Protein Separation Technology

Transcription

1 [ ] Protein Separation Technology

2 Protein Separation Technology The analysis and characterization of protein samples requires the detection of small chemical differences between large molecules. Most often these analyses have employed an array of analytical techniques, each sensitive to a different property of the protein. Reversed-phase HPLC has not been fully exploited in these tests because the separation of proteins often yields relatively broad and asymmetrical peaks with poor recovery and significant carryover. Waters new reversed-phase, ethylene bridged hybrid (BEH) Protein Separation Technology columns are specifically designed for the high resolution analysis of proteins. Introducing a New Family of BEH C Columns for Protein Separations Separates proteins of various sizes, hydrophobicities, and isoelectric points Available in. μm particles for HPLC and.7 μm material for UPLC and nano UPLC applications Maximizes recovery and minimizes protein carryover Tolerates extreme ph and temperature Quality-control tested with protein mixture Couples directly to ESI-MS for protein identification

3 Å C Columns Developed for Protein Chromatography... Column: Mobile Phase A: Mobile Phase B: XBridge BEH C,. µm,. mm.% TFA in water.7% TFA in 7.% acetonitrile. ml/min Column Temp.: ºC Injection: µl 8-% B in min nm Protein mixture prepared in.% TFA in % acetonitrile. Ribonuclease A (. mg/ml). Cytochrome c (. mg/ml). Bovine Serum Albumin (. mg/ml). Myoglobin (. mg/ml). Enolase (. mg/ml). Phosphorylase b (.9 mg/ml) 8 min BEH C columns can be used with proteins that have a wide range of properties. This protein mix was chosen to represent a range of isoelectric points, molecular weights, and hydrophobicities.

4 protein separation technology COLUMNS Designed for Protein Separations The new Protein Separation Technology material is based on Waters patented BEH Technology hybrid material. The combination of the hybrid particle, short chain bonded phase, and wide pore overcomes the performance limitations of traditional silica-based column packings. Å pores C ligand Minimal secondary interactions Long column lifetime at elevated temperature Minimal carryover and maximum recovery Effect of Pore Size on Separation of a Monoclonal Antibody Effect of Bonded-Phase Chain Length on Separation of IgG Heavy and Light Chains Intact mouse monoclonal antibody (IgG). mg/ml prepared in.% TFA in water Column: XBridge BEH C 8 (bottom) and XBridge BEH C 8 (top),. µm,. mm Mobile Phase A:.% TFA in water Mobile Phase B:.7% TFA in % acetonitrile. ml/min -7.% B in min Column Temp.: 8 ºC Injection:. µl nm Reduced and alkylated mouse monoclonal antibody (IgG). mg/ml prepared in.% TFA in water Column: XBridge BEH C (bottom) and XBridge BEH C 8 (top),. µm,. mm Mobile Phase A:.% TFA in water Mobile Phase B:.7% TFA in % acetonitrile. ml/min -7.% B in min Column Temp.: 8 ºC Injection:. µl nm. Å. C Light chain Å. Multiple forms of heavy chain reflecting partial alkylation and multiple glycoforms C min min A murine monoclonal IgG shows a small, badly tailing peak on a C 8 column with Å pores and a better recovered, symmetrical peak on the larger pore size. Generally, proteins are expected to give better reversed-phase chromatography on short chain bonded phases. For testing this effect, a murine monoclonal antibody was reduced and partially alkylated to create a sample that includes several large protein subunits that are distinguished by very small differences in structure. The light and heavy chain components of the mouse monoclonal antibody offer improved peak shape, resolution, and recovery on the XBridge BEH, C relative to the same particle and pore size with the longer chain C 8. [ ]

