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7:00am – 6:00pm Registration Open

7:30 Breakfast Workshop (Sponsorship Available)   

ANALYTICAL ADVANTAGE

8:15 Chairperson’s Remarks

8:20 Novel Dye Affinity Matrix Library for Protein Chromatography, Proteomics and Scale Up
Narayan Punekar, Ph.D., Professor, Biotechnology, Bioschool, Indian Institute of Technology Bombay
The success of Cibacron Blue 3G-A based matrices has made dye-ligand pseudo affinity chromatography an attractive proposition.  Specificity range of such matrices was expanded by generating a systematic library of ninety-six affinity resins using different dye structures and varying spacer lengths for immobilization.  This dye affinity matrix library displayed excellent discrimination for binding and elution with six model proteins including human serum albumin. Technology validation by twenty potential users shows interesting binding trends for hormones, antibodies and viral proteins.  In an automated 96-well multiple matrix testing format, a large number of chromatographic conditions such as pH, ionic strength and presence of ligands are easily and rapidly assessed.  Subsequently, promising dye-matrices could be selected for large-scale purification of desired therapeutic proteins.  Taken together, a novel proteomics and down-stream processing tool for isolating natural/expressed proteins/enzymes for diagnostics and therapy is described.

8:50 A Rapid Method for Determining Dynamic Binding Capacity of Resins
Laura Lerner, Ph.D., Director, Antibody Engineering, MacroGenics, Inc.
Bio-Layer Interferometry (BLI) offers a rapid, convenient method for determining the concentrations of macromolecules in solution.  We demonstrate the utility of BLI for determining the dynamic binding capacity of resins for the first capture step of a purification process, when the load is a crude mixture.

9:20 Quantitative Evaluation of His-Tag Purification and Immunoprecipitation of Recombinant Proteins
Heping Cao, Ph.D., Research Biologist, Human Nutrition Research Center, US Department of Agriculture
Histidine (His)-tag is widely used for affinity purification of recombinant proteins, but the yield and purity of expressed proteins are quite different.  Little information is available about quantitative evaluation of this procedure.  We evaluated His-tag procedure quantitatively and compared it with immunoprecipitation using radiolabeled proteins from transfected human cells. These proteins were expressed and localized in the cytosol of transfected cells by immunoblotting, immunocytochemistry and confocal microscopy.  The results showed that His-tag purification was more effective than immunoprecipitation for protein purification and suggest that bioengineering amino acid residues in proteins can increase the production of recombinant proteins.

9:50 Coffee Break in the Exhibit Hall

CASE STUDIES

10:45 Purification of Acetyl-CoA Carboxylases
Dong Cheng, Ph.D., Principal Scientist, Bristol-Myers Squibb Company
Acetyl-CoA Carboxylase 1 and 2 (ACC1 and ACC2) are critical for de novo fatty acid synthesis and for the regulation of beta-oxidation.  Emerging evidence indicates that one or both isozymes might be therapeutic targets for the treatment of obesity, type 2 diabetes and dyslipidemia.  One of the major obstacles in the field is the lack of a readily-available source of recombinant, human ACC enzymes to support systematic drug discovery efforts.  Here we describe an efficient and optimal immunoaffinity purification strategy for isolating recombinant mammalian ACCs with high yield and purity.  The resultant human ACC2, human ACC1 and rat ACC2 possess highly specific activities, are properly biotinylated, and exhibit kinetic parameters very similar to the native ACC enzymes.  We believe that the current study paves a road to a systematic approach for drug design revolving around the ACC inhibition mechanism.  The immunoaffinity protein purification strategy described in the current study can also be generally applied in any recombinant systems.

11:15 Two-Dimensional Chromatography and Affinity Chromatography Based on Boronate Stationary Phases
Xiao-Chuan Sean Liu, Ph.D., Associate Professor, Department of Chemistry, California State Polytechnic University, Pomona
A new type of two-dimensional (2-D) separation material was synthesized and studied. The material is suitable for two-dimensional chromatography utilizing both covalent and noncovalent interactions. The first dimension is boronate affinity chromatography, and the second dimension is reversed-phase chromatography (or vice versa). The polymeric media were prepared using p-vinylphenylboronic acid (VPBA) as the functional monomer.  Two crosslinkers were evaluated, namely, ethylene glycol dimethacrylate (EDMA) and divinylbenzene (DVB).  Several parameters were evaluated to define the optimum for polymer strength and column performance including crosslinker, porogen, initiator, and column-packing parameters. The polymer-based HPLC columns were successful in separating phenol, catechol, dimethylphthalate and hydroquinone under reversed phase conditions, thus can be used as a reversed-phase HPLC column. The columns were also successful in separating catechol and adenosine under boronate chromatography conditions, thus can be used as a boronate affinity column. Moreover, the two types of chromatography can be performed consecutively on the same column during one complete chromatographic run, making it a two-dimensional (2-D) chromatography. Under these 2-D conditions, catechol was separated from a mixture of phenol, catechol, dimethylphthalate and hydroquinone; adenosine ribonucleoside was separated from a mixture of adenosine ribonucleoside,  adenosine deoxyribonucleoside, and uridine deoxyribonucleoside. This type of single column 2-D HPLC eliminates the requirement of complex and expensive multidimensional HPLC instrument and provides increased peak capacity for separation.
An affinity matrix was also synthesized and studied using immobilized boronic acids as the affinity ligands. Hemoglobin A1c (GHb A1c) can be determined easily using this affinity matrix.  Using our method, we determined the commercial human whole blood we used contained 5.78% GHb A1c; while horse blood contained 2.56% GHb A1c. Both values are consistent with literature data. Recently, we also did the chromatography using "micro columns" with only 10 µL of the affinity matrix and 0.2 µL of blood sample. The test was done within 3 minutes. We obtained the same results as these of the 1 mL column. This indicates that we can quantify GHb A1c using as little as 0.2 µL of blood samples.  Matrix stability has been studied for 9 months so far. There is no change in the performance of the matrix after storage for 9 months at 4 ˚C.

