ICH Q6B provides a set of internationally recognized principles for the characterization of biotechnology products to support the market application of monoclonal antibodies. Before monoclonal antibody drugs are marketed, they must go through a detailed monoclonal antibody characterization process to ensure quality and safety.
Determination of amino acid composition and extinction coefficient
ICH Q6b recommends the use of a variety of hydrolysis and analytical methods to determine the total amino acid content and, if necessary, to infer the amino acid composition from the genetic sequence of the desired product or natural counterpart for comparison.
In many cases, amino acid composition analysis provides some structural information about peptides and small proteins. But for large proteins, the data are often less conclusive.
Commonly used methods are reversed phase high-performance liquid chromatography (RP-HPLC) and pre-column derivatization for amino acid composition analysis, or ion exchange chromatography with post-column derivatization treatment for determining the amino acid composition of biopharmaceutical products.
Determination of amino acid sequence and peptide
The EMA guidelines and ICH Q6B require appropriate methods (for example, peptide map analysis, amino acid sequencing, mass spectrometry) to deduce the amino acid sequence of the mAb from the DNA sequence and confirm the DNA derived sequence. The N-terminal amino acid sequence (whether free amino acid or pyroglutamic acid) and the C-terminal amino acid sequence (the presence or absence of C-terminal lysine) also need to be analyzed.
Peptide mapping analysis can release mAbs to form peptides by selected proteases. The data obtained by peptide map analysis can confirm DNA-derived sequences, but not mAb light chain and heavy chain sequences. In order to provide confirmation at the primary amino acid sequence level, many protease digests need to be combined with online RP-HPLC and tandem MS/MS (LC/ES-MS/MS) analysis. ICH Q6B does not require sequencing at the primary amino acid level, but some other regulatory agencies have taken the corresponding measures.
Mass spectrometric sequencing cannot (in most cases) distinguish between isoleucine and leucine (both have the same molecular weight). Therefore, to distinguish these two amino acids, automatic N-terminal sequencing of the purified peptides in the variable regions of the light and heavy chains is required.
Determination of terminal amino acid sequence
The terminal amino acid analysis is performed to identify the amino and carboxy terminal amino acid sequences of the mAb light and heavy chains. The Edman degradation method is used for automated N-terminal sequencing. There is no completely reliable method similar to Edman's degradation method to determine the C-terminal amino acid sequence of biological products. Currently, carboxypeptidase digestion or mass spectrometry strategies can be used to obtain information related to the C-terminal sequence of a peptide or protein.
Determination of sulfhydryl and disulfide bonds
Free sulfhydryl groups and disulfide bonds should also be considered in peptide mapping analysis. After digestion with a specific protease, online LC/ES-MS or LC/ES-MS/MS analysis before/after reduction can provide the data needed for a comprehensive assessment of disulfide bonds and free radicals in mAbs.
Determination of the glycosylation structure of monoclonal antibodies
Since mAbs are glycoproteins, the glycan portion of each product must be characterized. ICH Q6B requires that carbohydrate content (neutral sugars, amino sugars and sialic acid) need to be determined for glycoproteins. In addition, the structure of the carbohydrate chain polypeptide chain, oligosaccharide pattern (sugar chain distribution) and glycosylation sites also need to be analyzed as much as possible. MALDI-TOF and high-performance anion-exchange chromatography-amperometric detection (HPAEC-PAD) can be used for the analysis.
Advanced structural analysis
The EMA and ICH guidelines recommend the characterization of the advanced structure of the mAb by appropriate physicochemical methods, including circular dichroism and nuclear magnetic resonance (NMR).
Determination of molecular weight
Online LC/ES-MS analysis of intact and reduced mAbs using quadrupole time-of-flight (Q-TOF) instrument
Determination of isomers
Use imaging capillary isoelectric focusing (cIEF) method for mAb isomer analysis
Electrophoresis and liquid chromatography
Isoelectric focusing, SDS-PAGE (before and after reduction), and capillary electrophoresis are usually used to analyze mAbs to provide product distribution based on size and charge.
Chromatographic analysis using size exclusion chromatography (SEC), RP-HPLC and ion exchange liquid chromatography can provide product characterization distribution based on size, hydrophilicity, hydrophobicity and charge distribution.
Both electrophoresis and chromatography provide information about product identity, homogeneity and purity, and are often used for purification, using the aforementioned analytical techniques for identification and structural characterization.
Protein aggregation analysis
Multiple methods must be used to evaluate the presence of polymers and polymers in each monoclonal antibody. Currently, regulators typically require cross-checking using multi-angle light scattering coupled with size exclusion chromatography (SEC-MALS) and column-free techniques, such as analytical ultracentrifugation (AUC).
Since the aggregates may be lost due to non-specific binding to the SEC column or dispersed due to the dilution and shear forces of the product on the column, column-free technology is required to confirm the SEC results.
