Overcoming Challenges in Protein Molecule Analysis
In general, the main challenge in protein molecule analysis is that there is no single and universal method or approach for separation and characterization of proteins. As a first choice, peptide mapping methods are a preferred technique for the comprehensive characterization of biopharmaceutical products. Its applications include:
Identification of proteins based on the elution pattern of peptide fragments
Determination of post-translational modifications
Confirmation of genetic stability
Analysis of protein sequences when interfaced to a mass spectrometer.
In a peptide map, it is necessary to resolve each peptide fragment into a single peak. Therefore, peptide mapping represents a significant chromatographic challenge due to the inherent complexity of protein digests. In addition to the large number of peptide fragments that are generated from the enzymatic digestion of a protein, the number of alternative peptide structures (e.g., post-translational modifications, oxidations, etc.) can be significant. The comprehensive characterization of protein-based polymers as drug candidates also is a requirement for safety and for regulatory agencies. Challenges include the high molecular weight of biopolymers and the heterogeneous nature of protein drugs which require extensive characterization to achieve regulatory approval.
To better characterize the physiological functions of a therapeutic protein, knowledge of their expression patterns is essential. Post purification analysis is a difficult and time consuming process, typically requiring multiple orthogonal methods such as a hydrophilic interaction chromatography (HILIC), reversed-phase HPLC (RP-HPLC), gel filtration chromatography (GFC), capillary electrophoresis (CE), Western Blot, SDS-PAGE or ELISA adding significant cost and burden to an analytical laboratory. Furthermore, these conventional approaches are not able to resolve and quantitate many process-related impurities and degradent products that exist after primary purification of the protein.
In order to overcome these challenges, Ultra Performance Liquid Chromatography (UPLC) technology is a good alternative because it has demonstrated superior resolution and higher sensitivity for protein separation and peptide mapping as compared to traditional HPLC detection. The combination UPLC-UV-SEC and electrospray mass spectrometry UPLC-TQD can assist with the analysis of proteins and peptides and provides high levels of detail to aid characterization.
Additionally, a major hindrance has been, until now the lack of automated tools to complete the analysis. Therefore, the ability to routinely generate and interpret LC-MS data in timely manner has been challenging in the past because involvement of an academic expert was required. With newly designed combined methods using UPLC-UV, UPLC-SEC and UPLC-MS/MS detection can provide more sensitive, high performance, accurate and reproducible data analysis. The automated calibration and system monitoring, provides the very best chromatographic system performance in routine protein analysis. Peptide mapping methods developed in this manner dramatically reduce analytical testing time and effort compared to traditional Gel Electrophoresis methods. This methodology allows for expedited confirmation that the correct protein has been produced. The efficient use of UPLC-UV, GFC and MS scan of the samples assures that the specific protein fragments and overall mass matches the expected retention times and mass. Without this confirmation, the wrong protein may be tested in several expensive assays abd bioassays, increasing time and costs.
Many of the challenges facing researchers in the proteomics and biopharmaceutical fields are related to the need to obtain as much information as possible on very limited samples. UPLC technology with BEH columns address these challenges.
Protein molecules may aggregate simply by physical association with one another without any changes in primary structure (physical aggregation) or by formation of a new covalent bond(s) (chemical aggregation). Formation of such a bond(s) can either directly cross-link proteins (aggregation), or indirectly alter the aggregation tendency of the original protein. Both mechanisms can occur simultaneously in a protein during preparation that can lead to formation of either soluble or insoluble aggregates. Recombinant proteins can undergo covalent or non-covalent association that is highly dependent on the solution conditions, including pH, ionic strength and excipients. The protein solutions which undergo oxidation also lead to aggregation.
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