Protein A /G/ LIntroduction and antibody purification examples
Case presentation 1: alkali resistance test of at protein a sanarose 4FF
♦ Materials and methods:
Filler batch No.: at proteinA sanarose 4FF batch No.: 20230331;
Test load sample: human IgG;
Chromatographic column: 1ml pre installed column;
♦ Buffer solution
Cleaning solution: 0.5m NaOH;
Buffer solution A: 20mm sodium dihydrogen phosphate, 150mm NaCl, pH 7.4;
Buffer B: 0.1 M sodium citrate, pH 3.0;
Neutralization buffer: 1m Tris Cl, pH9.0
♦ RUN
0.5M NaOH，15CV，1mL/min
↓
wash，10CV，1mL/min
↓
Buffer a balance，10CV，1mL/min
↓
Buffer B elution，10CV，1mL/min
The dynamic load was measured after 0, 10, 20, 30, 40, 50, 65, 75, 105, 155, 197, 212, 242 and 250 cycles respectively;
♦ empirical conclusion
As shown in the figure below, the chromatographic medium at proteinA sanarose 4FF was cleaned online with 0.5 M NaOH. The contact time of a single cleaning was 15min. After 250 cycles (cleaning time 3750min, 62.5hr), the remaining binding load was 89% of the initial load. This experiment shows that at protein a sanarose 4FF has strong alkali resistance, which is very important for the cleaning of chromatography medium in large-scale purification.
Fig. 3 Relationship between at protein a sanarose 4FF 0.5m NaOH cleaning times and load change
Case display 2: purification of mouse serum using at protein a sanarose 4FF
♦material information
Sample information: 2ml mouse ascites (mouse IgG);
Filler batch No.: at proteinA sanarose 4FF batch No.: 20230331;
Chromatographic column: 1ml pre installed column;
♦ Buffer system
Equilibrium solution: 1*pbs, pH 7.4;
Eluent: 10mm NaAc HAC, pH 3.5;
Neutralization Solution: 1m Tris, pH9.0;
♦ RUN
Buffer a balance，10CV，1mL/min
↓
Sample loading，0.2 mL/min
↓
Buffer a balance，10CV，1mL/min
↓
Buffer B elution，10CV，1mL/min，Collect the target protein
↓
Detection of SDS-PAGE purification results
♦ Detection of SDS-PAGE purification results
Fig. 4 at protein a sanarose 4FF purified mouse serum
结Theory: from the perspective of purification results, the purification effect of at protein a sanarose 4FF is better than that of competitive product (lxpa).
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