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Monoclonal Antibody MS

Monoclonal Antibody Basics

Monoclonal antibodies (mAbs) are a rapidly growing class of drugs with recent successes in the treatment of cancers. mAbs are derived from identical immune cells and demonstrate a monovalent affinity toward a specific epitope, unlike polyclonal antibodies derived from multiple cell lineages and demonstrate affinity toward multiple epitopes.  The development of mAbs for therapeutic and research applications, demand thorough characterization of the molecule and production method.  Molecular mass, amino acid sequence, 2°, 3° and 4° structure, post-translational modifications (PTMs) including glycosylation, binding affinities and endotoxin limits are some of the important characterization parameters essential to development of a quality mAb.

mAbs belong to the larger immunoglobulin (IgG) protein family.  The basic structure of mAbs consist of a complex of two light and two heavy chains connected by disulfide bridges.  Each chain is divided into two major regions; a variable (V) region at the amino-terminal end and a constant (C) region at carboxyl-terminal end; the C region is frequently divided into smaller sub-regions.  The sequence differences in the V region accounts for a great deal of structural diversity responsible for the target specificity of the antigen-binding cleft.  Early fragmentation work by Rodney Porter and Gerald Edelman broke the IgG structure into 3 fragments using papain to cleave above the region later called the hinge region.  The result was 2 identical antigen binding fragments called Fab fragments (fragments of antigen-binding) and 1 Fc fragment (fragment crystallizable).  The Fc region of the mAb can interact with cellular receptors and additional proteins to form more complex multi-meric structures; depending on the sequence and glycosylation state.  The majority of glycosylation on mAbs is N-linked through conserved asparagine residues (N297) in the CH2 section of the Fc region on the heavy chain.  N-linked Glycans fall into 3 major types high mannose, complex and hybrid (an intermediate of high mannose and complex); with complex types being the most prevalent on mAbs.  The heterogeneity of N-linked glycans on the Fc region of mAbs modulates important biological parameters, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).  A thorough understanding of the glycosylation state of the Fc region is of critical importance to therapeutic mAb design and production.  The cartoon below illustrates the basic structure of an IgG discussed above.

Why should you consider Novatia and ESI/LC/MS for characterization of your mAbs?

At Novatia, we use electrospray ionization liquid chromatography mass spectrometry (ESI/LC/MS) to characterize your monoclonal antibodies.  Our expert team has more than 38 years of biological mass spectrometry experience.  We offer rapid turnaround of high quality analysis results to meet your product specific needs, at cost-effective prices.

mAb LC/MS Analysis Services

Features and Benefits of Novatia mAb LC/MS ServicesApplications
Excellent mass accuracy, typically +/- 0.01% (i.e., 1 Da in 10 kDa).Confirm MW of target mAb, with and free of glycans.
Methods for detailed profiling and high-throughput MW determination are available.Identification of attached glycan and site of attachment.
Unparalleled ESI spectral deconvolution technology: ProMassIdentification of sequence mutations and post-translational modifications.
mAbs can be analyzed in high concentrations of salts, buffers and other contaminants.Assess heterogeneity of the mAb protein and glycosylation.
Backed by over 38 years of experience in biological mass spectrometry.Compare mAbs from different production lots for consistency.

Select the service of interest for an expanded description of the analysis and sample results for MSQC4, standard mAb, and MSQC8, antibody-drug conjugate mimic.

Intact mAb analysis


Molecular weight determination or confirmation of the mAb, MSQC4, with glycans still attached.  The mass is accurately determined and displayed using ProMass software to deconvolute the raw ESI spectrum.

Zoom-in of the deconvoluted peak reveals the heterogeneity of the glycosylation state of MSQC4.  The table below is a screenshot of the ProMass accurate mass report including the breakdown of glycosylation state of the intact MSQC4 sample.

Deglycosylated mAb analysis


PNGase F is used to rapidly remove N-linked oligosaccharides on MSQC4 while leaving the heavy and light chain complex intact.  ProMass deconvolutes the ESI spectrum and produces an artefact-free accurate mass spectrum.  The table below is a screenshot of the ProMass accurate mass report.

