The Application Notebook
For the analysis of biotherapeutics, size-exclusion and ion-exchange chromatography are typically conducted under native separation conditions, requiring high ionic strength, 100% aqueous eluents.
For the analysis of biotherapeutics, size-exclusion and ion-exchange 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 proper set-up and care of a chromatographic system, robust and reproducible chromatography can be achieved with minimal down time (1,2).
Figure 1: SEC-PDA chromatogram of bovine serum album (BSA) (5 mg/mL in water) shows the effect of flow cell material on peak shape. BSA monomer exhibits extensive peak tailing.
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.
Solvent Delivery System:
The buffers used in SEC and IEX can favor 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. Some proteins, under native conditions, 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 contamination (3), 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.
(1) "Size-Exclusion and Ion-Exchange Chromatography of Proteins using the ACQUITY UPLC System", Waters User Manual (2010), Rev A, Part Number 715002147.
(2) "Size-Exclusion and Ion-Exchange Chromatography of Proteins using the ACQUITY UPLC H-Class System", Waters User Manual (2010), Rev A, Part Number 715002909.
(3) "Controlling Contamination in LC/MS and HPLC/MS Systems", Waters User Manual, Part Number 715001307.
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