• Application Note

Future-proofing the Biopharmaceutical QC Laboratory: Integrating Auto•Blend Technology to Improve Routine Peptide Mapping

Future-proofing the Biopharmaceutical QC Laboratory: Integrating Auto•Blend Technology to Improve Routine Peptide Mapping

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

Abstract

This application note demonstrates the ability of Auto•Blend Technology to control the trifluoroacetic acid (TFA) component of the mobile phase during routine peptide mapping analyses.

Benefits

  • Auto•Blend Technology for acidic modifier control
  • Transfer peptide map applications from HPLC to UPLC
  • Future-proof laboratory for UPLC methods

Introduction

As an initial step towards transferring peptide mapping methods from HPLC to UPLC, we previously presented an approach using the ACQUITY UPLC H-Class Bio System for legacy HPLC-based peptide mapping.1 Our method transfer discussion continues here, focusing on improving the consistency of peptide mapping separations during routine analyses.

Peptide mapping methods generally include an acidic modifier to improve peak shape. However, accurate and reproducible management of the modifier content within mobile phase solvents can be variable, consequently affecting the peptide map quality and reproducibility.

This application note demonstrates the ability of Auto•Blend Technology to control the trifluoroacetic acid (TFA) component of the mobile phase during routine peptide mapping analyses, thereby producing chromatograms of equivalent performance to HPLC-acquired chromatograms with conventionally modified mobile phases.

TFA is commonly used as a modifier in peptide mapping methods with optical detection because it provides peak shape and chromatographic resolution benefits. Concentrations of TFA are typically low in most applications, accounting for 0.02% to 0.20% of the final mobile phase. Subtle changes in the modifier concentration can have profound effects on peptide retention time, resolution, and elution order, causing concern over chromatographic reproducibility and the occurrence of out-of-specification results. Such issues ultimately affect productivity due to time-consuming resolution of QC issues as opposed to moving product to the marketplace.

The reason for this classical approach using TFA in peptide mapping chromatography has been to modify the two mobile phases, normally water and acetonitrile, with a pre-determined amount of TFA. Here, we demonstrate the benefits of allocating TFA to an independent solvent line using Auto•Blend to manage its contribution to the solvent composition throughout gradient delivery. The result is not only consistency in chromatographic performance but a significant benefit in terms of chromatographic reproducibility with minimal solvent preparatory requirements. Auto•Blend Technology in peptide mapping enables QC labs to spend less time in the prep labs, instead focusing on driving productivity.

Experimental

Sample Preparation

Two peptide preparations were used in this study: Ribonuclease B (Sigma Aldrich, USA), and infliximab, both prepared as follows. Five hundred μg of protein was reduced with dithiothreitol, alkylated with iodoacetamide, and isolated using NAP-5 columns (GE Healthcare, PA, USA). Sequence-grade trypsin (Promega, CA, USA) was added to each protein to a final composition 1:20 enzyme/ substrate with samples subsequently digested overnight at 37 °C. Following digestion, trypsin was deactivated by incubation at 70 °C for 15 minutes, and 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.

UPLC conditions

System:

ACQUITY UPLC H-Class Bio with Tunable UV Detector with 10-mm titanium flow cell

Extension loop:

100 μL (p/n 430002625)

Mixer:

250 μL (p/n 205000737)

Column:

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

Column temp.:

40 °C

Injection volume:

95 μL

Mobile phase A:

Water

Mobile phase B:

Acetonitrile

Mobile phase C:

1% (v/v) TFA in water

Detection wavelength:

214 nm

Gradient

Time (min)

Flow rate (mL/min)

%A

%B

%C

%D

Curve

0.500

85

5

10

0

6

5.00

0.500

85

5

10

0

6

45.00

0.500

40

50

10

0

6

47.50

0.500

0

90

10

0

6

52.50

0.500

0

90

10

0

6

52.60

0.500

85

5

10

0

6

60.00

0.500

85

5

10

0

6

Results and Discussion

Evaluation of TFA effect on peptide retention time

To illustrate the impact of changing TFA concentration on peptide retention time, a series of trypsinized Ribonuclease B peptide separations were performed with a TFA concentration ranging from 0.08% to 0.10% in 0.01% increments. The resulting chromatograms (Figure 1) show significant differences in peak retention times with changes in TFA concentration of 0.01%. Changing TFA concentrations is observed to affect not only the peak retention time but also selectivity, where various peaks are observed to change elution order based on TFA concentration (Figure 1). Based on such sensitivity to TFA concentration, accurate and reproducible preparation of solvents containing TFA must be considered critical for improving consistency in peptide mapping.

Figure 1. Effect of TFA concentration on peptide separation. Trypsinized Ribonuclease B peptides were separated with varying amounts of TFA ranging from 0.08% to 0.10%. Changes of as little as 0.01% TFA resulted in changes in peak retention times. Peaks within green enclosures indicate a loss of resolution while peaks contained within blue enclosures illustrate an increase in resolution. (A) 0.08% TFA. (B) 0.09% TFA. (C) 0.10% TFA. Adjustments to TFA concentration were obtained using Auto•Blend Technology.

Using Auto•Blend Technology to automate accurate delivery of TFA One approach for eliminating TFA concentration variability is to remove the additive as a component of each mobile phase, instead providing a stock concentration of TFA as its own solvent line. This results in three solvents contributing to a peptide map gradient, all of which can be accurately managed using Auto•Blend. To demonstrate the use of Auto•Blend in peptide mapping using this solvent arrangement, 1% TFA in water was prepared and configured on solvent line C beside MilliQ dH2O and acetonitrile as solvents A and B, respectively. Evaluation of Auto•Blend Technology for controlling TFA in peptide mapping was performed using trypsinized infliximab as a model protein therapeutic.

