Title page for ETD etd-05132010-123505


Type of Document Master's Thesis
Author Hey, Carolyn McKenzie
Author's Email Address chey@vt.edu
URN etd-05132010-123505
Title Antibody Purification from Tobacco by Protein A Affinity Chromatography
Degree Master of Science
Department Biological Systems Engineering
Advisory Committee
Advisor Name Title
Zhang, Chenming Mike Committee Chair
Barone, Justin Robert Committee Member
Vinatzer, Boris A. Committee Member
Keywords
  • Tobacco
  • Protein A
  • Affinity Chromatography
  • Antibody
Date of Defense 2010-04-29
Availability restricted
Abstract
Antibodies represent the largest group of biopharmaceuticals. Due to the nature of

their clinical applications, they often need to be produced in large quantities. Plants have

distinct advantages of producing large quantities of recombinant proteins, and tobacco is

arguably the most promising plant for plant-made-pharmaceuticals (PMP) due to its high

biomass yields and robust transformation technology. However, to produce proteins using

transgenic tobacco for human applications, purification of the proteins is challenging. On

the other hand, Protein A, a bacterial cell wall protein isolated from Staphylococcus

aureus that binds to the Fc regions of immunoglobulins, is useful to the isolation and

purification of antibodies. An affinity chromatography purification step utilizing Protein

A resin introduced early in the purification process can reduce successive unit operations,

thereby reducing the overall process cost. However, directly applying tobacco extract to

Protein A chromatography columns may be problematic due to the non-specific binding

of native tobacco proteins (NTP). In this project, three different Protein A resins, ProSepvA

High Capacity, ProSep-vA Ultra, and ProSep Ultra Plus, marketed by Millipore, were

studied to provide valuable information for future downstream processes for antibody

purification from transgenic tobacco. The efficiency of the post load wash buffer to

reduce non-specific binding of NTP to the ProSep A resins were evaluated by altering the

ionic strength and pH. Lower salt concentrations of sodium chloride (NaCl) in the post

load wash preformed best at reducing the non-specific binding of NTP to the ProSep A

resins, while higher salt concentrations were more effective at reducing the amount of

NTP contaminants present during elution of the columns. Using a post load wash buffer

with an intermediate pH between the binding buffer and the elution buffer was more

efficient at eluting our model antibody, human IgG. However, lowering the ionic strength

and the pH of the post load wash buffer resulted in a greater presence of IgG prematurely

eluting from the ProSep A resins. The non-specific binding of NTP to the resins reduced

the dynamic binding capacity (DBC) of the resins after repeated cycles of tobacco extract

samples were loaded onto the column. Nevertheless, cleaning the columns with

denaturing solutions, such as urea or guanidine hydrochloride, every 8-10 cycles was

effective in regenerating the DBC of the resins and prolonging the life cycle of the resins.

This is important to evaluating the economic feasibility of directly using Protein A

chromatography to recover antibodies from tobacco extract. Of the three Protein A resins

studied, ProSep Ultra Plus performed best for antibody purification from tobacco using a

PBS wash buffer with a lower ionic strength of 140mM NaCl and an intermediate pH of

5.

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