• Application Note

Future-proofing the Biopharmaceutical QC Laboratory: Using the ACQUITY UPLC H-Class Bio System for HPLC Peptide Mapping

Future-proofing the Biopharmaceutical QC Laboratory: Using the ACQUITY UPLC H-Class Bio System for HPLC Peptide Mapping

  • Eoin F.J. Cosgrave
  • Sean M. McCarthy
  • Waters Corporation

Abstract

The application of the ACQUITY UPLC H-Class Bio System for HPLC-based peptide mapping is described in this application note. By making a simple adjustment to the legacy HPLC method in the form of a gradient start offset, nearly identical chromatograms were obtained on the ACQUITY UPLC H-Class Bio System compared to the HPLC chromatogram.

Benefits

  • Transfer peptide map applications from HPLC to UPLC
  • Future-proof laboratory for UPLC methods

Introduction

This application note represents the first in a series focusing on transitioning HPLC-based biopharmaceutical separations to UPLC-based methods. The procedure for transferring a peptide mapping method from an HPLC quaternary system to the ACQUITY UPLC H-Class Bio System is described here. The goal of this first step is to perform the identical HPLC assay on the UPLC system operating as an HPLC. This approach enables the QC laboratory to employ UPLC technology for legacy HPLC methods while transitioning to UPLC methods at a later time. Improvements in assay reproducibility and, ultimately, incorporating UPLC based technology will be discussed later in the series.

Peptide mapping represents a typical assay for QC labs dealing with routine analyses of large molecule therapeutics. Peptide separation is a considerable challenge in a QC environment due to the numerous parameters that can influence retention time and selectivity. Consequently, adoption of new separation technology such as UPLC can be a challenge in the QC environment due to the qualification requirements for new instruments together with the need to establish and validate new methods for UPLC instrumentation. Regulatory agencies and pharmacopoeias are becoming increasingly aware of the advantages UPLC offers for product characterization. Correspondingly, regulatory guidelines and pharmacopoeial monographs are beginning to outline new analytical requirements for future drug applications. Companies that manufacture such drugs are, therefore, under pressure to begin the adoption process of new analytical technology.

As a first step in transitioning from HPLC to UPLC technology for routine biopharmaceutical analysis in the QC laboratory, a simplified process for running legacy HPLC peptide mapping methods on the ACQUITY UPLC H-Class Bio System is demonstrated. This allows the laboratory to implement UPLC technology without the burden of developing, validating, and qualifying new UPLC separation methods. HPLC-based peptide mapping performed on the ACQUITY UPLC H-Class Bio System generates comparable chromatograms compared to equivalent peptide maps acquired on HPLC instrumentation. With UPLC technology established in the lab, efforts can be directed to transferring legacy HPLC methods to UPLC without disruption to the HPLC-based analytical workflows. Users can then deploy the ACQUITY UPLC H Class Bio System for UPLC-based separations at a suitable time in the future, having previously qualified the instrumentation.

Experimental

UPLC conditions

System:

ACQUITY UPLC H-Class Bio

Detector:

ACQUITY UPLC Tunable UV (TUV)

Extension loop:

100 μL

Bio mixer volume:

250 μL

Column:

XBridge BEH C18 130 Å 4.6 x 100 mm, 3.5 μm

Column temp.:

40 °C

Flow rate:

0.500 mL/min

Injection volume:

95 μL

Mobile phase A:

H2O with 0.1% (v/v) TFA

Mobile phase B:

Acetonitrile with 0.1% (v/v) TFA

Detection wavelength:

214 nm

Gradient:

Time(min)

FlowRate(ml/min)

