Peptide pools are versatile tools in the development of immunotherapeutic solutions. The quality of the peptide pools is crucial for the accuracy of immunotherapies, but the quality control of these complex products poses a significant challenge. Gaby Bosc-Bierne and team at the Federal Institute for Materials Research and Testing (BAM) and peptides&elephants, both in Berlin, Germany, evaluated the effectiveness of ultra-high performance liquid chromatography (UHPLC) coupled with two different detectors. [1] They used the peptide pool CEF advanced from peptides&elephants for this approach to establish a cost-efficient and swift quality control method for peptide pools in immunotherapy.   

Quality control of peptide pools poses a significant challenge due to their complex composition, which sometimes includes more than a hundred individual peptides. In order to expand tumor-specific T cells for immunotherapy, each of these peptides must be verified to be present in the peptide pool. Potential synthesis errors, degradation processes, adsorption losses, dimerizations, and other unwanted chemical changes must be rigorously excluded. Moreover, the pool must not contain any unintended peptides.

Advantages and disadvantages of peptide quantification methods

Peptide quantification is typically performed using (U)HPLC-UV, (U)HPLC-MS/MS, or capillary electrophoresis mass spectrometry (CE-MS). Each method has its advantages and disadvantages when it comes to quality control of peptide pools. HPLC-UV is not ideal for complex samples due to a frequent lack of baseline separation, and synthesis byproducts and degradation products can further complicate matters. (U)HPLC-MS/MS is more suited to complex samples, offering excellent selectivity and serving as the gold standard for quantification. It can also confirm the identity and indirectly the structure of the analytes. However, ionization efficiency can vary among compounds in mass spectrometric methods, and co-eluting substances can suppress ions in the ion source, leading to matrix effects that can make individual peptides unidentifiable. While reduction or compensation strategies can mitigate matrix effects, they may not be practical due to high calibration efforts or the use of expensive isotope-labeled analyte analogues. 

UHPLC with two different detectors

Given these challenges, there is a need to develop enhanced methods for peptide pool quality control. Bosc-Bierne and her team have explored the potential of employing ultra-high performance liquid chromatography (UHPLC) equipped with two different detectors for cost-effective and rapid quality control of peptide pools. They utilized a standard UV detector at 214 nm for quantitative analysis and a high-resolution mass spectrometer (HRMS) for identity confirmation, achieving quantification and identification in a single chromatographic run. The team conducted their investigations with the frequently used CEF advanced peptide pool from peptides&elephants, which contains 32 peptides from cytomegalovirus, Epstein-Barr virus, and influenza virus.

Separation protocol and overlapping peak areas

The findings of the study indicate that a flat gradient, increased temperature, and trifluoroacetic acid at a concentration of 0.05/0.04% were beneficial for the chromatographic separation of the peptide pool. Applying this optimized separation protocol, 30 out of the 32 peptides in the pool were relatively quantified using UHPLC-UV/HRMS. The remaining two peptides were identified by their masses but not quantified due to poor peak shape. Several peaks in the experimental setup overlapped significantly, making it difficult to determine UV peak areas. However, when using simple perpendicular drop integration, relative quantification was possible by grouping peaks. More computationally intensive peak fitting methods could be employed in the future as an alternative.

Efficient for medium sized peptide pools

In summary, the study demonstrates that cost-effective and time-efficient quality control of synthetic peptide pools is possible, when using UV detection for relative quantification and high-resolution mass spectrometry for peptide confirmation in just one chromatographic run. The proposed method significantly improves the quality control of peptide pools in immunotherapy, particularly for pools up to medium size.

Peptides&elephants is currently investigating whether the method is also suitable for larger peptide pools. In addition, the scientists are refining the method by utilizing more recent UHPLC systems with enhanced resolution and longer columns that allow for longer separation times to increase peak capacity. The implementation of chromatography optimization software and artificial intelligence are further advancements that could contribute to optimizing separation and integration methods, as well as analyzing complex mass spectra.

Literature

1. Bosc-Bierne G, Ewald S, Kreuzer OJ, Weller MG. Efficient Quality Control of Peptide Pools by UHPLC and Simultaneous UV and HRMS Detection. Separations. 2024; 11(5):156. https://doi.org/10.3390/separations11050156