5 protein separation technology Minimal Secondary Interactions. Column: ACQUITY UPLC BEH C,.7 µm,. mm Mobile Phase A:.% TFA in water Mobile Phase B:.% TFA in % acetonitrile. ml/min -% B in min Column Temp.: ºC Injection:. µl nm. Protein mixture prepared in.% TFA in % acetonitrile. Ribonuclease A (. mg/ml). Cytochrome c (. mg/ml). Bovine Serum Albumin (. mg/ml). Myoglobin (. mg/ml). Enolase (. mg/ml). Phosphorylase b (.9 mg/ml) 8 min The surface chemistry of the BEH C particles gives symmetrical peak shapes for a variety of protein types. For example, the sharp peak for the basic protein cytochrome c supports the lack of non-desired secondary interactions with the BEH particle surface. Separations at Elevated Temperature... ºC ºC Reduced and alkylated mouse monoclonal antibody (IgG). mg/ml prepared in.% TFA in water Column: XBridge BEH C,. µm,. mm Mobile Phase A:.% TFA in water Mobile Phase B:.7% TFA in % acetonitrile. ml/min -7.% B in min Column Temp.: ºC (top), ºC (middle), 9 ºC (bottom) Injection:. µl nm. Light chain. Multiple forms of heavy chain reflecting partial alkylation and multiple glycoforms 9ºC min Elevated temperature is routinely used in protein separations. For testing this effect, a murine monoclonal antibody was reduced and partially alkylated to create a sample that includes several large protein subunits that are distinguished by very small differences in structure. The yield of the heavy and light chains improves the temperature is increased from -9 C. BEH Technology columns were developed for use at extreme temperatures. [ ]

6 protein separation technology Minimal Protein Carryover Ovalbumin Absence of detectable Ovalbumin carryover. Columns: Eluent A: Eluent B:. µg Ovalbumin injected ACQUITY UPLC BEH C,.7 µm,. mm mm.% TFA in % HPLC grade water.7% TFA in 7.% acetonitrile. ml/min Column Temp.: C 8-% B in min Hold at %B for min then re-equilibrate column at 8% B for 8 min Gradient restarted at min and at 9 min nm min st gradient without sample injection nd gradient without sample injection Column carryover was tested by running multiple gradients following a single injection. Protein peaks observed during the first gradient are not found in subsequent gradients. Effective Alternatives to Use of % Acetonitrile ACN 7: IPA:ACN IPA 8 min Protein Mixture prepared in.% TFA in % MeCN:. Ribonuclease A (.8 mg/ml),. Cytochrome c (. mg/ml),. Bovine Serum Albumin (. mg/ml),. Myoglobin (. mg/ml),. Enolase (. mg/ml),. Phosphorylase b (.8 mg/ml) Column: BEH C,.7 µm,. x mm Mobile Phase A:.% TFA in water Mobile Phase B:.% TFA in MeCN;.% TFA in 7: IPA:MeCN;.% TFA in IPA;. ml/min Column Temperature: 8 ºC Injection:. µl nm Gradient Table: Time (min) %A %B Curve Column Volumes (CV) % Change (CV) Initial * 9. 8 Acetonitrile is the preferred solvent for reversed-phase protein separations. Altered selectivity and improved recovery in reversed-phase protein separations can be achieved with different solvents. It is important to consider alternatives to acetonitrile during the recent worldwide shortage of this solvent. A protein standard mixture was analyzed on BEH C with % acetonitrile (top), pure isopropanol (bottom) and a 7: blend (middle). All three solvents give useful separations with similar selectivity. [ ]

