Bacterial sphingomyelinase from Bacillus cereus, bacterial ceramidase from Pseudomonas aeruginosa, and lysenin from Eisenia fetida were expressed as his-tag fusion protein in E. coli BL21, and protein was purified using affinity chromatography with a HisTrap FF 5 ml column and size-exclusion column (Superdex 200 HiScale 26/40) using a FPLC system Äkta Pure 25 from Cytiva (Marlborough, MA), as previously described (21 (link)).
Kta pure 25
Manufactured by Cytiva
Sourced in United States
The Äkta Pure 25 is a liquid chromatography system designed for purification of biomolecules. It provides automated control and monitoring of the purification process, including sample application, separation, and fraction collection.
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Lab products found in correlation
6 protocols using kta pure 25
1
Purification of Bacterial Enzymes
2
Optimizing Trastuzumab Purification Process
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The precipitate harvested during the integrated perfusion‐capture process was dissolved at low pH as reported in Section 2.6 . For process optimization, we applied a DoE approach. The software used for the design of experiments and statistical evaluation was Design‐Expert (Stat‐ease inc.). The solution pH was neutralized to the required pH by titration with 0.02 M NaOH. The conductivity was adjusted by addition of 1 M NaCl or by dilution with HQ‐water. For these experiments, an Äkta pure 25 (Cytiva) and a negatively charged membrane adsorber, the 3 M Polisher ST (1 cm2 area), were used. Prior to the loading, the device was flushed with 400 L/m2 of 50 mM phosphate buffer for which pH and conductivity were adjusted according to the feed solution. 50 mL (500 L/m2)of re‐dissolved trastuzumab was loaded on the device at a flow rate of 1 mL/min (600 LMH), as suggested from the manufacturer. The flow‐through was pooled and stored at 4°C before analysis.
3
Antibody Purification by Protein A Chromatography
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The antibodies were captured using preparative protein A chromatography with a conventional chromatographic workstation ÄKTA Pure 25 coupled with a fractionating module (Cytiva). The equilibration, wash 1, wash 2, and elution buffers were 50 mM phosphate buffer, pH 7.4; 50 mM phosphate buffer, pH 5.0; 50 mM phosphate buffer and 1 M NaCl, pH 7.4 as well as 100 mM Glycine buffer pH 3.5. Protein A MabSelect Sure resin (Cytiva) was packed in a HiScale 26 housing (Cytiva) resulting in a column volume of 24.4 ml. For purification, the column was equilibrated with 10 CV equilibration buffer and loaded with 450 ml of culture supernatant. Washes were performed with 10 CV of wash buffer 1, and 10 CV of wash buffer 2. The product was eluted with a linear gradient of 5 CV to the elution buffer and an additional 5 CV of elution buffer. The sample was collected using the fractionating system connected to the ÄKTA and was neutralized to pH 7. The process was monitored by the conductivity, pH, and absorbance at 280 nm through the Unicorn 7.0 Software (Cytiva).
4
Pulse Injection Experiments for Trastuzumab RTD
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The RTD for trastuzumab was determined by pulse injections experiments on all unit operations in which the antibody is in form of precipitate (the PEG6000 tubular reactor and the two stages of TFF). The precipitated antibody is retained in the retentate of the HF. To mimic the path followed by the antibody with salt, the permeate lines were kept closed during the experiments. The outlet of the system was connected to an Äkta pure 25 (Cytiva) for conductivity recording. First, the system was flushed with deionized water until the conductivity was below 0.3 mS/cm. Pulse injections with a total volume of 10 mL of 1 M NaCl were performed by switching the feed line from deionized water to the salt solution and back. We applied the tank‐in‐series model with increasing number (N) of continuously stirred tank reactors (CSTR) to fit our experimental data. The cumulative distribution function (F) for a series of ideal‐stirred tanks is given by equation 1 :
where θ refers to the total loading volume [mL] normalized by the system volume [mL].
where θ refers to the total loading volume [mL] normalized by the system volume [mL].
5
Production and Purification of AAV-DJ Vectors
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HEK293F (ThermoFisher, Waltham, MA, USA) cells were cultivated in 2.8 L shake flasks to prepare for vector production. Prior to transfection, cell counts were initially assessed using the Vi-Cell XR Cell Counter (Beckman Coulter, Pasadena, CA, USA) to ensure that viable cell concentrations fell within the range of 2.0 × 106 to 2.6 × 106 cells/mL, with viabilities exceeding 95%. The respective mass proportions of pHelper, pRep/Cap, and pGOI were 0.43, 0.35, and 0.22. The capsid variant was AAV-DJ [20 (link)], and the gene of interest (GOI) was human FIX driven by a liver-specific promoter. The transfection reagent employed was FectoVIR (101000004, Polyplus, SA, France). Cell harvesting was performed 72 h following transfection of the cultures. The harvested cells underwent treatment with a lysis buffer (1× PBS, 1 mM MgCl2, 0.5% Triton-x 100 (ThermoFisher, Waltham, MA, USA)) to release AAV particles, followed by the removal of cellular debris using a high-speed centrifuge. Subsequently, purification was carried out utilizing ÄKTA pure 25 (Cytiva, Malborough, MA, USA) to isolate AAV particles from cellular debris and impurities. The concentrated AAV underwent an additional step of CsCl ultracentrifugation to eliminate empty particles. The ultimate AAV product was then formulated in a suitable buffer and stored in a −80 °C freezer for preservation.
6
FGF-2 Purification by Cation Exchange
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FGF‐2 was purified by chromatographic purification on an Äkta Pure 25 (Cytiva) as described in Sauer, et al. (2019b (link)). In brief, E. coli homogenate was 0.22 μm filtered and 118 ml (10 CV) of the clarified homogenate were loaded on a column packed with weak cation exchanger Carboxymethyl‐Sepharose Fast Flow (Cytiva) with 11.8 ml CV (1 cm diameter, 15 cm bed height). The column was equilibrated before and washed after loading with 100 mM Na‐phosphate, pH 7.0. FGF‐2 was eluted by a linear gradient from 0 to 1 M NaCl in 100 mM Na‐phosphate pH 7.0. During the elution phase, the effluent was collected in 1 ml fractions. Fifteen fractions were analyzed around the peak center. The column was sanitized after each run with 1.0 M NaOH for 1 h (5 CV). For the case studies, proprietary purification protocols were used. For model training, 8–9 replicate runs were performed at each of the new sites, 6–7 of them as training runs, and 2 runs as test set.
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