Webinar 5: Affinity chromatography! How pure is your protein? June, 5 th 9-10 a.m. and June, 6 th 5-6 p.m. (CET)

Transcription

1 Webinar 5: Affinity chromatography! How pure is your protein? June, 5 th 9-10 a.m. and June, 6 th 5-6 p.m. (CET) 1

2 Summary webinar 4: Don t worry: Use HIC and HILIC! 2

3 Presteps in your purification process Characteristics of your sample (molecular weight, stability, purity -> extra filtration step necessary?) Resin: exclusion limit, flow rate, extra immobilization necessary? Classical affinity chromatography or IMAC? Pre-packed column or self-packing? How do you want to stay in progress during your run? 3

4 Affinity chromatography (AC) purification of one protein of interest from a mixed sample material: highly crooslinked agarose with special ligands (Protein G, Protein A or Heparin) or pre-activated agarose Step 1: preparation of an immunoadsorbent with BioFox ACT (activated via Bromohydrin Method) Matrix-OCH 2 CH(OH)CH 2 Br + Nu - (i.e. -SH, -NH 2 or -OH) Matrix-OCH 2 CH(OH)CH 2 -Nu special examples: coupling of special peptide sequences or antibodies for the isolation of a particular protein Special form: Immobilized metal-ion affinity chromatography 4

5 Which are the main characteristics for the BioFox ACT medium? Particle size: 40 +/- 10 µm Exclusion limits: 40/100; 40/1200; 40/10000 Pressure stable up to 40 bar ph range 1-14 BULK-material in different volumes (25 ml, 50 ml, 150 ml, 300 ml, 1000 ml, 5000 ml) 5

6 Important specs of pre-activated material BioFox media: for coupling of small molecules and peptides coupling groups: -OH, -SH, NH 2 competitors differentiate into coupling groups (they have special pre-activated media for): -NH 2 -SH -COOH -OH 6

7 Choice of different activated materials Example: CNBr-activated Sepharose 4 Fast Flow (GE Healthcare) BioFox 40/10000 ACT (Knauer) Goal: Comparison regarding: coupling yield Leakage of antibodies Interferon capacity 7

8 CNBr-activated Sepharose vs. BioFox 40/10000 ACT CNBr-activated Sepharose: basic material: cross-linked 4 % agarose beads particle size: 90 µm preactivated with cyanogen bromide designed for protein ligands contanining amino groups BioFox 40/10000 ACT: basic material: cross-linked % agarose bead particle size: 40 µm preactivated with bromhydrin exclusion limit: kd designed for protein ligands contanining amino-, hydroxy- and thiol groups

9 Coupling of polyclonal goat antibody Type of resin Eluate [ml] A 280 Coupling yield [mg/ml resin] BioFox 40/10000 ACT CNBr Sepharose 4 Fast Flow Coupling capacity 30 % greater for BioFox Reason: higher agarose content of BioFox-material 9

10 Leakage of polyclonal goat antibody Type of resin BioFox 40/10000 ACT CNBr Sepharose 4 Fast Flow Wash step at ph 8 [ng/ml] Wash step at ph 2 [ng/ml] Interferon fraction [ng/ml] Leakage much lesser for BioFox Reason: Bond between BioFox and antibody is secondary amine or thioether -> both are stable in a wide ph range 10

11 Interferon capacity Type of resin BioFox 40/10000 ACT CNBr Sepharose 4 Fast Flow Applied material titer [IU] Interferon capacity [IU/ml resin] 127 x x x x 10 6 Interferon capacity is comparable 11

12 Conclusion Antibody leakage significantly lower for BioFox Interferon capacities are comparable for the two gels Coupling capacity much higher (30 %) bonding chemistry in BioFox creates stable bonds This minimizes leakage of antibodies from the gel BioFox is suitable for the purification of pharmaceuticals This study was performed by Sven Lundberg at Bionative AB in Umea Sweden, in cooperation with the research laboratory 12 at Inovata AB in Stockholm, Sweden.