7 protein separation technology Waters UPLC Technology and Advanced Detection for Protein Characterization Waters introduced UPLC Technology in. This new class of separation technology brings improved resolution, sensitivity, and speed to a wide range of chromatographic analyses. With the BEH C packing material in.7 µm particles, these enhancements can now be realized with protein separations. Easily scaled. μm for HPLC and.7 μm for enhanced UPLC and nano UPLC separations Effective with MS compatible eluents to address the need for advanced detection techniques Improved Protein Resolution with UPLC Technology BEH C. µm BEH C.7 µm Column: ACQUITY UPLC BEH C,. mm,.7 μm (top),. μm (bottom) Mobile Phase A:.% TFA in water Mobile Phase B:.% TFA in % acetonitrile. ml/min -% B in min Column Temp.: 8 ºC Injection:. µl nm Protein mixture prepared in.% TFA in % acetonitrile. Ribonuclease A (. mg/ml). Cytochrome c (. mg/ml). Bovine Serum Albumin (. mg/ml). Myoglobin (. mg/ml). Enolase (. mg/ml). Phosphorylase b (.9 mg/ml) 8 min The protein test mixture was separated on two BEH C columns, one with. µm particles and the other with.7 µm particles. The UPLC separation provides sharper peaks for all the proteins in the test mixture. This translates into better resolution as shown by the multiple peaks around phosphorylase, at approximately min. This comparison, with both columns, was performed on a UPLC system to preserve the minimized band-broadening. The benefits of the small particle UPLC BEH C column would be lost without the optimized ACQUITY UPLC System. BEH C Columns for Protein Analysis with UPLC/MS % Fc/ LC Fd Lys-C digested (limited) reduced monoclonal antibody Column: ACQUITY UPLC BEH C,.7 µm,. mm mm Eluent A:.% FA in % HPLC grade water Eluent B:.% FA in % acetonitrile. ml/min % B for min Then -% B in. min. Then -9% B in min. Column Temp.: 8 C 9 Fc/ Clip Fc/ Clip min Waters LCT Premier XE MS Detection Capillary Voltage: V Cone Voltage: V Source Temp.: C Desolvation Temp.: C Desolvation Gas: L/h The large fragments obtained through LysC digestion of a monoclonal antibody can be separated on the UPLC BEH C column coupled directly to ESI/Tof MS for identification of the individual peptide products. [ 7 ]

8 protein separation technology BEH PA RT IC L E SYNT HESIS AND BONDING T EC HNOLOGY for Stable and Reproducible P rotein Separations Waters has developed a second generation hybrid particle synthesis. T he patented process ensures the highest purity and most consistent column properties. T he material can be prepared in particle sizes for HPLC and UPLC. T he pores can be enlarged as appropriate for protein separations. Well characterized, state-of-the-art bonding procedures for short-chain ligand Particle structure and bonding chemistry stable at low ph and at elevated temperature Quality-control tested with a diverse protein mixture Consistent protein separations from batch-to-batch E to CH CH OE t Si O Si Si E to O Si O O O OE t Si O E t O OE t Polyethoxysilane (BPEOS) Si O Et OEt EtO Si EtO OEt EtO + E to E to Si EtO OEt CH Si OEt CH OEt n Tetraethoxysilane (TEOS) Bis(triethoxysilyl)ethane (BTEE) Anal. Chem., 7, , U.S. Patent No.,8, B Accelerated Low ph Stability Testing on BEH C vs. Competitive Columns B - Å TP A - Å C XBridge BEH C % Change k' for Methyl Paraben % Change k' for Methyl Paraben XBridge BEH C C - Å C A - Å C D - Å C B - Å TP Waters innovative BEH C particle synthesis and bonding process yields-9 HPLC and -9 8 min UPLC columns that tolerate separation conditions of low ph and elevated temperature. 8 min Shown above are our available XBridge BEH C Columns. ACQUITY UPLC BEH C time (min) in.% aqueous C Exposure Exposure time (min) in.% aqueous C Columns are also available. [ 8 ] C-

9 protein separation technology Batch-to-Batch Reproducibility Quality Control Document for BEH C Columns Column: ACQUITY UPLC BEH C,.7 µm,. mm mm Eluent A:.% TFA in % HPLC grade water Eluent B:.7% TFA in 7.% acetonitrile. ml/min 8-% B in min Protein mixture prepared in.% TFA in % acetonitrile Column Temp.: C. Ribonuclease nm. Cytochrome c. BSA. β-lactoglobulin. Enolase. Phosphorylase b Batch A Batch B Batch C 8 8 min T his comparison shows the consistent batch-to-batch performance with a protein separation. Each batch of BEH C material is rigorously tested to ensure consistent performance. T he Certificate of Analysis included with each column reports physical, chemical, and chromatographic tests. Protein Separation Stability Comparison Between BEH C and Competitive Column. Å Silica C Column BEH C Column. Columns: XBridge BEH C,. µm (top), % silica-based, Å C,. µm (bottom),. mm mm Eluent A:.% TFA in % HPLC grade water Eluent B:.7% TFA in 7.% acetonitrile. ml/min 8-% B in min Column Temp.: C Detection: nm Protein mixture prepared in.% TFA in % acetonitrile. Ribonuclease. Cytochrome c. BSA. myoglobin. Enolase. Phosphorylase b min T he two columns were tested with an initial analysis of the protein standard mixture (black line). Each column was operated continuously for one week with the same gradient at 9 C. T he protein separation test at C was then repeated (green line). Note that the retention shifts earlier on the silica-based column while the separation is stable on the BEH C. [ 9 ]

