoresis (CE). Biophysical properties (circular dichroism [CD], nano differential scanning fluorimetry [nano‐DSF], size exclusion chromatography [SEC]) were determined from one run in duplicates.
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