Navigation Links
Improve Recovery and Reproducibility with Pre-Treatment of Analytical TSK-GEL G3000SWXL Size Exclusion Columns

T.J. Higley, Tosoh Bioscience LLC and Richard Cornell, Wyeth BioPharma


Abstract:
During a recent method optimization and investigation at Wyeth BioPharma, pre-treatment with bovine serum albumin improved reproducibility and recovery during the analytical SEC analysis of fractions pulled from a multi-step orthogonal purification of a 140 kDa apparent molecular weight protein from cell culture feedstock.


Introduction:
Ideally in size exclusion chromatography the only mechanism that provides separation of sample components is a sieving process based on the hydrodynamic radius of the sample components in combination with a porous and non-interactive stationary phase. For monodispersed samples such as proteins, spherical 5 micron silica-based materials derivatized with proprietary hydrophilic diol-type coatings have been widely used since their introduction by Kato et al. in 1987. Although exhaustively derivatized, unreacted silanol groups and the hydrophilic coating may contribute to unwanted ionic or hydrophobic interactions between the bonded phase and sample components. One technique to reduce the impact of such secondary interactions is to inject bovine serum albumin to occupy accessible reactive sites on the stationary phase.

At Wyeth BioPharma, a SEC analysis is employed at every stage of a three step cell culture purification to determine the purity and recovery of the high molecular weight species of the target protein. The cell culture is purified using affinity chromatography (protein A) and subsequent chromatographic polishing steps. The recovery and reproducibility of the peaks eluting in the region of high molecular weight species during SEC analysis of the post-protein A fraction was highly variable, signaling possible adsorption of the sample to the packing materials. A pre-treatment experimental protocol with BSA was administered to reduce the variability.


Conditions of SEC analysis:
Column: TSKgel G3000SWXL, 30cm x 7.8mm, 5 micron

Elution: Phosphate Buffered Saline, pH 6.5

Sample: 50 mL injection volume, 50 mg load

Detection: 280 nm

Instrument: Waters 2690 Alliance


Results:
As can be seen in Figure 1 , recovery of the high molecular weight region, expressed as a percent of total area recovery (% HMW), of the protein A purified sample was 1.1%. Upon injecting 10 aliquots x 20 mL of 5 mg/mL bovine serum albumin (BSA) onto the column, the recovery of the %HMW species increased to 3.5%. In the absence of BSA treatment, protein A purified samples historically returned %HMW values of 1.0% to 1.4% when a column was new and gradually increased to 3.5% - 4.0% after as many as 60 injections. Interestingly, this result was only apparent with the protein A purified sample. Table 1 shows that BSA pre-treatment had no effect on the samples obtained from the downstream chromatography steps. Further characterization work indicated that elevated levels of host cell proteins (HCP) were present in the protein A purified samples with respect to the post polishing samples. The HCP were found to elute in the HMW region of the analytical SEC profile. Furthermore, when the protein A purified samples were analyzed by analytical SEC after BSA treatment, the HMW region was found to be enriched in host cell protein when compared to the HMW region of the same sample run on analytical SEC prior to BSA treatment. This suggests that the association between sample and column packing was most prevalent with the HCP as opposed to target molecule HMW species.




Conclusions:
Pre-treatment of SEC columns with BSA is a viable option to reduce secondary interactions and improve consistency of results between analyses. In this particular protocol, the dramatic improvement in the Improve Recovery and Reproducibility with Pre-Treatment of Analytical TSK-GEL G3000SWXL Size Exclusion Columns T.J. Higley, Tosoh Bioscience LLC and Richard Cornell, Wyeth BioPharma Figure 1. Overlay plot of the Protein A sample pre and post-treatment with BSA Sample % HMW pre treatment % HMW post treatment Post Protein A 1.1 3.4 Post Polishing chromatographic step1 1.5 1.5 Post Polishing chromatographic step 2 (final product) 0.2 0.2 Table 1: Recoveries of all three types of samples pre and post-treatment with BSA consistency of results associated with the protein A purified fraction, in contrast to the lack of effect seen on recovery of HMW species from downstream purification steps (chromatographic polishing steps 1 and 2), has provided the opportunity to further characterize the high molecular weight fractions of the protein A purified sample. This characterization has led to the conclusion that the results of SEC performed on samples from early purification steps may be negatively influenced by non-product related impurities derived from the cell culture environment.


