This is an Application Brief and does not contain a detailed Experimental section.
This application brief outlines good practices for the routine maintenance and use of UltraPerformance LC Systems in conjunction with high ionic strength, 100% aqueous mobile phases, typical eluents used in size-exclusion and ion-exchange chromatography.
Given the complexity of biotherapeutics, full characterization typically requires a variety of orthogonal methods. While many chromatographic techniques are conducted under reversed-phase conditions, others are 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. The presence of bacteria and particulates in the LC system can affect chromatography quality and column lifetime.
The components of the chromatographic system are equally important. If the chromatographic system is not inert or bio-compatible, metal-protein adducts or undesired protein interactions can occur. Long-term use of high ionic strength, 100% aqueous mobile phases can also lead to rust formation if the chromatographic system contains steel components in the wetted path. However, with proper set-up and care of a chromatographic system, robust and reproducible chromatography can be achieved with minimal down time.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 favor microbial growth leading to contamination of the column and system (Figure 1). Recommendations include:
For short-term storage, maintain 0.1 mL/min of drawing an equal percentage of mobile phase from all lines in use.
If system will be idle for more than 2 days, prime each line for 10 minutes with high purity water. Thoroughly flush system. Repeat steps with 90/10 water/methanol.
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 affect on the sample syringe and/or needle. For variable flow through needle systems, remove salt deposit on a regular basis to minimize maintenance and repairs. Other 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 (Figure 2). 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, 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 , Rev A, Part Number 715002147.
2. “Size-Exclusion and Ion-Exchange Chromatography of Proteins using the ACQUITY UPLC H-Class System”, Waters User Manual , Rev A, Part Number 715002909.
3. “Controlling Contamination in LC/MS and HPLC/MS Systems”, Waters User Manual, Part Number 715001307.
4. “ACQUITY UPLC H Class Sample Manager Flow-Through Needle Operator’s Overview and Maintenance Information”, Waters User Manual , Rev B, Support Number USRM10144216.
5. Waters Corporation (Producer) (2011). Auto•Blend Plus Technology Tutorial [Video]. Retrieved from http://www.waters.com/waters/nav. htm?cid=134623262
6. Hong P., Fountain K.J., Wheat T. E., Morrison D. “IEX Method Development of a Monoclonal Antibody and Its Charge Variants”, Waters Application Note , Part Number 720003836EN.
720004182, January 2012