Capto mmc

Manufactured by GE Healthcare

Sourced in United States

Capto MMC is a multimodal chromatography resin developed by GE Healthcare. It is designed for the purification of biomolecules such as proteins, peptides, and other macromolecules. The resin utilizes a combination of ion exchange and hydrophobic interaction mechanisms to capture and separate target molecules from complex mixtures.

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Capto MMC ImpRes (3 citations)

12 protocols using capto mmc

Purification of Recombinant Human G-CSF

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Example 6

Start Material

Purified rhG-CSF was diluted in an equilibration buffer to lower the total protein concentration and to achieve a more convenient volume prior to be loaded on a CAPTO® MMC column. And also to achieve the pH that was requested for each experiment. The rhG-CSF was prior to dilution dissolved in 20 mM sodium acetate, 0.2M NaCl, 0.02% TWEEN® 20, pH 6.5.

Chromatographic Resin and Column

CAPTO® MMC, a mixed mode resin from GE Healthcare (cat no. 17-5317), was used as capture step for the rhG-CSF molecule. CAPTO® MMC is a weak cationic resin with hydrophobic and thiophilic interactions and hydrogen bonding. A TRICORN® 5/150 column (GE Healthcare) was packed with CAPTO® MMC resin to a bed height of 15 cm. The column volume (CV) of CAPTO® MMC was 3 ml.

Buffers

US10214575B2. Process for the purification of a growth factor protein (2019-02-26). OCTAPHARMA AG [CH]. Inventors: Gustav Gilljam [SE], Stefan Winge [SE], Maya Tiemeyer [DE].

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Capto™ MMC Protein Purification

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The MMC was carried out using different volumes of Capto™ MMC (GE Healthcare/Cytiva, UK) resin packed in columns from different sizes attached to Äkta Avant 150. The volumetric flow for all purifications was set at 2.26 mL/min·cm². Once again, two strategies were tested in this purification step. In both strategies, the pH and conductivity of the fraction recovered from the previous purification step were corrected to match both the pH and conductivity of the equilibration buffer of the strategy. In the first strategy, the system was equilibrated with 5 CV of 50 mM sodium acetate buffer, pH 6.5. The elution was performed by an isocratic elution using 5 CV of 250 mM L-arginine in 50 mM acetate buffer, pH 6.5. In the second strategy, the system was equilibrated with 5 CV of 50 mM sodium acetate buffer, pH 5.0. The elution was performed by an isocratic elution using 5 CV of 50 mM bis–tris buffer, pH 7.0.

da Costa Rodrigues T., Zorzete P., Miyaji E.N, & Gonçalves V.M. (2024). Novel method for production and purification of untagged pneumococcal surface protein A from clade 1. Applied Microbiology and Biotechnology, 108(1), 281.

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Purification of TIMP3 and Fusion Constructs

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Human TIMP3, modified TIMP3 with glycosylation mutations (v2, v82), and fusion constructs (HSA, Fc, or Ab) were expressed in a Chinese hamster ovary cell line, whereby conditioned media were concentrated by tangential flow filtration (TFF; Millipore,10 kDa MWCO) and filtered. The TIMP3 and fusion constructs were next purified through a three‐column chromatography procedure, each utilizing a specific capture column; TIMP3 and glycosylation mutants with Capto MMC (GE Healthcare, Freiburg, Germany): HSA fusions with Cibacron Blue (Merck KGaA, Darmstadt, Germany): and Fc or Ab fusions with MabSelect Sure (GE Healthcare, Pittsburg, PA). Each of these was then followed by Capto Adhere and SP HP (GE Healthcare, Freiburg, Germany) chromatography steps. The TIMP3 containing SP HP fractions were finally concentrated and buffer exchanged (10 mmol/L sodium acetate pH 5.2, 9% sucrose) by TFF. N‐TIMP3 (13.9 kDa N‐domain; aa 1‐120) was expressed as inclusion bodies in an E. coli system with a subtilisin prodomain fusion. After refolding and prodomain cleavage, the N‐TIMP3 was purified by a three‐column procedure using SP HP, CHT, and Butyl HP chromatography. The N‐TIMP3 containing Butyl HP fractions were also concentrated and buffer exchanged (10 mmol/L sodium acetate pH 5.2, 9% sucrose) by TFF (Pall, 5 kDa MWCO).