References:
Y. Kato et al; J. of Chromatrogr. 404 (1987) 333-339


More Infomation
TSKgel G3000SWxl



'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. The Best Enzyme for the Toughest PCR Challenges Improved with New Hot-Start Feature
2. Novel PCR Enhancing Factor Improves Performance of Pfu DNA Polymerase
3. Improved Sensitivity for Staining RNA with SYBR Gold Stain
4. Improve Lipid- or Calcium Phosphate-Mediated Transfection of Human Dermal Fibroblasts
5. Improved Vectors for PathDetect Trans-Reporting Systems
6. Improve Amplification Specificity with Hot Start PCR Enzyme
7. Improved Quantitative Selectivity of Clenbuterol in Human Urine using High Resolution on the TSQ Quantum Mass Spectrometer
8. Gene Expression Arrays: Highly Sensitive Detection of Expression Patterns with Improved Tools for Target Amplification
9. Improve RNA Isolation for Array Analysis and More
10. Protect RNA and Improve Target Cell Identification
11. The Worlds Best DNase Improved TURBO DNA-free
Post Your Comments:
(Date:12/17/2014)... December 16, 2014 “Never say, ... Philipps- Universität Marburg’s Department of Physics, advises young ... the newly released SPIE Women in Optics ... too shy to ask questions.” , In the ... and mathematics) occupations ranging from university professor and ...
(Date:12/17/2014)... December 17, 2014 Xeno Diagnostics ... following its most recent COLA inspection. Xeno has ... a national healthcare accreditation organization. Accreditation is earned ... quality in day-to-day operations, demonstrate continued accuracy in ... rigorous on-site laboratory survey. , While ...
(Date:12/17/2014)... Md. , Dec. 17, 2014 CASI ... company dedicated to the acquisition, development and commercialization of ... for the global market with a primary commercial focus ... it has been granted a Type C meeting with ... the meeting will be held in February 2015.  During ...
(Date:12/15/2014)... DIEGO , Dec. 15, 2014 ... three-dimensional biology company focused on delivering breakthrough 3D ... the industry and collaborators of its exVive3D TM ... made widely available in full commercial release on ... the field, resulting in several awards for innovation. ...
Breaking Biology Technology:Network, Multitask, Persevere: SPIE Women in Optics Planner Subjects Share from Experience 2Network, Multitask, Persevere: SPIE Women in Optics Planner Subjects Share from Experience 3Xeno Diagnostics Receives 3rd Award for Laboratory Excellence 2CASI Pharmaceuticals Granted Type C Meeting With U.S. FDA For ENMD-2076 In Fibrolamellar Carcinoma 2CASI Pharmaceuticals Granted Type C Meeting With U.S. FDA For ENMD-2076 In Fibrolamellar Carcinoma 3CASI Pharmaceuticals Granted Type C Meeting With U.S. FDA For ENMD-2076 In Fibrolamellar Carcinoma 4CASI Pharmaceuticals Granted Type C Meeting With U.S. FDA For ENMD-2076 In Fibrolamellar Carcinoma 5Organovo Celebrates a Positive 2014 Reception of its exVive3DTM Human Liver Tissue Product 2Organovo Celebrates a Positive 2014 Reception of its exVive3DTM Human Liver Tissue Product 3
... Chilcott,Limited (Nasdaq: WCRX ) announced today that LEO ... from the U.S. Patent and,Trademark Office (the "USPTO"). In ... Patent No. 6,753,013 (the "013 Patent") in,response to a ... TACLONEX,and certain of LEO,s products in development. Warner Chilcott ...
... 17 Diageo (NYSE: DEO ),the world,s leading ... sponsorship of a physician,s training program in screening and ... State of,New York (MSSNY). The program, funded entirely ... better prepare physicians to address and assess issues,related to ...
... LMNX ) today announced that management will present ... held September,17-20, 2007, at the St. Regis Hotel in ... Balthrop, president and chief,executive officer, will begin at 11:00 ... 18, 2007. The presentation will be webcast live,and may ...
