1. Bringing Biotech Product to Market Chapter 9

2. Objectives Purifying product Define chromatography and distinguish between planar and Column chromatography

3. Purification Techniques Column chromatography  Sample passed through a column packed with submicroscopic beads  Beads act as molecular sieves (separate mole. Based on characteristics ex. size, shape, charge)

4. Column chromatography Tube with membrane (frit) near the base Resin beads of particular type (in buffer) are poured into the column Resin beads settle onto the frit and form matrix Molec pass through it

5. Column chromatography Sample get added to top of resin bed Gravity pulls it into the matrix Depending on the resin, molec will either bind to beads or pass through them As sample drips out, collected at regular intervals (fractions) Before sample is passed through column it must be in the right buffer

6. Column chromatography Proteins Since most proteins are colorless their separation on column is not visible So are fractions collected To visualize which protein separate into which fraction samples are run on PAGE and ID by MW By comparing bands in original column load to the bands in the fractions, technicians can determine the amount of separation Often take more than one kind of column chromatography

7. Chromatography Types Column chromatography Planar chromatography  Paper  Thin layer Separation method for biomolecules Outcome of a chromatography experiment is a CHROMATOGRAM

8. Chromatography Basics All chromatographic systems contain:  Stationary phase  Mobile phase  Sample molecules (mixture for separation) Movement of molecules determined by balance between 2 forces:  1. Mobile phase  2. Stationary phase

9. Chromatography Basics 1. Mobile phase (impelling force)  Carries with it molecules for which it has affinity - favored by solubility (LC), volatility (GC) 2. Stationary phase (retarding force)  Holds back molecules with which it interacts Balance between forces differs for different molecules Different mobilities, separation of components

10. Column chromatography

11. Planar Chromatography Paper Thin-layer

12. Column chromatography Types 1. Gel filtration (size exclusion) chromatography  Sep molec based on size 2. Ion-exchange chromatography  Sep molec based on charge 3. Affinity chromatography  Sep molec based on shape and unique functional group 4. Hydrophobic- interaction chromatography  Sep molec based on hydrophobicity (not soluble in water)  Not very common (will not discus further)

13. Gel filtration (size exclusion) chromatography Preferred for high weight (>2000-3000) neutral molec  Polymers, high MW proteins Separation b/c solute permeation into solvent filled pores within column packing  Large: excluded from pores b/c physical size No retention  Small: permeate greater portion of pores Retained  In between: partial permeation Retention btw 2 extremes

14. Ion-exchange chromatography Ion exchanger surface  Cations, anions, water Cations, or anions chemically bond to insoluble matrix (organic and porous in nature)  Chemical bound ions = fixed ions  Ions of opposite charge = counter ions  Pores have water and sufficient [counter ions]  Exchanger electrically neutral Cations exchanger: fixed ions negative Anion exchanger: fixed ions positive  Counter ions replaced by ions of same charge from external solution M + E - + A - ↔ M + A - + E - Anion exchanger

15. Affinity chromatography Antibody recognizes only certain antigen Will bind them and pull them out of solution

16. High performance liquid chromatography HPLC

17. HPLC

18. Homework Sec 9.1 Questions 2 and 3 Sec 9.2 Questions 1, 2, and 3 This project is funded by a grant awarded under the President’s Community Based Job Training Grant as implemented by the U.S. Department of Labor’s Employment and Training Administration (CB-15-162-06-60). NCC is an equal opportunity employer and does not discriminate on the following basis: against any individual in the United States, on the basis of race, color, religion, sex, national origin, age disability, political affiliation or belief; and against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998 (WIA), on the basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in the United States, or his or her participation in any WIA Title I-financially assisted program or activity. This product was funded by a grant awarded under the President’s High Growth Job Training Initiative, as implemented by the U.S. Department of Labor’s Employment & Training Administration. The information contained in this product was created by a grantee organization and does not necessarily reflect the official position of the U.S. Department of Labor. All references to non-governmental companies or organizations, their services, products, or resources are offered for informational purposes and should not be construed as an endorsement by the Department of Labor. This product is copyrighted by the institution that created it and is intended for individual organizational, non-commercial use only.