Chintalgattu V., Greenberg J., Singh S., Chiueh V., Gilbert A., O'Neill J.W., Smith S., Jackson S., Khakoo A.Y, & Lee T. (2018). Utility of Glycosylated TIMP3 molecules: Inhibition of MMPs and TACE to improve cardiac function in rat myocardial infarct model. Pharmacology Research & Perspectives, 6(6), e00442.

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Recombinant Human Serum Albumin Production

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Approximately 1,500 kg of rice powder derived from the host variety TP309 was used to prepare the HCPs following a standard operation protocol of manufacturing OsrHSA (DMF No. 029648, https://www.fda.gov/drugs/developmentapprovalprocess). Briefly, rice seeds were ground into powder and homogenized in a phosphate buffer (PB) [25 mM PB with 50 mM NaCl (pH 7.5)] at a ratio of 1:5 (wt/vol) at room temperature for 1 h, and the mixture was precipitated for 2 h at pH 5.0. The crude extract was clarified by a filter press skid. The clarified extracts were carried out by three steps of chromatography, i.e. Capto-MMC, Q Sepharose Fast Flow and Phenyl HP (GE Healthcare, www.gelifesciences.com). The final residual HCPs from the final step of the chromatography was collected (Lot No. 201512001) and were concentrated to the appropriate concentration for future use. OsrHSA (Lot No. C201504001) was provided by Healthgen Biotechnology Co. Ltd, Wuhan, China (http://www.oryzogen.net), and its residual HCP content was 1.5 μg/g, as determined by Bradford assay. pHSA (Lot No. 201206A049) was purchased from the Institute of Chengdu Biological Products.

Abiri N., Pang J., Ou J., Shi B., Wang X., Zhang S., Sun Y, & Yang D. (2018). Assessment of the immunogenicity of residual host cell protein impurities of OsrHSA. PLoS ONE, 13(3), e0193339.

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Optimized Aggregate Removal via Mixed-Mode Chromatography

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Example 3

Purification with a Mixed-Mode Chromatography

The protein A purified mutIL15-Fc was adjusted to the required pH value and salt concentration by dialysis and/or adding of appropriate stock solutions and afterwards applied to the mixed-mode chromatography column previously equilibrated with 25 mM acetate buffer pH 5.5. As mixed-mode chromatography material Capto MMC has been used (GE Healthcare, Uppsala, Sweden). The column was loaded with up to 20 mg protein per ml column volume. A solution comprising 25 mM sodium phosphate at pH 6.5 was used for recovery of the protein from the column.

TABLE 2

Aggregate removal with a mixed-mode chromatography material.

HMWTotal

[%]HMWMonomer

123[%][%]

Load5.212.014.731.968.1

Eluate0.93.84.895.2

(Pooled)

Regenerate13.829.722.365.734.2

TABLE 3

HCP removal with a mixed-mode

chromatography material.

HCP [ng/mg]

Load112

wash<1071

pool23

Regenerate202

The mutIL15-Fc recovery was 58.3%.

TABLE 4

Purification with a mixed-mode chromatography.

loadingloadelution yield

buffer[mg/ml]modewithmonomermonomerHMWs

25 mMup to 20bind-25 mM58%95%5%

acetateand-sodium

buffer pHelutephosphate

5.5pH 6.5

US11168110B2. Method for separation of monomeric polypeptides from aggregated polypeptides (2021-11-09). HOFFMANN-LA ROCHE INC. [US]. Inventors: Monika Baehner [DE], Adelbert Grossmann [DE], Stefan Hepbildikler [DE].

