Novel Identification of mAbs by Raman Spectroscopy
Maoqin Duan1, Jun Liu2, and Lan Wang1,* Jialiang Du1,Jialing,Zhang1,
Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech, Division of Monoclonal Antibody Products, National Institutes for Food and Drug Control, Beijing 102629, China;
Thermo Fisher Scientific (China) Co., Ltd; 8011867@qq.com(J.L);
* Correspondence:wanglan@nifdc.org.cn (L. Wang)
Abstract: The identification of the critical quality attributes (CQAs) of monoclonal antibodies (mAbs) is a key component of quality by design. Traditional detection methods for the identification of mAbs, such as peptide mapping and weak cation-exchange chromatography (WCX-HPLC), require sophisticated equipment, experienced staff, and a considerable amount of time. In this study, the novel identification of mAbs was performed using Raman spectroscopy, combined with the evaluation of other CQAs, such as the appearance and purity of size-exclusion high-performance liquid chromatography(SEC-HPLC), charge heterogeneity of WCX-HPLC, and bioassay. Raman spectroscopy achieved comparable results to the conventional, but complex, approach to identifying anti-vascular endothelial growth factor antibodies. Raman spectroscopy was used to identify and distinguish between different antibody types. Additionally, the Raman technique with principal component analysis of multivariate algorithms is rapid, efficient, and accurate for mAb identification; this technique has great potential to support biopharmaceutical development and counterfeiters.
Keywords: Raman spectroscopy; identification; monoclonal antibody; appearance; SEC-HPLC; CEX-HPLC; bioassay; PCA
1. Introduction
The first monoclonal antibody (mAb) was licensed in 1986, and mAb therapeutics have become a predominant treatment modality to cure various diseases in fields such as hematology, oncology, and immunology [1,2]. The mAb market is growing rapidly. Biopharmaceutical companies have gradually developed a scientific and risk-based quality by design approach. A key concept of quality by design is to identify the critical quality attributes (CQAs) during early drug production and process development. Post-translational modifications [3,4], charge variants, and N-linked glycosylation profile of the mAb primary structure are CQAs. The potential CQA identity confirmation is a critical component of quality control and quality assurance used in manufacturing, distribution, and release of drug substances and drug products [5,6]. Peptide mapping is a bottom-up approach to analyzing enzymatically digested peptide mixtures of biotherapeutics, using a separation technique coupled with mass spectrometry or ultraviolet light detection, to identity the primary structure of biotherapeutic products [7-9].
However, all techniques require a considerable amount of time to separate the peptides. The charge variants of biotherapeutics are usually characterized by ion exchange chromatography [10], capillary isoelectric focusing [11], and capillary zone electrophoresis [12], with optical detection. However, these identification techniques depend on the reference migration times. Consequently, quick tools are required for analysis to expedite the investigations [13].
The analytical technology of Raman spectroscopy is nearly 100 years old [14]. In1928, the Indian physicist CV Raman illuminated a benzene liquid with a mercury lamp and found that the light elastically collided through the medium, i.e., the scattering frequency changed; this phenomenon was named Raman scattering [15], which led to the award of the Nobel Physics Prize to CV Raman in 1930. Raman spectroscopy is a spectroscopic analysis technique that enables rapid quantitative and qualitative analysis based on inelastic collision and Raman scattering intensity [16,17].
Raman spectroscopy has several advantages and is useful for non-destructive, simple, rapid, efficient, and accurate detection [18,19]. Therefore, Raman spectroscopy has been applied to the field of drug analysis over the past decade, including the identification of active pharmaceutical ingredients, qualitative and quantitative analysis of pharmaceutical preparations, detection of illicit and chemical drugs, identification of Chinese herbal medicines, and combination with other technologies [20].
MAbs, like other therapeutic proteins, are sometimes exposed to temperature variations during their processing, storage, and transportation. High temperatures can perturb native protein conformation to a degree sufficient to promote aggregation, and they can also accelerate chemical reactions such as deamidation and oxidation. Protein medications are exposed to light at many points during their life cycle, and their aromatic residues are especially sensitive to light. Exposure to light can thus induce photodegradation, mainly through photooxidation and the formation of oxygenated radicals, but also through fragmentation and crosslinking. Antibodies containing a large number of aromatic residues (especially tryptophan residues) are particularly susceptible to this phenomenon. Therefore, it is important to avoid high temperatures and light during the transportation and preservation of antibodies [21].
Raman spectroscopy has been used for identification of the formulated protein products, such as mAbs [22]. The widespread use of Raman spectroscopy for this purpose is due to the high level of molecular specificity, compatibility with aqueous solutions, and ability to acquire spectra of materials within seconds to minutes with little or no sample preparation. Time for analysis can be reduced by performing an initial screening of the sample activity. Therefore, in the present study, the accelerated degradation of anti-VEGF antibody was induced through high temperatures and exposure to light, and data on the accelerated degradation of the antibody were collected using a handheld Raman spectrometer and verified based on other key indicators [23].
2. Materials and Methods