%A

%B

%C

%D

Curve

0.500

95

5

0

0

5.00

0.500

95

5

0

0

6

45.00

0.500

50

50

0

0

6

47.50

0.500

5

95

0

0

6

52.50

0.500

5

95

0

0

6

52.60

0.500

95

5

0

0

6

60.00

0.500

95

5

0

0

6

Sample description

Three peptide preparations were used in this study: Waters MassPREP peptide mixture standard, ribonuclease B (Sigma Aldrich, USA), and infliximab with the latter two undigested protein samples prepared as follows. Five hundred μg of ribonuclease B or infliximab was reduced with dithiothreitol, alkylated with iodoacetamide, and isolated using NAP-5 columns (GE Healthcare, USA). Sequence-grade trypsin (Promega, USA) was added to each protein to a final composition 1:20 enzyme/substrate, and samples were digested at 37 °C overnight. Following digestion, trypsin was deactivated by incubation at 70 °C for 15 min whereupon 60 μL of digested protein material was reconstituted in 40 μL of 5% MeCN/0.1% TFA, generating a final peptide concentration of 0.6 μg/μL.

Results and Discussion

Adding a gradient start offset to an instrument method

As a first step in transferring the method from HPLC to UPLC, the ability of the ACQUITY UPLC H-Class Bio System to generate a comparable chromatogram using identical conditions outlined in a legacy HPLC peptide mapping method was evaluated. This study included the use of a MassPREP peptide mixture standard containing peptides of varying hydrophobicity eluting regularly across a delivered gradient. The MassPREP peptide mixture was analyzed using both the ACQUITY UPLC H-Class Bio System and an HPLC instrument, each fitted with an XBridge C18 130 Å, 3.5 μm column. As shown in Figure 1A, separation on the ACQUITY UPLC H Class Bio System resulted in early elution positions for all peaks in the peptide mixture standard. This result indicated that the reduced dwell volume of UPLC compared to the HPLC was causing a change in peak retention times.

Figure 1. A gradient start offset aligns chromatograms generated on instruments with different dwell volumes. A MassPREP peptide mixture standard was separated using either HPLC or UPLC. (A) Comparison of the peptide mapping method transferred directly across without any modifications to the ACQUITY UPLC H-Class Bio instrument method. (B) The resulting chromatogram after the addition of a 360 μL gradient start offset volume on the ACQUITY UPLC H-Class Bio instrument method. The asterisked peak (*) refers to a system peak  observed on the HPLC instrument.

A simple adjustment was made to the ACQUITY UPLC H-Class Bio System method to account for this difference, which worked to simulate a larger dwell volume. To implement this adjustment, the dwell volume was calculated for each instrument where a difference of 360 μL was determined to exist between the instruments. This value was then included as the sole change on the ACQUITY UPLC H-Class Bio instrument method as a gradient start offset volume, as shown in Figure 2.

The addition of a gradient start offset following sample injection acts to increase the length of the isocratic hold step, providing a means for simulating an instrument with a larger dwell volume. This offset does not affect void markers that may elute within the isocratic hold as the injection step occurs in a typical manner, ensuring all unretained molecules will appear at a similar position in the chromatogram. This allows for continuity in relative retention times should the reference peak(s) of interest be calculated against a void marker. As shown in Figure 1B, separation of the MassPREP peptide mixture standard following implementation of the volume correction yields nearly identical chromatographic performance to that of the original HPLC system.

Impact of a gradient start offset on trypsinized ribonuclease B

While the gradient start offset feature was able to align chromatograms generated on two separate instruments, the MassPREP peptide mixture standard contained a limited number of peaks. To challenge the applicability of this approach, a more complex sample in the form of trypsinized ribonuclease B was selected for analysis. With the gradient start offset enabled, the ribonuclease B peptide map produced a highly homologous chromatogram on the ACQUITY UPLC H-Class Bio System compared to the legacy HPLC, as shown in Figure 3. Slight differences were observed in retention times of similar peaks; however, these differences were limited to no greater than a two-second window, as shown in Table 1. Relative retention times were also calculated for all of the 33 monitored peaks on each instrument to determine the extent of comparability between the HPLC and UPLC instruments, as shown in Table 1. Peptide maps from each instrument  generated highly similar relative retention time values.

