C-terminal lysine clipping is a common phenomenon occurring during the bioproduction of mAbs and leads to variable amounts of final process-related charge variants. The methods used in this study can be widely applied to other IgG1 to define criticality of the C-terminal lysine clipping as a CQA.
What is charge variants in Mab?
The existence of multiple variants with differences in either charge, molecular weight or other properties is a common feature of monoclonal antibodies. These charge variants are generally referred to as acidic or basic compared with the main species.
What is antibody heterogeneity?
The concept of the heterogeneity of antibodies – that the antibody formed even to a single. antigen is not homogeneous but consists of populations of molecules with different physical, chemical or immunochemical properties – goes back in time to the discovery of cross reactions.
What is charge heterogeneity?
Charge heterogeneity is often evaluated during biosimilar development as it is a universal feature of monoclonal antibodies (mAbs). A common approach in the industry is to develop a biosimilar product with a similar overall charge profile as the reference product.
What is Cex HPLC?
We reasoned that a cation exchange (CEX) chromatography based HPLC method, capable of analyzing large molecules or particles based on their differences in surface charge would provide a starting point for such an assay. CEX separates ionic molecules based on their affinity with the cation exchanger in the column.
What is iCIEF?
Imaged capillary isoelectric focusing (iCIEF) is a powerful technique used in many biopharmaceutical laboratories responsible for evaluating the charge heterogeneity of proteins. Bottlenecks occur in the manual preparation of different samples and variability of test results is introduced by the technician.
Are monoclonal antibodies heterogeneous?
Heterogeneity of monoclonal antibodies is common due to the various modifications introduced over the lifespan of the molecules from the point of synthesis to the point of complete clearance from the subjects. The vast number of modifications presents great challenge to the thorough characterization of the molecules.
Are monoclonal antibodies naturally occurring?
These monoclonal molecules, termed naturally occurring antibodies (NAbs),1 are immunoglobulins present in the serum of healthy individuals in the absence of antigenic stimulation by foreign antigens.
What is a charge variant?
Charge variants are usually characterized by preparative ion exchange chromatography, collection of fractions and subsequent reverse-phase liquid chromatography with mass spectrometry analysis. While this process can be automatized by on-line two-dimensional chromatography, it remains often complex and time consuming.
What does RP HPLC do?
RP-HPLC is a commonly used method for the analysis and purification of peptides, proteins, and glycoproteins. Monosaccharide composition and content can be determined by using the RP-HPLC separation of p-aminobenzoic ethyl ester derivatives of neutral and amino sugars released from glycoproteins.
How does isoelectric focusing work?
IEF works by applying an electric field to protein within a pH gradient. The proteins separate as they migrate through the pH gradient in response to the applied voltage. When a protein reaches a pH value that matches its pI, its net electrical charge becomes neutral, and stops migrating.
What is C-terminal lysine clipping in mAbs?
C-terminal lysine clipping is a common phenomenon occurring during the bioproduction of mAbs and leads to variable amounts of final process-related charge variants.
Is the C-terminal lysine clipping of the present therapeutic IgG1 A CQA?
As a consequence, C-terminal lysine clipping of the present therapeutic IgG1 should not impact both FcRn-dependent pharmacokinetic profiles and FcγRIIIa-driven cytotoxic activities. The methods used in this study can be widely applied to other IgG1 to define criticality of the C-terminal lysine clipping as a CQA. 1. Introduction
What are the C-terminal Lys variants in recombinant antibodies?
C-terminal Lys variants are frequently observed in mammalian cell-derived recombinant antibodies, usually produced by proteolysis of endogenous carboxypeptidases during the manufacturing process. Fig.1 The C-terminal heterogeneities of mAbs. (Beck, 2013)
Is C-terminal Lys deletion a critical quality attribute (CQA)?
It has been reported that C-terminal Lys deletion has no impact on antibody structure, thermal stability, antigen binding and potency, FcRn binding, and pharmacokinetics in rats. Thus, it is not considered as a critical quality attribute (CQA).
