Agilent Analysis of oxidized insulin chains using reversed phase Agilent ZORBAX RRHD 300SB-C18
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1. the API Methods include size exclusion chro matography for the quantitation of dimers and aggregates and ion exchange for the identification of charge variants Both of these techniques use aqueous eluents and non denaturing conditions As part of the full characterization of a protein it is also necessary to look at the primary amino acid sequence and any post translational modifications to the sequence that may have occurred during the purification or formulation steps of manufacture To perform this type of analysis denaturing con ditions are required and so reversed phase HPLC is normally the technique of choice In this example we use Agilent ZORBAX Rapid Resolution High Definition RRHD columns which benefit from improved packing processes to achieve sta bility up to 1200 bar for use with the Agilent 1290 Infinity LC Materials and Methods The conditions in Table 1 were used throughout the investigation with variations as noted in the relevant chromatograms Table 1 Standardized Chromatographic Conditions Parameter Item Column Agilent ZORBAX 300SB C18 1 8 um 2 1 x 50 mm p n 857750 902 Agilent Technologies Wilmington DE Sample Insulin oxidized insulin chain A and chain B from bovine pancreas Sigma Aldrich St Louis MO Sample concentration 1mg mL Injection volume 3 uL Flow rate 1 0 mL min Pressure 650 bar 0 1 TFA in distilled water 80 ACN 0 01 TFA in distilled water UV 280 nm Agilent 12902. Author Phu T Duong Agilent Technologies Inc Wilmington DE USA Linda Lloyd Agilent Technologies Inc Essex Road Church Stretton S46 6AX UK Analysis of Oxidized Insulin Chains using Reversed Phase Agilent ZORBAX RRHD 300SB C18 Application Note BioPharma Abstract A new reverse phase media Agilent ZORBAX RRHD 300SB C18 1 8 um was used for the separation of a typical protein biopharmaceutical insulin The value of sub 2 um particles for protein separations was assessed under denaturing conditions The advantages of these particles in separating small molecules were also realized in protein separations The use of a 1 8 um column designed for UHPLC systems significantly reduced analysis time critical for increasing the efficiency of QC for protein primary structure analysis The separations also demonstrated how this technology achieved resolution of various insulin isoforms The eluents routinely used for reverse phase analysis are acidic containing trifluoroacetic acid or formic acid which can limit the lifetime of many HPLC columns Using StableBond technology it was possible to produce a 300A pore size media that was stable under acidic conditions to provide the robust reproducible separations required for protein QC RE Agilent Technologies Introduction Due to the heterogeneity of a protein biopharmaceutical it is necessary to use a number of chromatographic techniques to fully characterize
3. Figure 4 Two hundred injections reveal the reproducibility of the Agilent ZORBAX RRHD 300SB C18 1 8 um column Separation of Heat Degraded Insulin A forced degradation study can be performed by heating APIs and HPLC used to monitor the degradation products Heat treating insulin produces degradation products that can be quickly resolved by the column from the monomer insulin Figure 5 mAU 80 70 si treated insulin 55 C for 24 h 50 40 injection 4 uL gradient 35 50 0 5 min 0 1 2 3 4 5 min Figure 5 Heat treated insulin quickly resolved on an Agilent ZORBAX RRHD 300SB C18 1 8 um column Separation of Insulin Isoforms Figures 6 and 7 showed that the column can also separate isoforms of insulin in this case oxidized insulin chain A Figure 6 and the mixture of insulin and oxidized insulin chain A Figure 7 Once again different gradient systems can be selected with very similar results For fast analysis of insulin high gradient systems are usually used However these conditions force oxidized insulin chain A to be eluted rather quickly To analyze oxidized insulin chain A from its subspecies a shallow gradient system is required to ensure that the molecule can retain longer on the column mAU ow o 1 204 25 oxidized insulin chain A gradient 5 100 B 0 05 1 15 2 25 3 3 5 min peal o 2 125 oxidized insulin chain A gradient 5 50 B 0 05 1 15 2 25 3 35 4 45 m
