Maximizing ACQUITY UPLC Protein BEH SEC Column Life

With proper care in preparing and handling the SEC mobile phase and samples, ACQUITY UPLC Protein BEH SEC Columns can deliver stable performance for >1000 sample injections (Figure 9). However, the performance and lifetime of an ACQUITY UPLC BEH SEC Column will deteriorate if the samples and/or mobile phase introduced into the column contain particulates.

Figure 9. One hundred overlays from 1000 repetitive injections of commercial Vectibix mAb formulation on a Waters ACQUITY UPLC Protein BEH SEC, 200Å, 1.7 µm, 4.6 x 150 mm Column.

To maximize column lifetime, high quality filtered water (e.g., Milli-Q Millipak 0.22 µm filtered water) should be used, and mobile-phase buffers should be passed through a <0.2 µm filter. It is recommended that mobile-phase buffers be filtered using a sterile disposable filter. It is further recommended that sintered glass filter supports be avoided when filtering mobile phases due to the potential of introducing silicates which could alter column performance. Use of sterile 0.2-µm-filter units is recommended. 

a. Mobile Phase Preparation and Use

Mobile phases with pH values in the physiological range that do not contain organic solvent can support microbial growth. Implementation of the following recommendations will help minimize microbial contamination, thus avoiding premature column failure.

• Clean laboratory glassware properly and dry at >170 °C for at least 1 hour to avoid potential microbial growth in remaining water droplets. The use of sterile containers is advised.
• Use only high purity water (18.2 MΩ cm) when preparing SEC mobile phases. If bottled water is used, it should be opened the day of use. 
• Always filter prepared mobile phase through compatible 0.22 µm or smaller membrane filters. The use of sterile filters and containers is also recommended. 
• Never "top-off" mobile-phase bottles. Always change bottles when replacing the mobile phase.
• Replace low-ionic-strength mobile phases (<150 mM) every 2-3 days.
• High-ionic-strength mobile phases (>150 mM) should be replaced every two weeks. This longer interval is acceptable because high salt concentrations will inhibit microbial growth.
• All mobile-phase bottles should be visually inspected daily for microbial growth and/or particulates. Microbial growth may form a film on the bottle surface and may be observed by swirling the bottle. 
• It is highly recommended to remove and not use the Waters ACQUITY UPLC Mobile-Phase Line Sinkers when performing SEC with 100% aqueous mobile phases since they may become contaminated with microbes, thus contaminating freshly prepared solutions of filtered mobile phase. 

Note: Waters Corporation will honor an existing service contract at sites that have removed the UPLC Mobile-Phase Line Sinkers provided that the account's SOP states that all SEC mobile phases must be filterd and contained in sterile glassware.

• If microbial growth is observed, immediately discontinue SEC analysis and perform the LC cleaning procedure detailed later in this document.

Additional Considerations:

• To prevent potential precipitation of the buffer from the mobile phase in the LC system, good laboratory practice suggests maintaining a low flow rate (e.g., 0.1 mL/min) through the system after the SEC column has been removed and stored as recommended.
• If the UPLC System will be idle for more than 2 days, and after the SEC column has been removed and stored as recommended, prime each mobile-phase line for 15 minutes at 1 mL/min with high purity water followed by flushing and storage in 70/30 isopropanol (IPA)/water to prevent microbial growth.

b. Avoiding Microbial Contamination of the Solvent Delivery System

Microbial contamination of the UPLC System may cause BEH SEC column fouling, resulting in an unacceptable separation (Figure 10). The column pressure may also increase significantly. Note that injection of particulates (e.g., microbes) will result in premature failure of any HPLC or UPLC Column. However, failure may occur sooner on columns containing <3 µm particles. If the mobile-phase reservoir becomes contaminated with microbes, the microbes will enter the solvent delivery flow path, and may contaminate fresh mobile phase. The mobile-phase line sinkers are one location that commonly harbors microbes.  

Figure 10.  Effect of microbial growth on an ACQUITY UPLC Protein SEC separation of the BEH200 SEC Protein Standard Mix (p/n: 186006519). Contamination was confirmed by analysis of the column inlet frit (Figures 11 and 12).

