Tosoh Bioscience LLC is hosting a series of educational seminars, featuring column selection guidance, tips and tricks to extend column lifetime, and a focus on the analysis of biomolecules and monoclonal antibodies.

Wednesday, May 21, 2014
8:15 am - 4:00 PM

Courtyard Marriott O’Hare
2950 South River Road
Des Plaines, IL 60018

There is no course fee for this one-day seminar. Lunch will be provided. Registration deadline is May 14. Check back as additional talks are added to the schedule.

Who Should Attend:
This course is designed to benefit a range of HPLC users, from beginners to more advanced chromatographers.

What Will Be Covered:

"Importance of Column Selection in HPLC” - Atis Chakrabarti, Ph.D., Manager, Technical Service, Tosoh Bioscience

With the basic knowledge of HPLC and theory, selection of the right column is the next most important thing. This is like solving a puzzle considering the many different selection criteria to consider; some of which are straight forward, some not but complicated and interdependent. This requires critical identification of stationary phase based on the characteristics of the sample, complexity of the mixture, the difference in chemistry among the components of the mixture and goal of the separation. Selecting a right column is a science but the artistic skills a chromatographer gains through hands-on experience is important too. With the advancement of technology more choices of columns and packing materials are available for selection which is obviously appropriate and useful for ever expanding range of individual needs. This presentation is intended to assist the attendees in column selection and method development with a focus on biomolecules.
"Separation of Biomolecules using Different Modes of Analytical Chromatography Columns" - Justin Steve, Technical Service Specialist, Tosoh Bioscience

Antibodies and recombinant proteins are now widely used for the therapeutic treatment. The evaluation of the heterogeneity of the therapeutic antibody is essential for the development, stability study and quality control of the final product. Protein heterogeneity is generated by posit-translational modifications, decomposition or chemical modification. These increase the risk of anaphylaxis or immunoreaction if not separated. In this presentation we have shown the use of high resolution nonporous resin columns for Ion Exchange Chromatography (IEX) and Hydrophobic Interaction Chromatography (HIC) to detect even 1 residue difference of the proteins. Size exclusion chromatography (SEC) is suitably used to separate the monomer (150 kDa) and the dimer (300kDa) of a human monoclonal antibody to very good baseline resolution. Papain and pepsin digested antibodies could be resolved to separate Fc, Fab and intact protein to the baseline resolution by size exclusion chromatography. Analysis of Aggregates, fragments and PEGylated proteins and the analysis of hapten conjugated protein using SEC are also reported in this presentation. This presentation is focused on the overview of the columns available for the detection and separation of protein heterogeneity by HPLC using four different modes of chromatography such as SEC, IEX, RPC and HIC.
“Separation of Monoclonal Antibody Monomer from High Molecular Weight (HMW), Low Molecular Weight (LMW) and Other Heterogenic Impurities using Different Modes of Chromatography” - Atis Chakrabarti, Ph.D.

Protein aggregation is a biological phenomenon. There are many reasons why the aggregates or the high molecular weight (HMW) species form. Mostly the mis-folded proteins have a tendency to form aggregate. Both intra- or extra-cellular protein aggregates are found. These protein aggregates are often toxic; protein aggregates have been implicated in a wide variety of disease known as amyloidosis, including Alzheimer’s, Parkinson’s and prion disease.

Nowadays, monoclonal antibody proteins are widely being used in the field of bio therapeutics. These bio therapeutic proteins must be free from aggregates. Separation of the pure antibody monomer needs to be very well resolved from its dimer and higher order aggregates. Similarly for quality control and regulatory purpose the separation of antibody fragments or low molecular weight (LMW) species is also very much essential.

The species other than the monomer might induce toxic side effects to the body if not removed. Even if the impurities are non-toxic, the mere presence of these species may reduce the potency of the final formulation and hence need to be removed.

