Quality Control of Glycopeptides for Diagnostic Reagents: What Matters?

Why Analytical Characterization Is Critical in Diagnostic Reagent Quality?

(1) Assay Sensitivity and Specificity Depend on Glycopeptide Purity

The Glycopeptide (GP) produced for diagnostic reagents requires analytical characterization to verify its high purity level. Achieving high sensitivity and specificity in the assay relies on the use of GP with high purity. The higher the impurity level of GP, the more background and cross-reactivity there will be, resulting in less accurate and reliable results. Diagnostic assay performance suffers due to impurities that disrupt antibody binding which causes false positive or negative results. For these reasons, methods such as high-performance liquid chromatography (HPLC) and mass spectrometry (MS) are often used to confirm the purity and structure of the GP and meet the rigorous requirements for diagnostic reagents. These techniques allow for a more comprehensive analysis of GP composition by identifying and quantifying impurities that can affect the assay's performance. For example, A study reported the generation of a high purity of cyclic V1V2 glycopeptides containing defined N-linked glycans at the conserved glycosylation sites (N160 and N156/N173) using gp120 from two HIV-1 isolates. Binding studies with antibodies confirmed the requirement for a Man5GlcNAc2 glycan at N160 for PG9 and PG16 recognition and also identified a critical role for a sialylated N-glycan at the secondary glycosylation site (N156/N173) in the context of glycopeptides for antibody binding in diagnostic assays.

(2) Regulatory and Reproducibility Impacts of Structural Consistency

GPs utilized as diagnostic reagents require structural consistency to function properly. FDA, EMA and other regulatory agencies have very strict guidelines with respect to consistency and characterization of reagents for diagnostics. Depending on how the GP functions as a diagnostic, it may be required that the GP be structurally identical from batch to batch, as a diagnostic must be reproducible. Comprehensive structural information from analytical methods could potentially demonstrate that a GP meets certain specifications and will perform as expected in a diagnostic assay. Information such as this can provide the confidence that one batch of GPs is equivalent to the next, which is necessary for high quality consistent diagnostics. Glycan profiling and peptide sequencing are two analytical methods that can provide this detailed structural information. Consistent GP structure may also be important for reproducibility of diagnostic results in a regulated setting. Structural variation in GPs may result in variability in the performance of diagnostic assays, which may not be acceptable in a regulated setting.

Key Analytical Methods for Glycopeptide-Based Reagents

(1) LC-MS/MS for Molecular Identity

A primary molecular identification approach for GPs in diagnostic reagents utilizes liquid chromatography with tandem mass spectrometry (LC-MS/MS). This technique also allows detection and characterization of the complex structure of GP. The separation can also be used to separate GPs based on their hydrophobicity or hydrophilicity, which is then followed by MS analysis for determination of the molecular identity. Through LC-MS/MS analysis researchers can map GP glycosylation sites and characterize their glycan structures. LC-MS/MS can distinguish diverse glycoforms among peptides to help understand GP biological functions and diagnostic applications. Biological samples can be analyzed by LC-MS/MS for the identification and quantification of GPs, which in turn can reflect disease states and potential biomarkers. In addition to ionization methods like electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), LC-MS/MS has an even greater capability for detection. ESI-MS is compatible with LC-MS/MS, and it also forms multiply charged ions, which in turn results in a higher fragmentation efficiency for MS/MS analysis. LC-MS/MS with ESI can be used for in-depth GP characterization, including glycan composition and attachment sites.

Fig. 1 Workflow for the LC-MS analysis and glycan nomenclature.^1,2

(2) HILIC and RP-HPLC for Glycan and Peptide Purity

Hydrophilic interaction liquid chromatography (HILIC) and reversed-phase high-performance liquid chromatography (RP-HPLC) have been utilized in the characterization of the purity of GPs. Researchers utilize HILIC and RP-HPLC techniques to differentiate and purify GP molecules through their hydrophilic or hydrophobic properties. These methods enable researchers to measure impurities within GP samples that might otherwise affect diagnostic tests. The glycan composition of GPs can be distinguished by HILIC which reveals the heterogeneity of glycan structures within a GP sample. HILIC proved effective in the separation of GPs with varying glycan structures. RP-HPLC can be used to separate and purify GPs based on their hydrophobicity. The hydrophobicity of a GP is largely determined by the peptide portion of the molecule. RP-HPLC is a widely used technique for the purification and analysis of peptides and proteins, including GPs. RP-HPLC can be used to separate GPs with the same peptide sequence but different glycan structures. This can help to ensure that the final GP product is free from impurities. Both HILIC and RP-HPLC can be coupled with MS to provide a more detailed characterization of GPs.