For sample and separation conditions, reference "Waters ACQUITY UPLC Protein BEH SEC Columns and Standards Care and Use Manual".

Figure 11. Scanning electron micrograph of the inlet frit removed from an ACQUITY UPLC Protein BEH SEC, 200Å, 1.7 µm Column contaminated with bacteria.

Figure 12. Mobile-phase line sinker (figure a) from a microbe-contaminated SEC mobile-phase bottle. The sinker was aseptically removed, suspended in sterile PBS, then 500 µL was applied to a microbe supporting agar plate. Figure b confirms the presence of microbes contained in the removed SEC mobile-phase line sinker.

c. Cleaning ACQUITY UPLC Systems

To clean an ACQUITY UPLC System used in 100% aqueous SEC methods, the use of 100% IPA, as used in the standard UPLC cleaning protocols, should be replaced by the use of 70% isopropanol (IPA) or 70% ethyl alcohol (EtOH) due to their ability to kill bacteria. One hundred percent concentration of either IPA or EtOH does not penetrate the cell wall of bacteria and is therefore not an effective bactericide nor are methanol-containing solutions (Reference 3). Waters has experienced situations where "microbe-contaminated mobile-phase filters" (a.k.a. sinkers) have resulted in rapid particulate fouling of a new ACQUITY UPLC Protein BEH SEC Column even when freshly prepared and filtered mobile phase is used. This is why Waters recommends that mobile phase sinkers NOT be used for this SEC application as previously detailed.

There are three different protocols for cleaning/purging ACQUITY UPLC Systems that can be used under different circumstances.

1. For a system that is in continuous use with sterile filetered mobile phase:  The best practice for the daily use of buffers starts with fresh mobile phase in a new bottle (no topping off). After installing fresh mobile phases, wet prime (a function of Empower Software) the buffer lines for 4 minutes if the system was used the day before. If the system was not used the day before, wet prime all lines for 6-10 minutes. This may be accomplished by using the system startup function and changing the prime time as needed. This function also primes the seal wash, purge, and needle wash solvent lines. Keep in mind that when the ACQUITY UPLC System is wet primed, under the control of the ACQUITY UPLC console, the column can remain on the system connected to the pump and detectors. The Empower solvent priming function diverts the priming solvents directly to waste. This makes it very easy to purge your lines with mobile phase or the other solvent sequences described below. It does require the movement of your solvent lines, but if you catch a bubble during the process, the wet priming command will eliminate it.
2. For a system that has been left unused for more than 2-3 days:  If your system is left in buffer with or without flow for more than 2-5 days, the lifetime of your column may benefit by wet priming all buffer lines with water for 4 minutes, then with 70% IPA for 6-10 minutes before going back to water for 4 minutes and then your buffer for 4 minutes before starting another analysis. It is further recommended to leave your system in 70% IPA (after priming the buffer lines with water) when not in use. When restarting, wet prime your system with 70% IPA for 3-4 minutes then water for 4 minutes before wet priming your system with your desired buffer.
3. For routine periodic cleaning for preventive maintenance when the system is contaminated: Table 2 is a summary of the cleaning protocol and reagents required to execute this procedure, which can be used at regular intervals (every 3-6 months) for preventive maintenance.
   1. Disconnect the column and attach a V-Detail Zero Volume Union (p/n: 700002636) to the column inlet and outlet lines. 
   2. For each step of the cleaning protocol, monitor the system pressure to keep it <1000 psi to prevent damage to the detector flow cell. If necessary, replace the backpressure device with large bore waste tubing. 
   3. If the system has recently been used with mobile-phase buffers or salts, it should be flushed thoroughly with 100% high-purity water prior to the introduction of organic solvents. 
   4. Place A, B, C, D, seal wash, sample wash, and purge lines into a clean bottle containing 70% isopropanol as the cleaning solvent.
      Note: If there is an incompatibility between the mobile phase or wash solvents with 70% isopropanol, flush first with the appropriate intermediate solutions to ensure compatibility.
   5. Prime solvent lines A, B, C, and D for 5 minutes each. The Empower Prime command vigorously forces solvents through the lines and system at 4 mL/min to assist in the cleaning process.
   6. Prime the seal wash.
   7. Prime the wash solvent for 200 seconds and the purge solvent for 40 cycles.
   8. Purge the system at 0.2 mL/min for 10 minutes using 25% A, 25% B, 25% C, and 25% D.
   9. Repeat steps d through h using 100% methanol as the cleaning solvent, using a flow rate of 1.0 mL/min for step h.
   10. Repeat steps d through h using 100% high-purity water as the cleaning solvent, using a flow rate of 1.0 mL/min for step h.
       Caution: If a nebulization-based detector is connected (MS or ELSD), take it off line before performing step k. Direct the flow from the union outlet to waste.
   11. Repeat steps d through h using 10% aqueous phosphoric acid as the cleaning solvent, using a flow rate of 0.2 mL/min for step h.
       Caution: To avoid damage, do not place the seal wash line in this solution. Place the seal wash line in high purity water.
   12. Repeat steps d through h using 100% high-purity water as the cleaning solvent, using a flow rate of 1.0 mL/min for step h.
   13. If applicable, reconnect the MS or ELS detector.
   14. Repeat steps d through h using 100% methanol with a flow rate of 1.0 mL/min for step h. 

