Application in the Biotech Industry
The characterization of therapeutic proteins is an important analytical activity during process development and manufacturing. Product characterization is a requirement for precise recognition of a therapeutic protein’s biochemical structure. Biochemical characterization of therapeutic proteins is carried out using a range of techniques that fingerprint the native structure, conformation, purity, and product-related impurities and contaminants.
These techniques include peptide mapping using LC/MS for primary structure identification, MALDI-TOF-MS for molecular mass determination, and circular dichroism or FTIR for secondary structure analysis. Product-purity assays include size-exclusion chromatography and SDS-PAGE. Product isoform and immunodetection techniques such as isoelectric focusing and Western blot are also routinely used. Impurities such as host cell proteins are analyzed using ELISA and/or HCP Western blot.
At Lonza Biologics (www.lonza.com), we evaluated the application of dispersive Raman spectroscopy to determine the Raman spectral profile of a therapeutic protein. The spectra were further processed to estimate secondary structure composition of the therapeutic protein.
Specifically, we assessed dispersive Raman spectra for a therapeutic mAb (Figure 3). The spectra can be analyzed at several levels of detail and can be used to compare different therapeutic protein batches visually. The protein stability profile across time and forced degradation processes for therapeutic proteins can be analyzed in a timely manner.
The spectra can also be used for a detailed analysis to compare the structural components that have Raman vibrational modes, such as aromatic amino acids. These amino acids carry important structural details in terms of stability and integrity of the proteins. Therapeutic protein denaturation and structural perturbation can be fingerprinted in a Raman spectrum at the expense of changes in aromatic amino acids and compositional shifts to the secondary structures.
Tentative peak assignments constituting the amide I region of a mAb Raman spectrum are shown in Figure 3. The profile is significant in terms of the number of peaks obtained for the mAbs and the descriptive nature of the technique. The peaks are remarkable structural markers that are ideal for detailed and efficient characterization of novel therapeutic proteins.
The amide I region of a mAb was peak fitted and deconvoluted to generate underlying conformational information of the therapeutic protein. b-sheet was the main secondary structure component of the mAb. Other secondary structures identified included a-helix, loose b-sheet, and other structures.
Dispersive Raman spectroscopy can also be used to address the increased demand for more efficient product-development and manufacturing processes. This methodology has the potential to be applied as a PAT tool for the purpose of determining protein conformation or other process-related analytes at-line or in-line during the upstream or downstream process stages.