advantages of ultra performance liquid … · 2012-01-26 · to download a copy of this poster,...

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INTRODUCTION Size exclusion chromatography (SEC) is typically used to measure the product of interest, aggregates, and potentially other size variants present in biopharmaceuticals. Unlike reversed-phase and ion-exchange chromatographic separations the elution volume and resolution of a true size-exclusion separation, where the mobile phase composition has been appropriately optimized to minimize protein-column packing interactions, is predominantly dependent on the packing materials (pore size, pore volume, and particle size), geometry of the column, and performance of the chromatographic system used. Size-based HPLC separations have traditionally been performed at low pressure conditions with 5µm and larger sized silica-based packing materials. However, new advances in a 125 Å pore size, sub-2µm ethylene-bridged hybrid (BEH) silica packing material and Waters Ultra Performance Liquid Chromatography (UPLC) instrumentation have allowed faster, higher sensitivity, and higher resolving separations of peptides and small proteins to be achieved while at the same time greatly reducing waste-stream volumes. The performance characteristics of this column are presented. The advantages of using a packing-material with the best suited physical characteristics of pore and particle size for the size-exclusion chromatography separation of peptides and small proteins, including the PharmEuropa Insulin Method (Sept 1996, Vol 8, No. 3, Pages 359—360), is demonstrated. Additionally, the investigation of column geometry, as well as other physical variables including sample loading are also presented.

ADVANTAGES OF ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY USING 125Å PORE SIZE, SUB-2 µm, SIZE-EXCLUSION PARTICLES FOR THE ANALYSIS OF PEPTIDES AND SMALL PROTEINS

Figure 3. Shown is the improved resolution of sub-2 µm UPLC-SEC packing material. Insulin was analyzed on an ACQUITY UPLC BEH125 SEC 1.7 µm column and a 100% silica-based diol-coated 10 µm SEC particle column (Waters HMWP) tested to perform in the European Pharmacopoeial method. The UPLC column delivers a significant increase in HMW resolution(1.8 times greater), decreased run-times, and reduced acetonitrile containing mobile phase use.

METHODS UPLC-SEC Chromatographic Conditions

LC System: ACQUITY UPLC® H-Class Bio System with PDA or TUV detector (Titanium Flow Cell, See Waters Tech Note: 715002147) Detection: 214 and 280 nm aqueous, 276 nm insulin method Aqueous Mobile Phase: 25 mM Sodium Phosphate, pH 6.8, 0.15 M NaCl Insulin Mobile Phase: L-arginine (1.0 g/L) /acetic acid (99%)/acetonitrile; 65/15/20 (v/v/v) Wash and Injector Needle Purge: Water and 10% MeOH Insulin Wash and Injector Needle Purge: 10% acetonitrile Seal Wash: 10% MeOH Temperature: 25˚C Sample Diluent: 25 mM Sodium Phosphate, pH 6.8, 0.15M NaCl Insulin Sample Diluent: 0.01 M HCl Flow rate: 0.4 mL/min unless otherwise specified Injection volume: 5 µL unless otherwise specified. Samples: All samples were obtained from Sigma-Aldrich except for the Insulin Product samples. Insulin Product Samples 1 and 2 were analyzed past expiry. Columns: Waters: ACQUITY UPLC BEH125 SEC,1.7 µm, 4.6 x 150 mm (p/n 186006505) Waters: ACQUITY UPLC BEH125 SEC,1.7 µm, 4.6 x 300 mm (p/n 186006506) Waters: ACQUITY UPLC BEH200 SEC,1.7 µm, 4.6 x 300 mm (p/n 186005226) Waters: BioSuite 125 UHR SEC, 4 µm, 4.6 x 300 mm (p/n 186002161) Waters: Insulin HMWP HPLC, 10 µm, 7.8 x 300 mm (p/n WAT201549) flow rate = 0.5 mL/min.

References

1. Waters Poster PSTR10169860 (www.waters.com), “Efficient Development of UPLC Separation Methods for the Analysis and Structural Characterization of Biopharmaceuticals”

2. Arakawa, D. Ejima, T. Li, J.S. Philo, “The Critical Role of Mobile Phase Composition in Size Exclusion Chromatography of Protein Pharmaceuticals,” J. Pharm. Sci., 99 (2010), 1674-1692

CONCLUSIONS

• The 125Å pore-size Waters ACQUITY UPLC BEH125 SEC, 1.7µm columns can provide improved resolution of small proteins and peptides as compared to larger particle size 100% silica diol-coated SEC columns when configured to an appropriate UPLC System.

• The Waters ACQUITY UPLC BEH125 SEC, 1.7µm column was developed to provide batch-to-batch consistency and outstanding stability for increased confidence when used in validated product release methods.