13 Immobilized metal affinity chromatography (IMAC) Purification and detection of recombinant antibodies One step purification of (HIS) 6 -tagged proteins Structure of the medium: Chelating groups: Nitrilotriacetic acid (NTA) chelat agarose Iminodiacetic acid (IDA) metal-ion Tris(2-ethylaminoethyl) amine (TREN) metal-ions: Zn 2+, Ni 2+, Co 2+, Ca 2+, Cu 2+, and Fe 3+ 13

14 When should you use IDA and when TREN? What is a suggested workflow? Start with BioFox 40 IDAlow If low capacity: use BioFox 40 IDAhigh If difficult to elute: use BioFox 40 TRENlow If low capacity: use BioFox 40 TRENhigh Note: Ni bound to IDA (3 available positions binding of proteins Ni bound to TREN (2 indicating positions and milder elution conditions) 14

15 BioFox IMAC media compared to the main competitor BioFox BULK medium Metal ion capacity [µeqv Ni 2+ /ml Biofox 40 IDAhigh BioFox 40 IDAlow BioFox 40 TRENhigh BioFox 40 TRENlow BioFox 40 Ni competitor BULK medium Metal ion capacity [µeqv Ni 2+ /ml IMAC Sepharose HP IMAC Sepharose 6 FF Chelating Sepharose FF ~ 15 ~ (Cu 2+ ) Max. pressure [bar] Pre-packed BioFox 40 TRENhigh-Co Metal ion capacity [µeqv Ni 2+ /ml Max. pressure [bar] BioFox 40 TRENhigh-Ni HighTrap IMAC HP HighTrap IMAC FF HighTrap chelating HP ~ 15 ~ (Cu 2+ )

16 Automated 2-step seperation Goal: purification of b-galactosidase Main conditions: material: BioFox 40 IDAlow loaded with Ni 2+ column volume: 11 ml sample volume: 2.5 ml crude extract buffer: A) 50 mm Na/K-phosphate buffer (ph 6.5) mm NaCl B) 50 mm Na/K-phosphate buffer (ph 6.5) mm NaCl mm Imidazol C) 20 mm Tris-buffer (ph 7.2) detection: UV (280 nm) 16

17 Configuration of the system 17

18 1st choice: 5 mm Imidazol in binding buffer Very small amount of b-galactosidase 18

19 2nd choice: 20 mm Imidazol in binding buffer Increase of the amount 19

20 Automated purification process 1st step IMAC-separation 2nd step desalting process run time: 100 min flow rate: 2 ml/min for the elution and desalting process result: desalted stablized b-galactosidase 20

21 How can I check the identification and purity of my biomolecule of interest? 21

22 1st option: SDSpage results in ww2.chemistry.gatech.edu/.../page_protein.htmlframe entfernen Quality control of human IgE preparation from hybridoma (lane 2) vs. IgE sample purified from myeloma patient serum (lane 3). MW markers are shown in lane

23 2nd option: UHPLC PLATINblue UHPLC w/o MS coupling Results in 23

24 3rd option: ELISA Enzyme-linked immunosorbent assay Step 0: coating Step 1: Antigen-antibody-reaction Step 2: Antibody-enzyme-reaction Step 3: Measurement of enzyme activity Increase of the signal: sandwich-elisa (two antibodies bind on the same antigen) 24

25 Summary Comparison between BioFox and competitor material: Better coupling capacity and lesser leakage with BioFox BioFox suitable for the purification of pharmaceuticals Application for IMAC 20 mm Imidazole in binding buffer is necessary Methods to stay in progress with your run SDS-Page UHPLC ELISA 25

26 Next webinar before summer break: June, 12 th and 13th Webinar 6: Do you want to purify more? Fast Liquid Biochromatography!

27 Biochromatography applications: LC systems: 27