Mass Spectroscopy

Mass Spectroscopy: Mass spectrometry (MS) can provide information on the protein sequence and secondary modification of a biologically active protein. MS as a biosimilar exercise, in combination with other analytical techniques, can provide information on the structural similarity between the biosimilar and the reference product.

Mass spectometry example

Principle of Mass Determination: The molecules analyzed via mass spectrometry need to be ionized, that is, given a discrete electrical charge. This is necessary for their acceleration in an electric field and successive separation in a magnetic field on the basis of their mass to charge ratio (m/Q). The mass to charge ratio of these molecules, or of their fragments, can be matched with that of reference samples to identify the analytes.

Liquid Chromatography-Electrospray Ionization- Mass Spectroscopy: Electrospray provide the ionization of samples separated by liquid chromatography in a MS setup creating a positively-charged microspray. A tandem mass spec/mass spec setup can compensate for the low EI separation rate. Biosimilar exercises aiming to compare the sequence similarity between biosimilar and reference proteins have made extensive use of LC-ESI-MS/MS in the past.

Matrix Associated Laser Desorption Ionization Mass Spectroscopy: Matrix Associated Laser Desorption Ionization Mass Spectroscopy (MALDI-MS) is an alternative method to determine peptides mass in biosimilar exercises. MALDI-MS uses a laser-based ionization on samples embedded in a crystalline matrix to create ionized samples analyzed via MS. MALDI ionization works particularly well with large proteins. This technique is accurate in determining protein mass secondary modifications, such as glycosylation, both essential for the function of biologicals.

Liquid Chromatography

Liquid chromatography, and specifically high pressure liquid chromatography (HPLC), separates molecule on the basis of their physiochemical characteristics. This means that molecules with similar characteristics eluting at different times from chromatographic colums. The combination of elution times and abundance of eluate creates sample-specific elution patterns that can be used to the identification of the samples or of its components. Biosimilar development can take advantage of this technique to demonstrate the similarity between comparators and reference biologics.

HPLC analytical example

Reverse Phase-HPLC: Reverse phase-HPLC (RP-HPLC) represent the fist step of separation for peptides and proteins for MS applications, typically before electrospray ionization. This technique is based on the use of a stationary non-polar phase, fixed to a chromatographic column, and polar mobile phase that serves as eluent for the analytes bound to the stationary matrix. This result in a separation based on the polarity of the analytes, where those with higher polarity will elute sooner that those with high non-polar features.

Cation-Exchange-HPLC: Cation-exchange-HPLC (CEX-HPLC) separates molecule on their net surface charge. This is achieved using a cation exchanger stationary matrix that bound the protein of interest at a certain pH, and eluting the column with increasing salt concentrations. As a biosimilar exercise, CEX-HPLC provides information on the presence of different isoforms, protein variants derived from the same gene, in particular regarding post translational modification, in a protein preparation.

Size Exclusion HPLC: Size exclusion HPLC divides analytes based on molecular size using a physical filter, generally matrices composed of porous beads. This HPLC technique is used both for sample preparation for MS, but also for the detection of protein aggregates in pharmaceutical products, including biologicals.

Hydrophilic Interaction Liquid Chromatography (HILIC-HPLC): is particularly useful to separate complex carbohydrates, problematic for other HPLC techniques. As these carbohydrates include glycanes, also forming secondary protein modification in the antibody-antigen recognition, HILIC-HPLC is of central importance as a biosimilar exercise.

HILIC-HPLC uses an organic mobile phase in which water is introduced during the run, either gradually or in isocratic increases.

Boronate Affinity Chromatography: Boronate affinity chromatography is a quantitative technique to measure protein glycation. This technique exploits the binding of boronate residues in the stationary matrix to the ketamine structure in glycated proteins.

Electrophoresis

Capillary Electrophoresis: Capillary electrophoresis (CE) divides peptides on the basis of their size or electrical charge. CE utilized a thin silica tube subjected to hight voltage, separating proteins in a complex mixture. Previous reports of biosmilar exercises used this technique to determine the purity of protein preparations.