• Fixed SEC method development parameters (pore size, particle size, column length) should be carefully selected for optimal separations.

• Minimizing sample injection volumes results in better resolution for UPLC-SEC separations.

RESULTS AND DISCUSSION COLUMN PERFORMANCE COMPARISONS

This latest BEH-based sub-2 µm SEC packing material, developed for UPLC-based separations, was evaluated and shown to provide improved resolution, sensitivity and higher throughput as compared to 100% silica-based diol-coated particle SEC columns with pore sizes and volumes specifically designed for the analysis of peptides and small proteins (<80kDa). Separations using aqueous and organic mobile phases were evaluated.

Figure 1. Shown is the effect of packing material on myoglobin monomer/aggregate resolution. The sample (2 mg/mL) was analyzed on a 4 µm diol-coated silica column and a UPLC BEH125 SEC 1.7 µm column. Flow rates and injection volumes were equivalent. USP monomer/aggregate resolution was 1.7 times greater on the BEH125 1.7µm SEC column as compared to 4 µm pore diol-coated silica column.

Figure 2. Shown are the separations of several peptides performed under aqueous SEC separation conditions on an ACQUITY UPLC BEH125 SEC 1.7 µm column and a 100% silica-based, diol-coated, 4 µm SEC particle column. Analysis on the BEH125 column provides increased resolution throughout the lower end of the peptide mass range (132-29,000).

Resolution of Insulin and Insulin High Molecular Weight

COLUMN REPRODUCIBILITY AND STABILITY

The column-to-column and batch-to-batch reproducibility and stability of the ACQUITY UPLC BEH125 SEC column (4.6 X 300 mm) was evaluated by comparing the retention times of several peptides and proteins of three columns from on batch and two columns from a second batch. The stability of the column was evaluated using the European Pharmacopoeial insulin method.

Figure 4. The stability of the ACQUITY UPLC BEH125 SEC column (4.6 mm x 30cm) using organic mobile phase is shown. After over 800 injections the retention time of the insulin monomer peak and the resolution between insulin monomer and dimer peaks are maintained.

Table 1. Shown are the retention time reproducibilities for 5 ACQUITY UPLC BEH125 SEC 1.7 µm columns (4.6 mm x 30cm) using aqueous and organic (insulin separation method only) mobile phases.

Column Stability for Insulin Analysis

Stephan M Koza, Paula Hong, and Kenneth J Fountain Waters Corporation

Resolution of Small Protein (Myoglobin) Monomer and High Molecular Weight Species (Aqueous)

Resolution of Proteins and Peptides (Aqueous)

ACQUITY UPLC BEH125 SEC 1.7µm Column Reproducibility

UPLC-SEC METHOD CONSIDERATIONS

Column selection, mobile phase, flow rate, and sample load should be optimized during SEC method development. The advantages of using sub-2µm particle UPLC-SEC columns and some considerations for their use are presented.

Figure 5. Select the optimal pore size. Shown above are the results for insulin on two sub-2µm, BEH-based, diol-coated columns of differing pore size. The HMW and insulin fragments (observed in Product Samples 1 and 2) are better resolved on the 125Å pore diameter ACQUITY UPLC BEH125 column as compared to the 200Å pore diameter BEH200 column.

Particle Size (Insulin Analysis)

Figure 6. Select the optimal particle size. Above are the results for insulin samples on three SEC columns. The 1.7µm ACQUITY UPLC BEH125 SEC col-umn better resolves the HMW and Fragment peaks than the 4 and 10 µm particle size columns.

Column Length (Insulin Analysis)

Figure 7. Select the optimal column length. Above are the results for insulin samples on two BEH125 UPLC SEC columns. The insulin fragment and HMW peaks in the Product 1 and 2 samples are better resolved on the ACQUITY UPLC BEH125 1.7 µm, 300mm column than on 150mm column regardless of flow rate.

Sample Load (Myoglobin Analysis)

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Figure 7. Optimize injection volume. Increased injection volumes can result in a significant loss of resolution in UPLC-SEC analyses. For increased sensitivity minimize sample dilution when possible based on the complexity and molecular weight differences of the components in the sample.

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ACQUITY UPLC BEH125 SEC 1.7 µm 4.6 x 300 mm column

USP Rs = 1.94 USP Rs = 3.29

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Rs = 2.1USP Plate Count = 3KFlow Rate = 0.5 mL/min

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ACQUITY UPLC BEH125 1.7µm(4.6 x 300 mm)

HMWP 10µm(7.8 x 300 mm)

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ACQUITY UPLC BEH125 1.7µm(4.6 x 300 mm)

HMWP 10µm(7.8 x 300 mm)

advantages of ultra performance liquid … · 2012-01-26 · to download a copy of this poster,...

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