Previous peptide mapping of trypsinized infliximab monitored a total of 56 peaks.1 For comparative purposes between the HPLC and ACQUITY UPLC H-Class Bio instruments, and instrument methods (with or without Auto•Blend), the same 56 peaks were monitored. Trypsinized infliximab was separated using either a standard configuration of solvents modified with 0.1% TFA or in an Auto•Blend configuration with three solvents (pure acetonitrile, pure water, and 1% formic acid in water) used for gradient formation. Each of these configurations was compared to the legacy method generated on an HPLC instrument (Figure 2a).

In the conventional mobile phase delivery using the ACQUITY UPLC H-Class Bio System, comparable chromatography can be observed to that obtained on the HPLC instrument (Figure 2b). Using the Auto•Blend configuration with 1% TFA as a separate solvent line, no difference in selectivity and nearly identical retention times are observed (Figure 2c). Importantly, no difference was observed between the chromatogram obtained using Auto•Blend Technology and the chromatogram obtained on the HPLC instrument; a result that supports the use of ACQUITY UPLC H-Class Bio and Auto•Blend for running legacy HPLC peptide mapping methods.

Figure 2. Incorporation of Auto•Blend Technology for peptide mapping produces chromatograms comparable to conventional solvent. The challenge for Auto•Blend control of multiple solvent lines is to replicate chromatographic performance of a legacy HPLC peptide mapping method. A trypsinized infliximab peptide map was produced on an HPLC using a legacy HPLC method with the acidic modifier as a component of two solvents (A), establishing the benchmark for separation on the ACQUITY UPLC H-Class Bio System. The same approach for solvent use was set up on the ACQUITY UPLC H-Class Bio System to establish comparability to the HPLC chromatogram (B). TFA was then removed as a component and provided its own solvent line, where the resulting chromatography was found to be significantly comparable to both the HPLC method and the HPLC method run on the ACQUITY UPLC H-Class Bio System.

Adoption of Auto•Blend Technology for peptide mapping methods allows more consistent mobile phase composition and delivery, which ultimately benefits chromatogram reproducibility over wider time spans and reduces analyst bench time preparing stock solvents adjusted with acidic modifiers.

Auto•Blend control of TFA results in reproducible peptide mapping

For QC labs performing peptide mapping on a routine basis, reproducibility and reliability are key factors that ultimately drive productivity. To determine the reproducibility, and hence consistency, of the ACQUITY UPLC H-Class Bio System with Auto•Blend for peptide mapping applications, five injections of trypsinized infliximab were run on the ACQUITY UPLC H-Class Bio System. Results of the evaluation demonstrated comparable chromatograms with standard deviations no greater than 0.011 (Figure 3a and Table 1). This finding was confirmed by measuring the relative peak area across each chromatogram, where low standard deviation was found with the relative peak area of each monitored peak (Figure 3b).

Measurement of reproducibility within a peptide map is best reported in terms of percent relative standard deviation (%RSD) of peak retention times. The performance of the ACQUITY UPLC H-Class Bio System in terms of retention time reproducibility was also evaluated through determination of relative standard deviation (RSD) with calculated retention times indicating a maximum %RSD value of 0.089% (Table 1). This represents a value significantly lower than that required by existing regulatory guidelines. Despite the inclusion of an additional solvent line, Auto•Blend Technology demonstrated a capacity to generate highly consistent and reproducible chromatograms in both retention time (Figure 3a) and peak area values (Figure 3b).

Figure 3. Auto•Blend Technology provides accurate and consistent reproducibility for peptide mapping on the ACQUITY UPLC H-Class Bio System. To ascertain the reproducibility of the ACQUITY UPLC H-Class Bio and Auto•Blend Technology, a total of five injections were performed with retention times tabulated for a total of 56 common peptide peaks identified in the infliximab peptide map. The resulting five chromatograms were overlayed to illustrate the reproducibility of a separation using Auto•Blend (A). Reproducibility of relative peak area was also assessed, with average relative peak areas and associated standard deviations provided as a column chart (B).
Table 1. Auto•Blend control of TFA delivery generates highly reproducible retention times. Five individual injections of trypsinized infliximab were separated on the ACQUITY UPLC H-Class Bio System using Auto•Blend Technology, where retention times of 56 peaks were monitored. Relative standard deviation (%RSD) was calculated across all chromatograms with a maximum value of 0.089 determined.

Conclusion

Reliability, robustness, and reproducibility are cornerstones of QC laboratories. For routine analysis of complex peptide maps, LC instrumentation needs to generate consistent chromatography over extended periods of time to adhere to specifications outlined in SOP documents. Modification of mobile phases with acidic modifiers introduces the potential for loss of reproducibility due to sensitivity of peptide maps to subtle changes in modifier concentration. Auto•Blend Technology using the ACQUITY UPLC H-Class Bio System circumvents this issue by simplifying solvent preparation and automating the formulation of the peptide map mobile phase throughout gradient delivery.

As a result, Auto•Blend improves the reproducibility of complex peptide separations, thereby reducing time dedicated to reviewing instrument-related separation issues.

References

  1. Cosgrave EFJ, McCarthy SM. Future-proofing the Biopharmaceutical QC Laboratory: Using the ACQUITY UPLC H-Class Bio System for HPLC Peptide Mapping. Waters Application Note 720004614EN. 2013 June.

720004742, June 2013

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