Many chromatographic modes can be coupled to separate complex mixtures, variants or impurities otherwise inseparable by a single mode of chromatography. A very favorable combination is Size Exclusion Chromatography (SEC) and Reversed Phased Chromatography (RPC). SEC and RPC are some of the most frequently used chromatographic modes for analytical separations of biomolecules, particularly monoclonal antibodies. The separation mechanism is orthogonal and the mobile phases are compatible.

Analytical SEC columns particularly one packed with 30 nm wider pore size, 3 μm particles with larger molecular exclusion limit can be used to separate as well as quantitate the aggregates or HMW species as a function of their hydrodynamic volume. A well resolved symmetric and apparently pure protein peak obtained from size exclusion chromatography need to be checked by another mode for the presence of any variants. A reversed phase chromatography using analytical column with wide pore size of 30 nm for maximum mass transfer is expected to be useful for the separation of large biomolecules such as proteins as well as their hydrophobic variants.

Here we discuss the use of analytical columns from two different modes of chromatography (SEC and RPC) in separation of monomer of proteins and monoclonal antibodies from their HMW and LMW impurities and variants.
"Separation of Antibody, F(ab’)2 and Fab’ Species Using Size Exclusion Chromatography" - Larissa Harwick, Senior Scientist, Abbott Laboratories

There is a constant need for increased sensitivity of antibody-based assays which in turn increases the need for high affinity molecules. Use of Fab’ antibody fragments can increase both the specificity and sensitivity of immunoassays. We have developed a simple SEC method that can be used to characterize Fab’ material produced following reduction of F(ab’)2 antibody fragments.

Reduction of F(ab’)2 is accomplished using cysteamine HCl. The resulting Fab’ fragments were alkylated with or without iodoacetamide prior to analysis. Fab’ fragments were analyzed by gel filtration chromatography using a Waters HPLC system. Tosoh TSKgel G2000SWxl and TSKgel SuperSW2000 columns were utilized to obtain chromatographic patterns and purity determinations.

The SEC method developed provides reproducible profiles for purity determinations and is capable of detecting low molecular weight impurities. Use of this method provides a quick analytical protocol that can be used to characterize reduced F(ab’)2 material prior to conjugation and use in immunoassays.

"Separation of Half-mAb and Half-mAb Equivalents with High Resolution Using Size Exclusion Chromatography Packed with a Unique Controlled Pore Technology" - Justin Steve

Monoclonal antibody research continues to grow in an effort to develop effective biotherapeutics for a wide range of diseases. Recent research has shown an interest into mAb half-bodies as therapeutic vectors as they can be further targeted for conjugation, enzyme labeling, or antibody immobilization. Monoclonal antibody half-bodies can be generated through genetic engineering of cells or by selective reduction of hinge-region disulfide bonds present in the monoclonal antibody by mild reducing agents such as 2-MEA, DTT or TCEP. TCEP (tris(2-carboxyethyl) phosphine) is a mild reducing agent commonly used in the reduction of hinge region disulfide bonds. Unlike 2-MEA or DTT, TCEP is odorless and more resistant to oxidation in the presence of air making it a more stable reducing agent and a commonly used reagent in mAb half-body formation. As expected, mAb half-bodies have a molecular weight of approximately 70 kDa, or one half of that of the intact monoclonal antibody. Bovine Transferrin, with a molecular weight of 78 kDa is expected to simulate the half-mAb species when chromatographically separated using SEC and can be used as a molecular weight marker. This presentation will show the use of new TSKgel SW mAb columns.

"Troubleshooting, Column Lifetime Tips and Tricks, Questions" - Atis Chakrabarti

HPLC system and analytical chromatography columns are costly. Method development is time consuming and costly too. It is important to employ an HPLC system that is optimized with regards to extra-column band broadening to take full advantage of the high column efficiency that can be obtained on analytical columns. This presentation is about common but often ignored method development and troubleshooting issues, discussion of column lifetime issues encountered in different modes of analytical chromatography in general. At the end of this presentation, the floor will be opened for open discussion among the attendee column chromatographers.

Space is limited, so reserve your seat today!

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