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Recombinant RBD Protein Purification

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Fermentations in 50 L bioreactors were conducted according to the methods described earlier [17] (link). In brief, the seed culture contained 200 mL of seed culture in yeast extract–peptone–dextrose (YPD) media. The fermentation culture contained 10 g yeast extract, 20 g peptone, 40 g glycerol, 4 × 10^–3 g biotin, 4.47 g Na₂HPO₄, 8.22 g NaH₂PO₄, and 13.4 g yeast nitrogen base (YNB) per liter of culture medium. The fermentations were carried out using the following conditions: The pH was controlled to 6.4 with NH₄OH and the temperature was set at 25 °C. After a 10 min starvation phase, the methanol feed (100% methanol with 12 mL·L^–1Pichia trace minerals (PTM1) salts) was initiated. Methanol induction was continued for 45–50 h.
The fermentation supernatant was recovered by centrifugation at 8000 revolutions per minute (rpm) for 25 min and then collected for RBD purification. The samples were sequentially subjected to a multimodal weak cation exchange (Capto MMC; GE Healthcare, USA), hydrophobic chromatography (Phenyl Sepharose High Performance; GE Healthcare, USA), strong anion exchange (Source 30Q; GE Healthcare, USA), strong cation exchange (Source 30S; GE Healthcare, USA), and size-exclusion chromatography (SEC; Superdex-200; GE Healthcare, USA) to finally obtain high-purity RBD protein.

Liu B., Yin Y., Liu Y., Wang T., Sun P., Ou Y., Gong X., Hou X., Zhang J., Ren H., Luo S., Ke Q., Yao Y., Xu J, & Wu J. (2021). A Vaccine Based on the Receptor-Binding Domain of the Spike Protein Expressed in Glycoengineered Pichia pastoris Targeting SARS-CoV-2 Stimulates Neutralizing and Protective Antibody Responses. Engineering (Beijing, China), 13, 107-115.

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Purification of Recombinant RimJ from E. coli

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Transformed E. coli BL21 was allowed to grow in liquid LB medium overnight at 37°C and 200 rpm and then transferred into 50ml AIM medium in 500 ml shake flask. 12h after cultivation at 37°C and 200 rpm, cell pellet was obtained after centrifugation at 4000rpm for 20 min at 4°C and re-suspended in phosphate buffer (pH7.0). After washing for three times, the wet cell mass was sonicated for 20 min at 10°C. Supernatant was harvested by centrifugation at 12,000×g for 20 min at 4°C, and loaded onto a weak cation exchanger (Capto™ MMC, GE Health, 2cm×25cm) pre-equilibrated with PBS buffer (50 mM, pH 8.0). After elution with Na₂CO₃- NaHCO₃ buffer (pH 10.0) containing 1.0M NH₄Cl, fractions containing RimJ were collected and loaded onto a 10ml Ni²⁺ chelating Sephrose Fast Flow column. After washing with the binding buffer (50 mM sodium phosphate buffer, 500 mM NaCl, 50 mM imidazole, pH 7.5) to baseline, the bound protein was eluted by a washing buffer (50 mM sodium phosphate buffer, 500 mM NaCl, 500 mM imidazole, pH 7.5). Imidazole was then removed through dialysis against 50mM PBS (pH7.0).

Chen J., Li H., Wang T., Sun S., Liu J, & Chen J. (2017). Production of Nα-acetyl Tα1-HSA through in vitro acetylation by RimJ. Oncotarget, 8(56), 95247-95255.

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Protein Purification Protocol via Chromatography