Cached Biology Technology:Warner Chilcott Announces New Information in USPTO Reexamination of Taclonex Patent 2Warner Chilcott Announces New Information in USPTO Reexamination of Taclonex Patent 3Diageo Sponsors Medical Society of the State of New York's Physicians Training Program 2Diageo Sponsors Medical Society of the State of New York's Physicians Training Program 3Luminex Corporation to Present at ThinkEquity Partners Investor Conference 2
(Date:12/10/2014)... Dec. 08, 2014 Research and Markets ... of the "Biometrics Market in Japan 2014-2018" ... http://photos.prnewswire.com/prnh/20130307/600769 The integration ... as rural banking and upgradation of the driver,s ... in the market. Besides the aforementioned projects, biometrics ...
(Date:12/4/2014)... , Dec. 3, 2014  Crossmatch™, a ... announced that its DigitalPersona ® Pro Enterprise ... have been deployed throughout First Bank branch locations ... South Carolina and Virginia. First Bank, ... Southern Pines, North Carolina , selected ...
(Date:11/21/2014)... -- Earlier this year Donald Spector , Chairman of ... the most prolific inventors in the world, got a ... to third party agencies. Spector envisioned this technology even ... earliest known patents in this area. Now major technology ... care, elder care and hospital applications. This technology has ...
Breaking Biology News(10 mins):Biometrics Market in Japan 2014-2018: Key Vendors are DDS, Fujitsu, Hitachi and NEC 2First Bank Partners with Crossmatch to Increase Log In Security 2First Bank Partners with Crossmatch to Increase Log In Security 3Patented Biosensor Wearable Technology Provides a More Civilized Method of Quarantine 2Patented Biosensor Wearable Technology Provides a More Civilized Method of Quarantine 3Patented Biosensor Wearable Technology Provides a More Civilized Method of Quarantine 4
... (BMC) and Boston University School of Public Health (BUSPH) ... at home are less likely to receive colorectal cancer ... English at home. The findings, which currently appear on-line ... patient-provider language barriers play a role in health-care disparities, ...
... to Secretary Kathleen Sebelius at the U.S. Department of ... called out 10 specific challenges to proposed modifications to ... on behalf of its membership of 4,000 informatics professionals, ... of Proposed Rulemaking (NPRM) on HIPAA modifications, along with ...
... frigatebird may be the least likely animal on the ... turns out the Galapagos population of this tropical seabird ... new conservation status, according to a paper by researchers ... Museum of Natural History and the University of Missouri-St. ...
Cached Biology News:Study finds language barriers may play role in health care disparities 2AMIA cites concerns about proposed HIPAA modifications 2AMIA cites concerns about proposed HIPAA modifications 3AMIA cites concerns about proposed HIPAA modifications 4Smithsonian researchers find differences between Galapagos and mainland frigatebirds 2
BD Falcon 850 cm Smooth-surface TufRol Roller Bottle, tissue-culture treated polystyrene, blow-molded one-piece design, with easy on/off non-vented cap. (2/sp, 20/ca) Packaging: 2 / pack, 20 /...
... Display Vector is specifically designed to target ... of Saccharomyces cerevisiae. Displayed proteins can be ... known or putative ligands. pYD1 uses the ... two domains, Aga1 and Aga2, to display ...
... are used for heterologous protein expression in the ... S2 cell line was derived from a primary ... Drosophila melanogaster embryos (1). This versatile ... CO2 and is easily adapted to suspension culture. ...
ACS Reagent Grade C 19 H 14 O 5 S Formula Weight: 354.38 PRODUCT SPECIFICATIONS Form: Rust/red crystalline powder Clarity of Solution: Passes test Visual Transition Interval: pH 6.8 - Yellow pH 8.2...
Biology Products: