Navigation Links
Mobile Phase Optimization for the Analysis of an Antibody-Based Fusion Protein and Aggregates using a TSKgel Super SW3000 Size-Exclusion Column

T.J. Higley and Roy Eksteen, Tosoh Bioscience LLC, Montgomeryville, Pennsylvania and Scott Lauder, Lexigen Pharmaceuticals, Lexington, Massachusetts


Introduction:
During method development, many variables are examined to ensure method robustness. Factors such as elution profile, peak shape, and recovery are required to be consistent by GMP/GLP protocols. During a recent method re-qualification at Lexigen Pharmaceuticals, several variables were investigated to eliminate non-specific binding and increase the robustness of an established antibody separation method. Previous protocol required an unacceptable two-week "break-in" period to equilibrate the column with the mobile phase. Reduction of non-specific binding of proteins to SEC stationary phases can be achieved by exploring variables that affect either the ionic strength or hydrophobicity of the mobile phase. Typically, changes in mobile phase composition, such as buffer type and pH, salt concentration, the addition of organic modifiers, as well as the evaluation of competitive columns are the usual strategies to correcting non-specific adsorption. In this case, these options did not result in an improvement. Thus, an alternative approach to increase the chaotropic nature of the mobile phase was investigated. Concurrently, the buffer concentration was reduced to a minimum buffering capacity and the modifying salt was changed to NaClO4. A drastic improvement on the performance of the method was evident.


Experimental Conditions:
Gel filtration was performed with a 4.6mmID x 30cm TSKgel Super SW3000 column packed with 4m particles containing 250 pores. The original run ning buffer consisted of 0.1M NaH2PO4 (pH 6.0) to which was added 15% acetonitrile and 0.2M NaCl. In the redesigned buffer system, NaCl was replaced by NaClO4 and the phosphate buffer concentration was reduced to 0.05M. Initially, the concentration of sodium perchlorate investigated was 0.2M, however, best results were obtained at 0.4M. The flow rate of 0.35mL/min was left unchanged. Note that recent work has shown that acetonitrile can be omitted from the mobile phase without affecting separation quality or method robustness.


Results:
As shown in Figure 1 , excessive peak tailing of "fusion protein 1" is evident with the use of 0.2M NaCl. Additionally, the expected protein dimer and trimer aggregates are not visible in the chromatogram. By switching from 0.2M sodium chloride to 0.2M of the more chaotropic sodium perchlorate salt, together with a two-fold reduction in the buffer concentration, less peak tailing and distinct peaks for the dimer and trimer species of MAb1 resulted. Doubling the perchlorate concentration to 0.4M provided further improvement in the peak shape of fusion protein 1 and associated aggregate species. Additionally, the re-development increased the methods' robustness allowing for a substantial decrease in column "break-in" time from two weeks to 4 days as well as better peak area reproducibility and linearity of both monomer and aggregate species.


As a precaution, a more rigorous cleaning protocol consisting of flushing the system with DI water was implemented during periods of instrument inactivity. Although perchlorate is slightly less corrosive to stainless steel components relative to chloride, it is considered more aggressive to chemically bonded stationary phases.


Conclusions:
The selection of sodium perchlorate as a mobile phase component substantially improved the analysis and quantification of an experimental monoclonal antibody and its aggregates by SEC, thus providing the required level of method robustness.


More Infomation
TSKgel SuperSW3000



'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. Protocol for RNA Isolation Using TRIzol Reagent with Phase Lock Gel-Heavy
2. Protocol for RNA Isolation Using TRIzol Reagent with Phase Lock Gel-Heavy
3. Easy preparation of total RNA from cultured cells with TRIzol and Eppendorf Phase Lock Gel
4. Direct Monitoring of Solid-Phase Peptide Synthesis by MALDI-TOF MS
5. Separation and Quantitation of R and S Enantiomers using Normal Phase Chromatography on an API 3000 LC/MS/MS System
6. Identification of Phase I and Phase II Metabolites of Buspirone on the Q TRAP LC/MS/MS System
7. Strategies to Identify and Confirm Phase I Metabolites of Glyburide Using the 4000 Q TRAP System
8. Novel Bonding Chemistry Imparts Enhanced Polar Selectivity to TSK-GEL ODS-100V Reversed Phase Columns
9. Two-dimensional Optimization of a Semi-nested PCR for Detecting Listeria monocytogenes
10. PCR Optimization
11. PCR Optimization Kit
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:1/19/2017)... ... January 18, 2017 , ... The American Medical ... update its Data Sharing Policy. Specifically, the nation’s leading informatics experts, said data ... the existing policy. AMIA recommended that NIH earmark funding for researchers to produce ...
(Date:1/19/2017)... Ca (PRWEB) , ... January 18, 2017 , ... ... events for tech innovators, engineers, and scientists from around the world, was today ... News. The awards program is based entirely on merit and decided upon by ...
(Date:1/19/2017)... ... January 19, 2017 , ... FireflySci Inc. is a ... The tremendous growth is accounted to two main factors. The first is ... expanding network of vendors supplying FireflySci products all around the world. , 2016 was ...
(Date:1/19/2017)... ... January 19, 2017 , ... DaVita Clinical Research ... drug and device development, and Prism Clinical Research , a leader in ... Verified Clinical Trials (VCT) has been selected by both companies as ...
Breaking Biology Technology:
(Date:1/13/2017)... 2017 Sandata Technologies, LLC, a leading ... industry, including Electronic Visit Verification™ (EVV™), announced today ... as Senior Vice President of Product Management. ... homecare experience to Sandata, where he will be ... align Sandata,s suite of solutions with the needs ...
(Date:1/11/2017)... 2017 Intoxalock, a leading ignition interlock provider, ... its patent-pending calibration device. With this new technology, Intoxalock ... upload data logs and process repairs at service center ... "Fighting drunk driving through the application of cutting-edge technologies ... but also for the customer who can get back ...
(Date:1/6/2017)... 2017  Delta ID Inc., a leader in consumer-grade ... automotive at CES® 2017. Delta ID has collaborated with ... the use of iris scanning as a secure, reliable ... in a car, and as a way to elevate ... Delta ID and Gentex will demonstrate (booth #7326 ...
Breaking Biology News(10 mins):