Quantification of residual hydrophobic fusion peptide with monomer and dimer forms using reversed-phase liquid chromatography

https://doi.org/10.1016/j.jchromb.2020.122073Get rights and content

Highlights

  • A simplified approach to measure the total amount of residual FP8 down to 2 µg/mL.

  • Establish a correction factor of UV peak areas between FP8 monomer and dimer.

  • Overcome the peptide low solubility issue with two-step solubilization approach.

Abstract

A fusion peptide mimicking a part of the sequence of HIV-1 envelope glycoprotein with an additional cysteine at its C-terminus (FP8: AVGIGAVFC) was conjugated to a carrier protein through a linker for development of an HIV-1 vaccine. Since this fusion peptide is very hydrophobic with poor solubility and can self-dimerize via a disulfide bond, co-existence of monomeric and dimeric forms presented a major challenge for residual unconjugated FP8 quantification. A reversed-phase liquid chromatography (RPLC) with UV detection was developed to monitor residual FP8 using an experimental correction factor of 0.85 for UV peak area measurement between FP8 dimer and monomer. Therefore, both forms of unconjugated residual FP8 can be measured based on a single FP8 monomer reference curve. Overall, this study demonstrated that the current purification process can remove free residual FP8 to a low level, <20 µg/mL, which showed negligible impact (<10%) for the conjugated FP8 ratio measurement using another method, amino acid analysis.

Introduction

An immunogenic peptide conjugated to an appropriate carrier protein can boost B-cell response to the immunogen; therefore, application of peptide-protein conjugates has been an important strategy for peptide-related vaccine development [1], [2]. An 8 amino acid peptide (AVGIGAVF) that mimics a part of the sequence of HIV-1 envelope glycoprotein with an addition of cysteine at the C-terminus (FP8: AVGIGAVFC) was conjugated to a carrier protein through a linker for the development of a vaccine candidate against HIV-1 [3], [4]. After the conjugation process, unconjugated fusion peptide and other impurities are removed during subsequent purification steps. Quantification of the residual FP8 peptide level is necessary for process clearance monitoring. Furthermore, the unconjugated FP8 peptide may affect the reported results of the peptide-to-protein conjugation ratio since amino acid analysis (AAA) applied to conjugation ratio measurement [5], [6] can only detect the total amount of FP8, including both conjugated and free FP8, in the sample matrix.

Reversed-phase liquid chromatography (RPLC) coupled with UV detection has been applied for residual quantification due to its simplicity and robustness in operation [7], [8]. However, FP8 is very hydrophobic and poorly soluble in most aqueous and organic solvents, so it was a challenge to build a FP8 reference curve for quantification in aqueous solution. In addition, a cysteine residue appended to the C-terminal of this FP8 can cause peptide dimerization over time [9]. Given the above considerations, this newly developed method needed to be able to report the total amount of monomeric and dimeric free residual FP8.

In this report, we describe how the solubility issue was addressed for FP8 monomer. In addition, through an innovative experimental design, we were able to measure the total amount of both FP8 forms (monomer and dimer) via a single FP8 monomer calibration curve using a correction factor.

Section snippets

Chemicals and reagents

The following reagents were LC-MS grade: water was purchased from Omnisolv (Billerica, MA), acetonitrile (ACN) was purchased from J. T. Baker (Center Valley, PA), and formic acid was purchased from Thermo Fisher Scientific (Rockford, IL). All other chemicals were analytical grade: dithiothreitol (DTT) was purchased from G Bioscience (St. Louis, MO), trifluoroacetic acid (TFA) was purchased from Thermo Fisher Scientific (Tempe, AZ), dimethyl sulfoxide (DMSO) was purchased from J. T. Baker, while

Dissolving hydrophobic FP8 monomer

The FP8 (monomer) lyophilized powder did not dissolve completely in most commonly used organic solvents, such as methanol, acetonitrile (with or without 0.1% TFA or formic acid) and isopropanol, or in aqueous solutions, such as water, PBS and Tris buffer (pH 7.6), except in 100% formic acid. However, dissolving FP8 in formic acid produced a split peak for FP8 via RPLC-UV chromatogram and was therefore deemed not suitable for quantification. DMSO was the only solvent capable of completely

Conclusions

In summary, a fit-for-purpose RPLC-UV assay was developed to measure the total amount of residual free FP8, including both monomeric and dimeric forms in the FP8-protein conjugate samples. We were able to overcome the low solubility issue for this highly hydrophobic peptide FP8 monomer by a two-step solubilization approach. Due to the unavailability of FP8 dimer standard, a conversion factor of 0.85 was applied for FP8 dimer measurement against a single standard curve of FP8 monomer. Overall,

CRediT authorship contribution statement

Cindy X. Cai: Conceptualization, Methodology, Validation, Formal analysis, Writing - original draft. Nicole A. Schneck: Conceptualization, Methodology, Validation, Formal analysis. Vera B. Ivleva: Writing - review & editing, Supervision. Krishana Gulla: Resources, Writing - review & editing. Yaqiu Zhang: Resources. Daniel Gowetski: Data curation, Project administration. Q. Paula Lei: Conceptualization, Data curation, Writing - review & editing, Supervision, Project administration.

Acknowledgment

The authors are grateful to Jason Gall, Kevin Carlton, Peter Kwong, Chris Barry and Nathan Barefoot for their organizational and scientific support. This work was supported by the intramural research program of the Vaccine Research Center (VRC), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH).

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