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1. Information Waters User Manual 2010 Rev B Support Number USRM10144216 5 Waters Corporation Producer 2011 AutoeBlend Plus Technology Tutorial Video Retrieved from http www waters com waters nav htm cid 134623262 6 HongP Fountain K J Wheat T E Morrison D IEX Method Development of a Monoclonal Antibody and Its Charge Variants Waters Application Note 2011 Part Number 720003836EN Waters THE SCIENCE OF WHAT S POSSIBLE UALITY Q MANAGEMENT Waters UltraPerformance LC ACQUITY UPLC UPLC and ACQUITY are registered trademarks of Waters Corporation The Science of What s Possible is a trademark of Waters Corporation All other trademarks are the property of their respective owners 2012 Waters Corporation Printed Produced in the U S A January 2012 720004182EN IH PDF Waters Corporation 34 Maple Street Milford MA 01757 U S A T 1 508 478 2000 F 1 508 872 1990 www waters com
2. OGY BRIEF Guidelines for Routine Use and Maintenance of Ultra Performance Size Exclusion and lon Exchange Chromatography Systems GOAL To outline 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 BACKGROUND 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 comp
3. all system recommendations include m lf using asteel system modify according to manufacturer s recommendations For use with a Waters UPLC System detailed guidelines are available m Clean laboratory glassware properly m If chronic loss of prime check valve problems reproducibility of retention time pressure or quantitative reproducibility are observed clean the system following a standard protocol m If possible use mobile phases containing a bacteriostat i e 0 02 sodium azide to prevent microbial growth m Retention time or pressure fluctuation can be indicative of problems with the pump m Area or peak variability without retention time variation can be indicative of an injector problem 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 Figure 1 Recommendations include m Always filter aqueous mobile phase through compatible 0 22 um or smaller membrane filters The use of sterile filters and containers is also recommended m Use only high purity water 18 2 MQ cm Bottled water should be opened the day of use m Never top off mobile phase bottles Always change bottles when replacing mobile phase m High ionic strength eluents gt 100 mM should be replaced every tw
4. ize 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 References I Size Exclusion and lon Exchange Chromatography of Proteins using the ACQUITY UPLC System Waters User Manual 2010 Rev A Part Number 715002147 2 Size Exclusion and lon 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 4 ACQUITY UPLC H Class Sample Manager Flow Through Needle Operator s Overview and Maintenance
5. ly flush system Repeat steps with 90 10 water methanol Sample Manager 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 m Ensure the sample is soluble in the mobile phase and sample manager washes m Follow the manufacturer s recommendations for wash solvents For example washes containing less than 500 mM salt s are recommended for the ACQUITY UPLC H Class Bio System m lf visible salt deposits appear clean the surfaces If salt deposits reappear check connections and system for problems m f the sample manager is idle for more than 2 days purge needle and or wash lines with high purity water minimum of 20 cycles or 200 seconds Repeat steps with 90 10 water methanol The sample manager should never be left idle for longer than 2 days in lines containing high salt washes gt 100 mM TECHNOLOGY BRIEF UV Detectors 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 su
6. o weeks m Low ionic strength eluents lt 100 mM should be replaced every 2 3 days m Water 100 bottles should be replaced daily m Higher salt concentrations which inhibit microbial growth would reduce the frequency of solvent replacement While it is not always practical to change mobile phases a quaternary solvent mixing system can accomplish the same effect by combining high concentration buffers gt 100 mM to produce SEC and IEX mobile phases m Alleluent bottles should be visually inspected daily for microbial growth and or particulates Microbial growth can be a film on the bottle surface or may be observed by swirling the bottle m f microbial growth has occurred in the eluent bottle replace the mobile phase filter or flush it with a 0 isopropanol solution Microbes can contaminate mobile phase filters m Use compatible mobile phase filters such as titanium Clean mobile phase filters weekly to reduce microbial contamination Sonicate or flush with 70 isopropanol solution m Recommended seal wash cycle time of 0 10 minutes 6 seconds m Seal wash recommendation of 90 10 water methanol m he solvent manager should never be left idle in either high salt mobile phases or 100 water 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 Thorough
7. onents 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 Proper Set Up and Maintenance of an ACQUITY UPLC System Allows For Robust and Reliable SEC and IEX Separations Mobile Phase 25 mM sodium phosphate 150 mM sodium chloride pH 6 8 Flow Rate 0 4 mL min Injection Volume 4uL Wavelength 280 nm Time 1 hour Figure 1 Effect of microbial growth on a SEC chromatogram of protein mix Contamination confirmed by analytical analysis of column frit Mobile Phase 25 mM sodium phosphate 150 mM sodium chloride pH 6 8 Flow Rate 0 4 mL min Titanium Injection Volume 4uL 5 mm flow cell 1 hour Wavelength 280 nm Analytical Teflon AF 10 mm flow cell Figure 2 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 Waters THE SCIENCE OF WHAT S POSSIBLE TECHNOLOGY BRIEF THE SOLUTION 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 Over
8. rface resulting in peak tailing and sloped baseline Figure 2 Recommendations for detectors include m Use titanium or stainless steel flow cells to reduce protein surface interactions Other flow cell material i e Teflon may cause peak tailing m Never leave the detector idle in high salt eluents Flush thoroughly with water 60 minutes at 0 2 mL min followed by 90 10 water methanol or higher organic eluent Column Storage To maintain long column lifetimes and minimize the risk of microbial contamination the following recommendations should be followed m Columns should never be stored in high salt aqueous mobile phase or 100 water m Before switching to recommended storage conditions flush columns with 10 20 column volumes of water m Flush and store columns following the manufacturer s recommendations Typical recommendations are 10 20 methanol or with a bactericide i e 0 1 sodium azide m Consider the use of guard columns to extend column lifetimes Regular replacement may be required Frequency of guard column replacement may be dependent on sample cleanliness m Size exclusion columns can typically be stored at 4 8 C to reduce microbial growth lon exchange columns are usually stored at room temperature Check the manufacturer s recommendations for details SUMMARY SEC and IEX chromatography are performed under native conditions requiring high ionic strength 100 aqueous eluents To minim

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