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When the fermentation process was completed, the culture medium was harvested and centrifuged at 4°C and 8000 rpm for 20 min. The supernatant was concentrated by ultrafiltration using Millipore Cogent M1 Tangential Flow Filtration System (molecular weight cutoff, 30kDa) and then loaded onto a SephadeG-25 column(2.6cm×60cm) to remove pigment. A weak cation exchanger (Capto™ MMC, GE Health, 2cm×25cm) pre-equilibrated with sodium acetate -acetic acid buffer (25mM, pH 4.6) was then applied. The bound protein fractions were eluted using Na₂HPO₄-NaH₂PO₄ buffer (50mM, pH7.2) containing 1.0 M NH₄Cl. Fractions containing the target protein were pooled and further loaded on a Blue-Sepharose™ 6 Fast Flow (GE Health, 2cm×25cm) column pre-equilibrated with 0.05M citric acid-0.1M Na₂HPO₄ (pH 7.0). The column was washed with the same buffer to baseline and the bound protein was eluted with 0.05M KH₂PO₄ containing 1.5M KCl (pH 7.0). The collection of target protein was stored at 4°C for further analysis. SDS-PAGE was carried out to determine the homogeneity of purification and the molecular mass of the recombinant fusion protein as previously described [25 ]. Coomassie brilliant blue R-250 was used for staining.

Chen J., Li H., Wang T., Sun S., Liu J, & Chen J. (2017). Production of Nα-acetyl Tα1-HSA through in vitro acetylation by RimJ. Oncotarget, 8(56), 95247-95255.

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Purification and Characterization of Recombinant Protein

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Briefly, the PCR product was cloned into pGEX-6p-2 (Sangon Biotech, shanghai) and transformed into the Escherichia coli strain Xl1-blue (Biovector Scienc Lab, Beijing). Escherichia coli strain Xl1-blue containing the recombinant plasmid was induced with isopropyl-b-D-1-thiogalactopyranoside (IPTG) at a final concentration of 0.1 mM for GST fusion protein expression. We purified the GST-tagged proteins from the cleared lysates by Capto MMC (GE), and the GST tag was cleaved by preScission protease (GE). Then, we removed the endotoxin from the protein eluate using Triton X-114 phase separation as described elsewhere31 (link). The resulting protein was analysed by gel-filtration using the Superdex^TM 200 10/300GL column (GE). Protein purity was determined using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and using high-performance liquid chromatography (HPLC) with a C3 column. The concentration of the resulting protein was determined using the bicinchoninic acid (BCA) method (Pierce). The endotoxin content was detected using the tachyplens ameboyto lysate assay (Houshiji cod Inc., Xiamen, China).

Yang L., Cai C., Feng Q., Shi Y., Zuo Q., Yang H., Jing H., Wei C., Zhuang Y., Zou Q, & Zeng H. (2016). Protective efficacy of the chimeric Staphylococcus aureus vaccine candidate IC in sepsis and pneumonia models. Scientific Reports, 6, 20929.

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Expression and Purification of SARS-CoV-2 RBD Beta

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The SARS-CoV-2 RBD gene (GenBank accession number MN908947.3) was synthesized, ligated into the pPICZαA vector, and subsequently transferred into glycoengineered P. Pastoris cells and RBD-wild type protein (RBD-WT) was successfully expressed and purified (Liu et al., 2022 (link)). The gene sequence of K417N, E484K, and N501Y sites were mutated, and the target gene was cloned to generate expression vector pPICZαA-RBD-Beta. BglII-linearized pPICZαA-RBD-Beta was transferred into glycoengineered P. pastoris cells by electric shock, and expression of the target protein was induced by addition of methanol. Positive clones were screened by SDS-PAGE and western blot. The primary antibody was anti-RBD-WT rabbit serum (produced and kept in our laboratory) and the secondary antibody was goat anti-rabbit-HRP (1:2500, SAB3700885, SIGMA).
The positive strain highly expressing RBD-Beta was fermented in a 5 L biofermenter, and the target protein was induced with methanol for expression. The supernatant was passed through the following chromatographic columns: Capto MMC, Phenyl Sepharose Fast Flow, Source 30Q, Source 30S, and Superdex-G75 (all from GE Healthcare, Cal., United States) to obtain high purity RBD-Beta protein.

Xu H., Wang T., Sun P., Hou X., Gong X., Zhang B., Wu J, & Liu B. (2023). A bivalent subunit vaccine efficiently produced in Pichia pastoris against SARS-CoV-2 and emerging variants. Frontiers in Microbiology, 13, 1093080.

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