Figure 3. Trypsinized ribonuclease B peptide maps acquired on an HPLC and the ACQUITY UPLC H-Class Bio System are aligned using the gradient start offset feature. A peptide mixture generated from trypsinization of ribonuclease B was used to evaluate the robustness of the gradient start offset feature. Using the same 360 μL offset, the peptide map produced on the HPLC (A) can be generated on the ACQUITY UPLC H-Class Bio System (B) with nearly an identical separation. Peaks denoted by an asterisk (*) refer to the peak used for calculating relative retention times.
Table 1. Trypsinized ribonuclease B separated on HPLC and the ACQUITY UPLC H-Class Bio System generates nearly identical chromatographic data when the gradient start offset feature is employed. Retention times (RT) and relative retention times (RRT) were recorded for the 33 monitored peaks observed in the ribonuclease peptide map. The difference in relative retention time (D) between the HPLC and the UPLC separations was calculated and recorded.

Gradient start offset approach aligns a trypsinized monoclonal antibody peptide map

To further challenge the applicability of the gradient start offset approach, infliximab, a therapeutic monoclonal antibody, was selected for analysis as it represents a typical protein used in routine peptide mapping analyses in QC labs. A total of 56 infliximab peptide peaks were selected for monitoring between the UPLC and HPLC systems, despite approximately 90 peaks identified within each chromatographic space. Peak selection was based on a number of factors including signal intensity, elution position, and relative resolution. This approach was chosen to simplify the comparative analysis. When separated using the ACQUITY UPLC H-Class Bio System, the infliximab peptide map illustrated significant chromatographic alignment compared to its equivalent acquired on the HPLC instrument, shown in Figure 4. Assessing the relative retention times, virtually no differences were observed with values of less than 0.005 reported, as shown in Table 2. These data provide convincing evidence that employment of a gradient start offset is a suitable strategy for transferring legacy HPLC peptide mapping methods to the ACQUITY UPLC H-Class Bio System.

Figure 4. The gradient start offset feature aligns chromatograms for complex trypsinized infliximab. Infliximab was used to evaluate the performance of the gradient start offset option for dwell volume adjustment. The chromatogram generated on the HPLC system (A) demonstrates extensive similarity to the chromatogram generated on the UPLC system (B). Peaks denoted by an asterisk (*) in each chromatogram refer to the peak selected for calculating relative retention times.
Table 2. A gradient start offset produces significantly similar chromatographic data for complex monoclonal antibody peptide maps. Retention times (RT) and relative retention times (RRT) were recorded for the 56 monitored peaks observed in the infliximab peptide map. The difference in relative retention time (D) between the HPLC and UPLC separations was calculated and recorded.

Conclusion

Migration from HPLC to UPLC for large molecule-based separations represents a key challenge for the QC laboratory. While the advantages of UPLC are apparent, many labs are reluctant to make the migration due to the time required to qualify and validate both the instrument and new UPLC-based separations. Here, we have illustrated the application of the ACQUITY UPLC H-Class Bio System for HPLC-based peptide mapping. By making a simple adjustment to the legacy HPLC method in the form of a gradient start offset, nearly identical chromatograms were obtained on the ACQUITY UPLC H Class Bio System compared to the HPLC chromatogram. It is important to note that the approach outlined here should be used on a case-by-case basis as various proteins will contain peptides that respond differentially to changes in separation conditions. By using the gradient start offset to account for differences in dwell volumes between instruments, QC labs can adopt UPLC instrumentation without the need to move directly to UPLC separations, choosing instead to run legacy HPLC methods while efforts are made to transfer methods to UPLC. This enables QC labs to align instrument technology with other divisions within their organization, future-proof their own laboratories, and migrate to UPLC-based technology in a step-wise manner with minimum disruption to analytical workflows.

720004614, February 2013

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