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