Waters Application Note
For the analysis of biotherapeutics, size-exclusion (SEC) and ion-exchange (IEX) chromatography are typically conducted under native separation conditions, requiring high ionic strength, 100% aqueous eluents. For high performance liquid chromatography systems, these conditions can be problematic: in the absence of bactericides, lack of proper maintenance can lead to bacterial contamination within hours. The presence of high salt concentrations increases the potential of particulates in the mobile phases. However, with the proper setup and care of a chromatographic system, robust and reproducible chromatography can be achieved with minimal downtime.1,2
The care and use of a size-exclusion and/or ion-exchange chromatographic system requires many of the same standard practices as any other system. However, there are some additional protocols that are required for high salt, aqueous mobile phases. While the practices outlined in this document are described for ACQUITY UPLC Systems, the principles apply to any chromatographic system. Overall system recommendations include:
Additional recommendations are listed below by component. These considerations are for microbial growth, system suitability and/or protein stability.
The buffers used in SEC and IEX can favour microbial growth leading to contamination of the column and system. Other recommendations include:
SEC and IEX conditions often require high-ionic-strength solutions in the sample manager wash lines (wash/purge, strong and weak needle). However, these eluents may have a detrimental effect on the sample syringe and/or needle. Recommendations include:
Waters recommends titanium or stainless steel optical flow cells when performing SEC or IEX under aqueous conditions. The standard ACQUITY optical flow cell contains Teflon AF in the fluidic path. Under native conditions, some proteins may interact with the flow cell surface, resulting in peak tailing and sloped baseline. Recommendations for detectors include:
To maintain long column lifetimes and minimize the risk of microbial contamination, the following recommendations should be followed:
SEC and IEX chromatography are performed under native conditions, requiring high-ionic strength, 100% aqueous eluents. To minimize protein-surface interactions, these conditions may require the use of a bio-compatible chromatographic system specifically designed for these applications. Precautions must be taken to prevent and minimize bacterial contamination. Signs of such contamination3 , which can occur within hours include: deteriorating peak shape, resolution and column lifetime. Unfortunately, once the column has been contaminated, regeneration is difficult. To decrease the frequency of system repairs and contamination, a series of steps have been outlined for maintenance and care of a chromatographic system and columns used for the analysis of biomolecules. These recommendations include maintenance for the solvent manager, sample manager, detector and column. Using these procedures in combination with good laboratory practices ensures a robust, reproducible system for ultra-performance-size-exclusion and ion-exchange chromatography.
Figure 1
1. "Size-Exclusion and Ion-Exchange Chromatography of Proteins using the ACQUITY UPLC System", Waters User Manual, Rev A, Part Number 715002147 (2010).
2. "Size-Exclusion and Ion-Exchange Chromatography of Proteins using the ACQUITY UPLC H-Class System", Waters User Manual, Rev A, Part Number 715002909 (2010).
3. "Controlling Contamination in LC/MS and HPLC/MS Systems", Waters User Manual, Part Number 715001307.
2012 Waters, Waters, ACQUITY UPLC, ACQUITY, UPLC and The Science of What's Possible are registered trademarks of Waters Corporation. All other trademarks are the property of their respective owners
Waters Corporation
34 Maple Street, Milford, Massachusetts, USA
tel: +1 (508) 478-2000 fax: +1 (508) 478-1990
Website: http://www.waters.com
AOAC International Awarded NIST Grant for Developing Drug Testing Standards
October 31st 2024The grant will be part of a new collaborative scientific initiative to address the need for standards that define the desired performance of lateral flow immunoassay test strips to detect illicit drugs in tablets and powders.
Supercritical Fluid Chromatography for Separating Chemotherapy Anti-Nausea Medication
October 30th 2024Scientists from Peking Union Medical College & Chinese Academy of Medical Sciences created a new method for separating palonosetron hydrochloride, which is frequently used to prevent acute or delated nausea and vomiting caused by chemotherapy.