A filler containing human serum albumin, a construction method and applications thereof

Abstract

The present invention relates to a coupled filler for purifying multivalent nanobodies that specifically bind to human serum albumin, as well as its preparation method and use. The present invention specifically provides a filler containing human serum albumin and a preparation method, comprising the steps of washing the filler using a specific method, mixing it with a human serum albumin solution for coupling, and then performing subsequent treatment with a blocking solution. The present invention also provides a method for purifying multivalent nanobodies using the filler. This method has high specificity, mild operating conditions, high nanobody purity and recovery rate, and is conducive to commercial large-scale production.

Claims

1. A method for purifying multivalent nanobodies using affinity chromatography filler based on human serum albumin (HSA), characterized in that: The method comprises the following steps: Column cleaning: Use 0.1 M NaOH cleaning solution to clean; Balance: Use 10 mM PBS, pH = 7.4 balance solution; Sample loading: loading samples containing multivalent nanobodies; Column cleaning: Use 10 mM PBS, pH = 7.4 cleaning solution; Elution of target product: 20 mM sodium acetate, pH = 3.5 eluent; Column cleaning: Use 0.1 M NaOH cleaning solution to clean; Preparation before column loading: wash with 20% ethanol and store the affinity chromatography column; Wherein, the multivalent nanobody is a trivalent nanobody Anti-EGFR & Albumin Nanobody, and the amino acid sequence of the Anti-EGFR & Albumin Nanobody is shown in SEQ ID NO: 3; Wherein, the affinity chromatography filler based on human serum albumin HSA is prepared by the following method: (1) Take the required amount of agarose filler medium and drain it into a sand core funnel of corresponding specifications; (2) Add 2 times the volume of medium to pre-cooled 1 mM HCl solution, stir evenly and then drain; repeat 2 times; (3) Transfer the medium to a reactor of corresponding specifications; (4) Dissolve human serum albumin sample at a concentration of 0.5-10 mg / ml in 0.1 M NaHCO3, 0.5 M NaCl, pH 8.3 solution, add it to the reactor, and stir gently at room temperature for 4 h; (5) Transfer the medium to a sand core funnel and drain it, saving the coupled solution in the collection container; (6) Add 2 times the volume of medium 0.1M Tris-HCl, pH 8.0 solution, stir evenly and drain; repeat twice; (7) Transfer the medium to a reactor of corresponding specifications, add 0.1 M Tris-HCl, pH 8.0 solution (2 times the volume of the medium), and gently stir at room temperature for 4 h; (8) Transfer the medium to a sand core funnel and drain; (9) Add 2 times the volume of medium 0.1 M Tris-HCl, 0.5 M NaCl, pH 9.0 solution, stir well and drain; (10) Add 2 times the volume of medium containing 0.1 M sodium acetate, 0.5 M NaCl, pH 3.0 solution, stir well, and drain; (11) Repeat steps (9)-(10) twice in total; (12) Add 20% ethanol twice the volume of the medium, stir evenly, and then drain. Repeat twice; (13) Transfer the medium to a container of corresponding specifications, add an equal volume of 20% ethanol and store for later use.

2. The purification method according to claim 1, wherein The purification method comprises the following steps: (1) Column cleaning: 0.1 M NaOH was used at a rate of 1 ml / min until A280 dropped to a constant value. (2) Equilibration stage: 10 mM PBS, pH = 7.4, washing at a rate of 1 ml / min until A280 drops to a near constant value and equilibrium is completed; (3) Sample loading: The sample was loaded at a rate of 1 ml / min, with a retention time of 5 min, until the A280 value dropped to a constant value and the loading was completed; the flow-through sample was collected for testing; (4) Column cleaning: 10 mM PBS, pH = 7.4, at a rate of 1 ml / min, until A280 drops to a constant value; (5) Elution stage: 20 mM sodium acetate, pH = 3.5, elution at a rate of 1 ml / min until A280 drops to a constant value; collect the eluted samples for detection; (6) Column cleaning stage: 0.1 M NaOH was used for cleaning at a rate of 1 ml / min until A280 dropped to a constant value, and the CIP was terminated. The CIP samples were collected for testing. (7) Preparation before column: Wash with 20% ethanol at a rate of 1 ml / min for 10 CV, and store the affinity chromatography column containing human serum albumin filler at 4°C.

3. The purification method according to claim 1 or 2, characterized in that The multivalent nanobody is obtained by fermentation, and the fermentation includes the steps of primary seed culture, secondary seed culture, fermentation culture, and preparation of fermentation supernatant.

4. The purification method according to claim 3, wherein The fermentation comprises the following steps: (1) Primary seed culture: Activate the stored recombinant Pichia pastoris and culture at 25-32°C and 100-250 rpm for 16-18 h; (2) Secondary seed culture: inoculate the primary seed solution into the seed culture medium at a volume ratio of 1-5%, and culture at 25-32°C and 100-300 rpm until OD600 = 6-8; (3) Fermentation culture: inoculate the seed liquid into the fermentation medium, culture in the fermenter, control the DO at 10%-50%, the stirring speed at 200-800 rpm, the ventilation volume at 0.5-2.5 vvm, and add a certain concentration of pure oxygen to maintain the DO of the fermentation liquid within an appropriate range; (4) Preparation of fermentation supernatant: After fermentation for at least 76 h, the fermentation broth was centrifuged to remove the bacteria, and the supernatant was vacuum filtered and collected to obtain the fermentation broth.

5. The purification method according to claim 4, characterized in that The controlled DO was 30%.

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