11:45 Purification of a Monoclonal Antibody using Crystallization
James Wilkins, Ph.D., Senior Scientist, Bioprocess Development, Genentech, Inc.
Although scattered reports have appeared in the literature from time to time, crystallization of full length monoclonal antibodies has not been commonly observed. This report describes the crystallization of a purified monoclonal antibody with high efficiency using relatively simple buffer conditions. The goal of the project however was to determine whether purification of the same antibody could be achieved from “upstream” feedstocks. We therefore applied conditions similar to those that had been successfully employed in crystallization of the pure molecule to the purification of the target antibody from harvested cell culture fluid (HCCF) collected from a bioreactor. We found that not only could the antibody be successfully and efficiently crystallized form this complex milieu, but that antibody purity equivalent to or better than that obtained from two traditional chromatographic steps (including protein A) was achieved. Yields from the crystallization step were 90% or better. In summary, this represents the first report to our knowledge of the successful purification of a monoclonal antibody from complex mixtures such as HCCF using the simplicity, power and specificity of crystallization. The current results suggest that crystallization may represent a viable alternative for the purification of certain therapeutic monoclonal antibodies. Further work in our laboratory will focus on determining the molecular basis of these interactions.

12:15pm Close of Morning Session

12:30 Luncheon Workshop or Lunch on your Own

HIGH THROUGHPUT REVOLUTION

2:00 Chairperson’s Remarks

2:05 Parallel Preparative Protein Purification
Robin Clark, Ph.D., Director of Protein Research, deCODE biostructures
The presentation covers a protein production pipeline that reaches from gene design to production of mg-quantities of crystallization grade protein in sets of 24.  The core of this technology consists of a protein purification robot that runs 24 chromatographic purifications in parallel, in a single ca. 40 min run. Applications have been developed to facilitate comparative surface mutagenesis studies, Antibody purification, immune-depletion preparations and for supplying purified protein samples in quantities suitable for functional characterization, biophysical and immunological analysis, including protein crystallization.

2:35 Purification of Recombinant Proteins Derived from Shewanella oneidensis: Developing a Rapid, High-Throughput Strategy
Chiann-tso Lin, Ph.D., Staff Scientist, Fundamental Science, Pacific Northwest National Laboratory
Protein crystallography, generating antibodies, mapping protein interactions, and other functional genomic approaches require a large quantity of purified proteins. Here, we cloned a 6x histidine tag into the C-termnus of selected genes of Shewanella oneidensis MR1 and over-expressed in Escherichia coli.  For purification, we developed and compared two alternative approaches for automated purification of recombinant proteins based on expression of bacterial genes in E. coli. The first one is a filtration separation protocol in which proteins of interest are expressed in a large volume (800 ml) of E. coli cultures, following isolation by affinity filtration using NiNTA agarose (Qiagen). The second approach involved a smaller scale magnetic separation method. For these purposes, proteins of interest were simultaneously expressed in a small volume of E. coli cultures (25 ml) following isolation in a 96-deep well purification system with MagneHis Ni2+ agarose (Promega). Both approaches were performed fully or semi-automatically with the aid of a robot-driving platform. Both approaches provided comparable average yields of proteins: ~8 mg of purified protein per optical density unit of bacterial culture measured at 600 nm. We discuss advantages and limitations of these automated workflows, which can provide proteins with more than 90% purity and yields in the range of 100 µg to 45 mg per purification run, and discuss strategies for optimizing these protocols.

3:05 High-Throughput Process Development on Tecan Freedom EVO Robotic Workstations
Jürgen Hubbuch, Ph.D., Professor, Institut of Process Engineering in Life Sciences, University of Karlsruhe
The demand for biopharmaceutical products is increasing rapidly; however, development timelines of about 12 years leave little time for return on investment before a patent expires.  Fast process development is therefore crucial to speed up the market entrance of new biopharmaceuticals.  Here we present a comprehensive overview of the tools - with a focus on chromatographic process development - available on the Tecan Freedom EVO Robotic workstation.  The application range from solubility studies, aqueous two-phase systems...to batch chromatography systems and packed bed applications.  We further provide an outlook over strategies using mathematical tools for directed process development and statistical evaluation of the operational space.

3:50 Refreshment Break in the Exhibit Hall

4:30 BuzZ Sessions—Moderated Small-Group Breakout Discussions
Have a topic idea?  Please send to Mary Ann Brown at mabrown@healthtech.com
Click here for a listing of BuzZ Session topics

5:30 Close of Day

Overview | Short Courses | Day 1 | Day 2 | Day 3 (Joint Session) | Download Brochure

For more information, please contact:
Mary Ruberry, Conference Director
Phone: 781-972-5421 
E-mail: mruberry@healthtech.com 

For exhibit and sponsorship information, please contact:
Suzanne Carroll, Manager-Business Development
Phone: 781-972-5452 
E-mail: scarroll@healthtech.com

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