Table 2. ACQUITY UPLC System Cleaning Protocol Summary

d. Minimizing Sample Particulate Contamination of Protein BEH SEC Columns

The performance and lifetime of an ACQUITY UPLC Protein BEH SEC Column can deteriorate due to the accumulation of particulates. These include insoluble protein aggregates contained in the injected sample. As a result, it is recommended to filter all protein-containing samples using sterile, low protein binding, 0.2 µm syringe filters. Alternatively, ACQUITY UPLC Sample Vials containing sample can be centrifuged so that any insoluble particulates form a solid pellet at the bottom of the vial.  

Note:  Be sure to adjust the ACQUITY UPLC needle depth to prevent sampling any particulates located at the bottom of the centrifuged vial. For more information on setting the needle depth and minimum volume in vials, reference "Sample Vials and Accessories".

e. Preventing Sample Formulation Constituent Contamination of Protein BEH SEC Columns

Studies have found a variety of chemical and physical conditions that can cause the creation of biotherapeutic protein aggregates. To minimize this occurrence, excipients are typically added to therapeutic protein formulations. However, these excipients can cause inaccurate SEC quantitation due to changes in retention, peak shape, or resolution. While a variety of SEC column cleaning protocols may be used to restore a column's performance, this can be time consuming and ultimately ineffective. As a last resort, column replacement is often required, resulting in higher costs and delays. As indicated in Figure 13, the use of guard columns may protect Protein BEH SEC columns from deterioration due to chemical contamination.

Figure 13. Effect of using an ACQUITY UPLC Protein BEH SEC Guard Column (4.6 x 30 mm) on column efficiency. The arrows indicate where the guard column was changed, noting how the column life was extended (top/red) vs. a control that did not use a guard column (bottom/blue).

For more information, reference "Improving the Lifetime of UPLC Size-Exclusion Chromatography Columns Using Short Guard Columns."

f. Column Storage

To prevent the potential of microbial growth in SEC columns that will be removed from your system and stored, we recommend the following:

1. Start by using a solvent line that is already in 100% water. Then using this line, flush the existing mobile phase from the column with approximately 20-column volumes of HPLC grade and filtered water (e.g., 50 mL for 4.6 x 150 mm columns) to remove any buffer and salts from the SEC column.
2. After preparing the UPLC System as needed, flush the SEC column with approximately 20-column volumes of filtered 20% methanol/80% water to prevent bacterial growth. The column can then be safely stored at room temperature. 

Note: It is important that all salt and/or protein be flushed from the BEH SEC column BEFORE storage in 20% MeOH. Failure to remove salt and/or protein may cause precipitation in the column when it is exposed to the 20% organic storage solvent.