Liquid chromatography example

SDS-PAGE: SDS-page employs polacrylammide gels to separate reduce or non-reduced proteins on the basis of their size. SDS-PAGE can reveled host cell-related impurities in protein samples.

Western Blotting: Wester blotting, the transferring of a SDS-PAGE on a nitrocellulose or PVDF membrane. Antibody-based detection can identify proteins transferred on the membrane, identifying the presence of a specific protein in a complex mixture.

Structural Evaluation

These methodologies provide data on the tridimensional protein structure of biologics. This includes both quantification of conformational motifs like alpha-helices, beta-sheets, and random-coils, as well as descriptive analysis of tertiary and quaternary protein structures.

GMP facility sample preparation

Circular Dichroism employs both left- and right-hand circularly polarized light to obtain structural information. Mathematical analysis can transform the different in absorbance in the two kind of polarized light in quantitative information on structural motifs. Previous biosimilar characterizations made extensive use of circular dichroism for structural characterization.

Fourier-Transformed Infra-Red Spectroscopy: Fourier-transformed infra-red (FTIR) spectroscopy uses infrared to generate a broad spectrum of wavelengths that provide and absorbance spectrum typical of the samples analyzed. In protein studies in specific, FITR can provide information on both secondary and tertiary protein structures. FITR is a common technique in structural biochemistry and previous biosimilar characterizations used it extensively.

Differential Scanning Calorimetry: Differential scanning calorimetry (DSC) measures changes in heat capacity of proteins when they are subjected to increasing temperature. These changes depend in turn on the specific structural organization and melting temperature of proteins. This provides information on the stability of proteins, and by extension on its tridimensional structure, and it can be an informative biosimilar exercise.

Hydrogen Deuterium Exchange Mass Spectroscopy: Hydrogen deuterium exchange mass spectroscopy uses the mass difference between protein enriched with the heavy hydrogen isotope deuterium and with their regular counterpart to infer structural information. Mass spectrometry analysis indicates which peptides derived from the protein of interest have a higher tendency to incorporate deuterium. This provides information on the position of hydrogen bounds and on solvent accessibility in the protein of interest.

X-ray Crystallography: X-ray crystallography is the golden standard for structural protein studies, in particular when interested in large proteins or complexes. This technique relies on the analysis of x- ray diffraction patters from proteins crystals bombarded with x-rays. Comparison of these patters can identify divergent tridimensional structures in two proteins, as for example a biosimilar and its reference product, usually indicative of difference in their biological functions.

Binding Assays

Surface Plasmon Resonance Binding Assays (Biacore): This technique provides a quantitative measurement of the binding between ligand and receptor. Surface plasmon resonance measures the reflection angle of incidence light on a metallic surface that carries the ligand of interest. This angle changes with the biding of the receptor, in proportion to the amount of receptor bound to the surface, allowing for a quantitative analysis in a tag-free assay. In case of antibodies, like most biologicals, antigen binding responsible for their biological activity. When used as a biosimilar exercise, surface plasmon resonance gives a quantitative comparison of the activity of biosimilars and of their reference product.

Enzyme-Linked Immunosorbent Assay (ELISA): ELISA uses tagged secondary antibodies that recognize primary antibodies specifically bound to a ligand fixed to the wells of a microtilted plate, giving qualitative information on antigen-antibody interactions. The tagged bound to the detection antibody is generally an enzyme that catalyze a colorimetric reaction, providing a semiquantitative estimate of bound proteins using spectroscopically methods.

Classic ELISA methods are fast and easy to repeat, but require specific antibodies and cannot detect the strength of the binding itself. However, in recent years the development of new ELISA methods made possible to measure absorbance direly in solution, allowing for measuring equilibrium binding constant.

ELISA can of course measure the binding of pharmaceutical antibodies to target antigens, representing then a potential method to measure the activity of biosimilars and to compare it with their reference biologicals.