4. Infinity HPLC Mobile phase A Mobile phase B Detector System Results Speed The system separated the test mixture very quickly distin guishing insulin a small molecule from its impurities in less than five minutes Figure 1 Using multiple gradients achieves the same fast analysis time Figure 2 Rapid equilibration is evident even with the screening gradient which starts from a highly aqueous eluent demonstrating that the column is suitable for use with a wide range of organic content mAU gradient 25 100 B 0 4 min 40 35 30 25 20 15 10 5 3 164 0 0 5 1 15 2 25 3 35 4 45 min base line expansion 4 2 0 2 4 0 5 1 1 5 2 2 5 3 3 5 min Figure 1 Fast resolution of insulin and some impurities on an Agilent ZORBAX RRHD 300SB C18 1 8 pm column 2 099 mAU 200 2 042 0 5 mL min 2 394 0 3 mL min gradient 3 100 B 0 4 min Norm gradient 5 100 B 0 4 min 1 727 1 5 mL min 1 789 1 0 mL min 0 5 1 1 5 2 25 3 3 5 4 45 min pr Figure 3 Different flow rates can be selected to separate insulin on the Agilent ZORBAX RRHD 300SB C18 1 8 pm column 120 me gradient 5 100 B 0 4 min Reproducibility Two hundred consecutive injections were done to examine the column s reproducibility The results show that the integrity of peak shape asymmetry retention time and efficiency remained the same after 200 injections of i
5. in Figure 6 Analysis of oxidized insulin chain A with different gradients mAU 30 1 208 1 798 Injection 5 pL 0 0 5 1 1 5 2 25 3 3 5 4 45 min Figure 7 Analysis of insulin and oxidized insulin chain A on Agilent ZORBAX RRHD 300SB C18 1 8 um Similarly Figures 8 and 9 demonstrate that the column sepa rates oxidized insulin chain B Figure 8 Again different gradi ent systems can be selected with very similar results The column also discriminates degradation products of oxidized insulin chain B Figure 9 oxidized insulin chain B gradient 3 100 B As 0 93 Plates 9343 0 5 1 1 5 2 2 5 3 3 5 min gradient 5 50 B 0 05 1 15 2 25 3 35 4 45 min Figure 8 Optimized conditions for fast analysis of oxidized insulin chain B on the Agilent ZORBAX RRHD 300SB C18 1 8 um column Norm amp 8 heat treated oxidized insulin B chain 6 55 C for 24h 4 gradient 3 100 B 0 4 min 2 S 0 PN 0 5 1 15 2 25 3 3 5 mi Figure 9 Oxidized insulin chain B and some degradation and impurities are well resolved on Agilent ZORBAX RRHD 300SB C18 1 8 um oxidized insulin chain B x 5 mAU S injection 2 uL 175 z pressure 650 700 bar oxidized insulin chainB 2 gradient 33 50 B 0 4min 33 B 4 5 min 15 insulin 12 5 AF E of oxidized insulin chain B 0 0 5 1 1 5 2 2 5 min Figure 10 Insulin and oxidized insulin A and B chains are resolved quickly bu
6. nsulin without cleaning the column Table 3 and Figure 4 0 0 5 1 1 5 2 25 3 3 5 4 45 min Table 3 Two Hundred Injections of Insulin Demonstrates the Figure 2 Multiple gradient systems can be selected to separate insulin Reproducibility of Agilent ZORBAX RRHD 300SB C18 1 8 um in less than 5 minutes using the Agilent ZORBAX RRHD 300SB C18 1 8 um column including eluents Run Pressure Retention time with high water content no bar min Asymmetry Plate count 3 i 680 520 1 789 0 86 9758 Flow rate can also be manipulated to provide a fast separation 50 680 520 1 790 Wo aed Figure 3 Peak asymmetry and efficiency remain unchanged 100 680 520 1 788 Aes ae Table 2 a feature of sub 2 um particles that facilitates rapid 200 680 520 1 789 0 87 9741 separations Table 2 Effect of Flow Rate on Retention Time Asymmetry and Efficiency in the Analysis of Insulin Flow rate Pressure Retention time mL bar min Asymmetry Plate count 0 3 230 150 2 39 0 80 8815 0 5 350 250 2 04 0 82 8390 1 0 680 520 1 78 0 88 8034 1 5 890 670 1 72 0 88 8060 mAU 1 789 70 1 run gradient 25 50 B 0 4 min o 05 1 15 2 25 3 35 4 45 mi n 60 Overlay of 200 runs Norm 30 80 70 0 TO ag 60 o 05 1 15 2 25 3 35 4 45 mi n 50 40 30 50 20 0 05 1 15 2 25 3 35 4 45 min o 05 1 15 2 25 3 35 4 45 mi n 0 G 0 05 1 15 2 25 3 35 4 45 min
7. t insulin and oxidized chain B often co elute Conclusions Analyzing small molecule protein biotherapeutic insulin together with its isoforms and breakdown products is fast and simple with the Agilent ZORBAX RRHD 300SB C18 1 8 um column The column s rapid resolution high definition technology permits high pressure UHPLC while the StableBond 300A pore sized particles are robust when analysis requires acidic condi tions Reproducibility is excellent with good resolution asym metry and efficiency The column is well suited to the needs of QC when assessing the structure of primary proteins For More Information For more information on our products and services visit our Web site at www agilent com chem www agilent com chem Agilent shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this material Information descriptions and specifications in this publication are subject to change without notice Agilent Technologies Inc 2011 Printed in the USA April 28 2011 5990 7988EN 7 Agilent Technologies
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Agilent Analysis of oxidized insulin chains using reversed phase Agilent ZORBAX RRHD 300SB C18