10 protein separation technology MassPREP On-Line Protein Desalting Devices Waters MassPREP On-Line Desalting Cartridge (. x mm) MassPREP on-line desalting columns can effectively desalt proteins prior to LC/MS analyses. Since non-volatile salts (e.g., NaCl) can suppress ionization of intact proteins leading to poor detection sensitivity, it is important to remove, or significantly minimize, the introduction of these compounds into the mass analyzer. The reversed-phase, phenyl material contained in MassPREP on-line columns successfully traps proteins, allowing the salts to be washed to waste prior to protein elution into the mass spectrometer. With an optimized LC/MS method, cycle times as low as minutes (for intact antibody) and minutes (for reduced antibody) are achievable. LC System: Alliance 79 Separation Module MS System: Q-Tof micro, ESI Positive Eluent A:.% Formic Acid (H O) Eluent B:.% Formic Acid (ACN) % % % Inject # = µl PBS Blank Inject #99 = µl mab ( µg/ µl PBS) Inject #98 = µl BSA ( µg/ µl PBS) Inject #97 = µl PBS Blank % m/z Effectively desalts proteins yielding improved LC/MS results Fast on-line method for high throughput applications Excellent protein recoveries and no detectable carryover Over a series of injections, satisfactory results were obtained for BSA and a mab, as shown for injections #97- on a MassPREP on-line desalting cartridge. Reference Desalting of Proteins Using MassPREP On-line Desalting Cartridges Prior to Mass Spectrometry. Waters Application Note 777EN injections from a single cartridge Waters ACQUITY UPLC System and Xevo Mass Spectrometer with Protein Separation Technology BEH C columns and MassPREP On-Line Desalting Devices Waters nanoacquity UPLC System with nanoacquity BEH C columns

11 [ ACQUITY UPLC System Accessories ] protein separation technology Ordering Information Ordering information for Waters BEH C offerings for traditional HPLC and advanced UPLC protein separations are shown below. UPLC Columns Part Number ACQUITY UPLC BEH C,.7 µm,. x mm 89 ACQUITY UPLC BEH C,.7 µm,. x mm 89 ACQUITY UPLC BEH C,.7 µm,. x mm 897 ACQUITY UPLC BEH C,.7 µm VanGuard Pre-Column 8 Note: ACQUITY UPLC BEH C,.7 μm columns are designed for use with the ACQUITY UPLC system. The benefits of the small particle packing in ACQUITY UPLC BEH C,.7 µm columns are only realized with the low system volume and low detector dispersion of an ACQUITY UPLC system. nanoacquity UPLC Part Number nanoacquity UPLC BEH C,.7 µm, 7 µm x mm 89 nanoacquity UPLC BEH C,.7 µm, µm x mm 8 nanoacquity UPLC BEH C,.7 µm, µm x mm 8 For use with nanoacquity UPLC systems rated to, psi only. Not for use with nanoacquity UPLC systems rated to, psi. HPLC Columns Part Number XBridge BEH C,. µm,. x mm 898 XBridge BEH C,. µm,. x mm 899 XBridge BEH C,. µm,. x mm 8 XBridge BEH C,. µm,. x mm 8 XBridge BEH C,. µm,. x mm 8 XBridge BEH C,. µm,. x mm 8 XBridge BEH C,. µm,. x mm 8 XBridge BEH C,. µm,. x mm 8 Custom BEH C 8 MassPREP On-Line Protein Desalting Devices Quantity Part Number MassPREP Micro Desalting Column /pk 8 MassPREP On-line Desalting Cartridge (. x mm) /pk 878 UPLC Intact Mass Analysis Application Kit* (Includes MassPREP Micro Desalting Column and ACQUITY Tubing Kit) Sentry. x mm Guard Cartridge Holder. Required for use of MassPREP On-line Desalting Cartridge /pk 79 /pk WAT9798 * See: UPLC Intact Mass Analysis Application Kit Manual (7) More information can be found at [ ]

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