Fig. 2 Analysis of pre- and post-operative N-glycans in CRC by RPLC-FD-MS.^3,4

(3) Structural Validation by NMR or Capillary Electrophoresis

Structural validation is one of the important strategies for quality assurance of GPs in diagnostic reagents. Nuclear magnetic resonance (NMR) spectroscopy is a structural validation method that can be used for the characterization of GPs. Using NMR spectroscopy scientists obtain details about the complete GPs structure which includes glycan residue identification and connectivity as well as linkage configuration between residues. NMR serves to verify that GPs are structurally identical to their intended designs by confirming their identity and structure. NMR spectroscopy enables researchers to examine the structural conformation and dynamic behavior of GPs which yields important information about their biological roles and diagnostic applications. Capillary electrophoresis (CE) is a separation technique that can be used to analyze GPs with different glycan structures. CE achieves separation of analytes through their charge-to-size ratio while delivering high-resolution and high-sensitivity analysis. CE enables researchers to analyze both purity levels and structural consistency of GPs while offering vital detection tools for low-abundance GPs which hold significant value in diagnostic applications. Both NMR and CE can provide complementary information on the structure and purity of GPs, and can be used to ensure that diagnostic reagents meet the highest standards of quality and reliability.

Our QC Offerings for Diagnostic Glycopeptides

(1) Standard vs Enhanced QC Packages Based on Application

The QC of GPs can be extensive and can be adjusted to your needs. We offer both standard and extensive QC packages. This includes GPs in general, but also diagnostic GPs. The QC package can be adjusted per your requirements for your application. This means we can give you the relevant analytical data for your GP, so you have the correct quality and consistency of your GPs for your applications. We offer a standard QC package which is tailored to give you the basic but essential quality control. The analytical methods in this package are HPLC, to determine purity, MS for molecular identity and monosaccharide analysis to determine glycan composition. Together this gives you an essential and broad overview of the quality of your GPs. We also offer an extensive QC package which can be adjusted to your needs for your application. In addition to the standard package, this includes extra analysis to fully validate your GPs such as NMR and CE for further structural and purity validation, and extended stability and functionality tests under different conditions.

(2) Certificates of Analysis with Every Batch

Certificates of analysis represent essential tools which serve regulatory purposes and quality assurance needs. A COA is a means to verify the identity, potency, and purity of diagnostic reagents. Here at Creative Peptides, we provide a COA with each batch of GPs we produce. A COA is a certificate that provides information about the identity, potency, and purity of a specific batch of compounds. It is used to track and trace the GPs, as well as to ensure that they meet the required specifications. Our COAs include extensive information from our QC analyses such as HPLC and MS analyses that validate the GP's purity and molecular identity, as well as structural and compositional information about the glycan. This not only ensures batch-to-batch consistency of the GPs, but also gives researchers confidence that their diagnostic reagents are the highest quality.

Glycoamino acids we can provide

References

1. Image retrieved from Figure 1 "Workflow for the LC-MS analysis and glycan nomenclature," Reyes C D G.; et al., used under [CC BY 4.0](https://creativecommons.org/licenses/by/4.0/). The original image was not modified.
2. Reyes C D G.; et al. "LC-MS/MS isomeric profiling of N-Glycans derived from low-abundant serum glycoproteins in mild cognitive impairment patients." Biomolecules, 2022, 12(11): 1657.
3. Image retrieved from Figure 1 "Analysis of pre- and post-operative N-glycans in CRC by RPLC-FD-MS," Reyes C D G.; et al., used under [CC BY 4.0](https://creativecommons.org/licenses/by/4.0/). The original image was not modified.
4. Moran A B.; et al. "Serum N-glycosylation RPLC-FD-MS assay to assess colorectal cancer surgical interventions." Biomolecules, 2023, 13(6): 896.
5. Mastrangeli R, Satwekar A, Bierau H. Innovative Metrics for Reporting and Comparing the Glycan Structural Profile in Biotherapeutics[J]. Molecules, 2023, 28(8): 3304. https://doi.org/10.3390/molecules28083304.
6. Chia S, Teo G, Tay S J, et al. An integrative glycomic approach for quantitative meat species profiling[J]. Foods, 2022, 11(13): 1952. https://doi.org/10.3390/foods11131952.