IN-HOUSE Laboratory Service

PROTEIN CHARACTERIZATION


Detailed characterization of therapeutic biological molecules is required before they can be considered acceptable for its intended use. As suggested in the ICH Q6B guidance, extensive characterization usually should include determination of physicochemical properties, biological activity, immunochemical properties, purity and impurities. The guidance also suggests that as new analytical technology and modifications to existing technology are continually being developed, and they should be utilized when appropriate to allow relevant specifications to be established.

A well characterized product requires characterization of product contaminations as well as establishing specifications for each step of process. Setting specification to in-process stages ensures a consistent drug substance, drug product or materials that can be regarded as safe for its intended use.
 
The team of scientists at Zelle Biotechnology has experience in Upstream and downstream development, manufacturing as well as assay development and quality control. We have worked with a broad spectrum of molecules, including small molecules, peptides, enzymes and glycoproteins.
 
A range of analytical services are available in support of:
 
Method development and transfer and qualification
 
  • Drug substance and drug product analysis
  • Impurity analysis
  • Stability testing
  • Biosimilar testing

Testing Methods

Intact Mass Analysis

Monoclonal antibodies are one of the most common types of biopharmaceuticals. Knowledge of an antibody’s molecular weight is essential to confirm that the antibody has been expressed correctly; if the molecular weight differs from predicted weight then this indicates that there is a change to the primary structure. One way of screening for these changes is through intact mass analysis by liquid chromatography-mass spectrometry (LC-MS). Analysis of the Intact Mass of a deglycosylated Monoclonal antibody can be done to determine the primary structure molecular weight. N-Deglycosylation of monoclonal antibody is performed using PNGase F.

?Monoclonal antibody (Glycosylated) with no prior treatment (only buffer exchange if required) is resolved from formulation components and detected, using reverse phase chromatography and UV detection on UPLC H-class Bio followed by mass analysis in positive sensitivity-MS only mode using Waters Xevo G2-XS QTOF with UNIFI. 

For biosimilarity studies a comparability of innovator and biosimilar is determined in terms of molecular weights and % relative abundance of monoclonal antibody glycoforms.

Subunit Mass Analysis

The analysis of antibody subunits provides additional and complementary information to intact and peptide mapping analysis with the advantage of requiring only very little sample preparation. Also, mass accuracy and resolution of mass spectrometers usually increase with decreasing mass providing additional advantage to the subunit analysis approach. Analysis of the Subunit Masses of a deglycosylated monoclonal antibody can be done to determine the individual unit molecular weight. N-Deglycosylation of monoclonal antibody is performed using PNGase F.
 
Monoclonal antibody (Glycosylated) is denatured and reduced using Rapigest™ and DTT to break inter-chain disulfide bonds and yield separate light and heavy chain subunits. The subunits are resolved and detected using reverse phase chromatography and UV detection on UPLC H-class Bio followed by mass analysis using Waters Xevo G2-XS QTOF with UNIFI.
 
For biosimilarity studies a comparability of innovator and biosimilar is determined in terms of molecular weights of light and heavy chain (glycosylated) subunits and % relative abundance of heavy chain glycoforms.

Reduced Peptide Mapping Analysis

Peptide mapping is an essential step in biotherapeutic characterization. It is a powerful technique which provides important information at numerous stages within the biotherapeutic developmental process. It is used as an orthogonal tool to support primary structure analyses performed at the intact or protein subunit level and to provide additional site-specific information. The analysis is used to confirm that the correct amino acid sequence has been expressed for the protein and to check for presence, location, and quantification of several post-translational modifications. High-resolution mass spectrometry is coupled to chromatography for peak identification and quantitation.
 
The monoclonal antibody is  denatured, reduced, alkylated and digested (using appropriate digestion system). The resulting peptides are resolved using reverse phase chromatography on Acquity UPLC H-class Bio. Chromatographically resolved peptides is further be fragmented, using collision induced dissociation (CID) and mass analyzed in positive sensitivity-MSe mode ( MS and MS/MS data acquisition) using Waters Xevo G2-XS QTOF with UNIFI. The  % amino acid sequence coverage, including product-specific complementarity-determining regions (CDRs), is determined.
 
Modification Analysis
  • Oxidation: 
    Methionine and tryptophan oxidation is investigated in the reduced peptide mapping analysis of monoclonal antibody. Unmodified and oxidized peptides are compared for relative quantification of modified peptides.
 
  • Deamidation:
    Asparagine and glutamine deamidation is investigated in the reduced peptide mapping analysis of monoclonal antibody. Unmodified and deamidated peptides is then compared for relative quantification of modified peptides.

Non Reduced Peptide Mapping Analysis / Disulfide Linkage Analysis

Disulfide bond formation between Cysteine residues, a posttranslational modification, is essential for the folding and stability of proteins. Such bonds increase the conformational stability of the protein both by lowering the entropy of the folded state and by forming stabilizing interaction in the native state.  Hence determination of disulfide bond arrangements of proteins is an important aspect of protein characterization.
 
The monoclonal antibody is denatured, alkylated and digested (using appropriate digestion system). The resulting disulfide linked peptides are resolved using reverse phase chromatography on Acquity UPLC H-class Bio. Chromatographically resolved disulfide linked peptides are further fragmented, using collision induced dissociation (CID) and mass analyzed in positive sensitivity-MSe mode (MS and MS/MS data acquisition) using Waters Xevo G2-XS QTOF with UNIFI. Disulfide linked peptides are confirmed based on low mass error and MS/MS fragment confirmation (% Matched 1st Gen Primary Ions) wherever possible.

Glycan Analysis

N- Glycan release and labelling is performed using Waters GlycoWorks RapiFluor MS Kit. The labelled glycans are resolved and detected using HILIC chromatography and fluorescence detection on UPLC H-class Bio followed by mass confirmation of glycan (MS Level) using Waters Xevo G2-XS QTOF with UNIFI.
 
O- Glycan release is performed using appropriate enzymes followed by tagging using the Waters GlycoWorks RapiFluor MS Kit.
 
GPTS (Glycan Performance Test Standard) is used as positive control and Intact mAb mass check standard for system suitability.

Differential Scanning Calorimetry (DSC)

DSC measures the heat capacity, Cp, of a molecule as a function of temperature. It measures the difference in heat flow between the sample and reference chamber, as both chambers are slowly heated. For some mAbs certain formulation conditions show changes in the thermograms. DSC analysis is done using a nano DSC  instrument.


Circular Dichroism (CD)

Circular Dichroism is performed to study far & near UV spectra, protein folding & thermal denaturation of the protein. The secondary and tertiary structure of the monoclonal antibody is characterized using a J-1500 automated CD spectrometer (Jasco). Samples will be dialyzed into buffer and diluted to obtain suitable concentrations for far- and near-UV measurements. The spectra will be obtained for multiple independent replicates, corrected for the dialysate baseline and normalized by absorbance at 192 nm in the far-UV and 280 nm in the near-UV.