Reduced mAb analysis


MSQC4 is rapidly reduced while leaving the glycosylation modifications intact.  The mass of the heavy and light chains are accurately determined using ProMass software to deconvolute the respective ESI spectra.

Heavy chain of MSQC4

 

Zoom-in of the deconvoluted peak reveals the heterogeneity of the glycosylation state of  the MSQC4 heavy chain.  The table below is a screenshot from the ProMass report; including a breakdown of the MSQC4 glycosylation state of the heavy chain subunits and their accurate mass.

Light chain of MSQC4

The single deconvoluted peak confirms that the light chain of MSQC4 is not glycosylated.

Deglycosylated and reduced mAb analysis


MSQC4 mAb standard is rapidly reduced and deglycosylated to produce the light and glycan-free heavy chains.  ProMass software is used to deconvolute the ESI spectra and determine the accurate mass.  The table below is a screenshot of the ProMass accurate mass report of the deglycosylated heavy chain subunits.

Deglycosylated heavy chain of MSQC4

Light chain of MSQC4

mAb Fab and Fc fragment analysis

Rapid Ide S only

MSQC4 mAb standard is rapidly digested in the hinge region, below the interchain disulfide bridges.  The result is a large Fab fragment, consisting of a portion of the heavy chain and the light chain, and two smaller Fc fragments, containing the glycosylated portion of the heavy chain.  ProMass software is used to deconvoluted the ESI spectra and determine the accurate masses of the fragments.

Fab fragment

 

Fc fragments

Zoom-in of the deconvoluted peak reveals the heterogeneity of the glycosylation state of  the MSQC4 Fc fragment.  The table below is a screenshot from the ProMass report; including a breakdown of the MSQC4 glycosylation state of the Fab and Fc fragments and their accurate masses.

 

Rapid Ide S and PNGase

MSQC4 mAb standard is rapidly digested in the hinge region, below the interchain disulfide bridges.  This is followed by removal of N-linked glycans by rapid PNGase F.  The result is a large Fab fragment, consisting of a portion of the heavy chain and the light chain, and two smaller Fc fragments, a portion of the heavy chain devoid of N-linked glycans.  ProMass software is used to deconvolute the ESI spectra and determine the accurate masses of the fragments.

Fab fragment

Fc fragment

Deconvoluted spectrum above shows the intact Fc fragment of MSQC4 and the minor loss of the C-terminal glycine residue of the heavy chain.  The table below is a screenshot from the ProMass report; consisting of the MSQC4 Fab and Fc fragments and their accurate masses.

Antibody-drug conjugate DAR analysis

MSQC8 is a commercially available antibody-drug conjugate mimic, consisting of the MSQC4 standard mAb with dansyl fluorophores attached to cysteine residues. DAR calculation can be determined following mAb analysis under standard antibody treatments such as reduction, Deglycosylated only, Deglycosylated and reduction as well as intact. The reported average DAR for MSQC8 is 4.2 +/- 0.8, while the average DAR per our methods is 4.17 +/- 0.08. For more information regarding the specific mAb treatment, please click on the appropriate drop-down menu, located above.  Below are sample LC/MS chromatogram, ESI and deconvoluted spectra from an analysis of MSQC8, following deglycosylation and reduction treatments.

The heterogenous addition of the drug, to produce MSQC8 (top), yields a more complex chromatogram compared to the MSQC4 standard (bottom).

The most intense peak, consists of the 1X drug addition and the unmodified heavy chain of the MSQC4 mAb.  Below is the ESI and deconvoluted spectra of the peak at 8.737 min.

 

The next intense peak, consists of the 2X and 1X drug additions to heavy chain of the MSQC4 mAb.  Below is the ESI and deconvoluted spectra of the peak at 9.051 min.

The final heavy chain peak, consists of the 3X, 2X, 1X drug additions and unmodified, in descending order.  Below is the ESI and deconvoluted spectra of the peak at 9.235 min.

The most intense light chain peak, consists of the 1X drug addition and unmodified forms.  Below are the ESI and deconvoluted spectra of the peak at 8.087 min.

The final light chain peak consists largely of the unmodified form.  Below are the ESI and deconvoluted spectra from the peak at 7.819 min.