Physicochemical Properties

Protein-based therapeutics are produced in living cells, which results in an inherent degree of heterogeneity. Characterizing the inherent structural heterogeneity is an essential requirement for the development of a therapeutic monoclonal antibody.  High and low molecular weight species, charge variants and post-translational modifications can all affect in vivo stability, cause loss of biological activity and unwanted immune effects. 

By verifying protein structure and physicochemical analyses at appropriate stages of the development of a therapeutic protein Sartorius’s team helps ensure these risks are effectively managed.  

Sartorius offers a comprehensive range of methods to characterize and confirm protein structure, carbohydrate profile, post-translational modifications and impurities. We use state-of-the-art techniques to ensure the ICH Q6B scientific guidelines are met resulting in an efficient, streamlined development process.

Orthogonal techniques

Advance protein characterization with high resolution orthogonal analysis of key physicochemical properties

Data rich

Intact mass analysis not only provides preliminary assurance of identity, but can inform on secondary structure and post translation modifications

Holistic characterization

Structural and physicochemical characterization combined with complex biological analysis enables a thorough understanding of your protein

Ready-to-use assays

For standard molecules such as IgGs we have pre-qualified assays ready for use

ICH Q6B Requirements

Our Solutions

Molecular weight and size analysis

  • Intact and reduced mass
  • Size exclusion Chromatography (SEC)
  • Capillary Electrophoresis SDS (CE-SDS)

Isoform pattern

  • Ion exchange chromatography (IEX)
  • Capillary Isoelectric focusing (cIEF)
  • Reverse phase LC/MS profiling for IgG2 isoforms

Extinction coefficient

Protein concentration analysis

Electrophoretic patterns

  • Capillary Electrophoresis SDS (CE-SDS)
  • Capillary Isoelectric focusing (cIEF)

Liquid chromatographic patterns

  • Size exclusion Chromatography (SEC)
  • Ion exchange chromatography (IEX)

Spectroscopic profiles

  • We offer CD, NMR and FTIR through an approved partner laboratory
  • Reverse Phase Chromatography


Determining molecular weight is essential for preliminary assurance that the recombinant product is synthesized as expected. Molecular weight measurement can indicate potential sequence variation and provide a high-level assessment of post-translational modification, including glycosylation. Our high-resolution LC-MS methods provide accurate molecular weight information and complements our size exclusion chromatographic and capillary electrophoresis SDS methods.

Our dedicated and experienced team has methods to assess the mass of the intact protein, deglycosylated protein and reduced protein. 

Intact mass analysis is the process of determining the molecular weight of a protein. Comparison of the observed mass to the mass expected from the primary sequence provides a preliminary assurance of amino acid composition. Intact mass analysis also provides information on the nature of the main protein glycoforms, as well as other major modifications. 

Deglycosylation of an IgG prior to analysis by LC/MS removes the heterogeneity associated with N-glycosylation. This is employed to assess the mass of the protein backbone without the glycans attached. Together with the intact mass data, this method provides indirect validation of the primary structure. 

LC/MS analysis of the reduced monoclonal antibody is used to confirm the molecular weight of the individual light and heavy chains. In addition to confirming the data for the intact and deglycosylated mass, the analysis of smaller protein fragments means that the method is more sensitive to less abundant modifications. 
 

Our team can also provide complementary analyses on size variants using CE-SDS and SEC.  These techniques provide an orthogonal approach to assessment of molecular weight and size isoforms

Capillary electrophoresis (CE) has become an effective replacement for manual slab gel electrophoresis due to its automation, quantitation, speed, and high efficiency. Sartorius’ CE-SDS technique provides information on the size variants of your protein. 

Size Exclusion Chromatography uses low dispersion, high resolution chromatographic methods to identify high and low molecular weight variants of your monoclonal antibody while evaluating the consequence on binding and functional activity. This also provides information on chromatographic patterns, which is required by ICH Q6b for identity, homogeneity and purity. 

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Protein aggregation and degradation products, as well as heterogeneity in charge variants, are fundamental characteristics implicated in the stability, biological activity and immunogenicity of the therapeutic monoclonal antibody. These characteristics can be indicative of the nature of the clonal cell line, the manufacturing process, or the stability of the drug – information that is needed throughout the biological development cycle. 

Size Exclusion Chromatography uses low dispersion, high resolution chromatographic methods to identify high and low molecular weight variants of a monoclonal antibody while evaluating the consequence on binding and functional activity. This also provides information on chromatographic patterns, which is required by ICH Q6b for identity, homogeneity and purity. 

Using chromatographic methods, it is possible to separate proteins according to their overall net charge based on the isoelectric point (pI). Ion Exchange Chromatography (IEX) is a powerful technique to distinguish between molecules that have minor differences in net charge. Due to the amphoteric properties of several amino acid side chains, this characteristic is fundamental to understanding heterogeneity of the biologic. 

Performing reversed-phase (RP) chromatography supports the characterization of monoclonal antibodies by revealing important information about product purity. Separation is based on hydrophobicity and therefore RP chromatography offers orthogonal selectivity to other techniques, such as size-exclusion chromatography and ion exchange chromatography. It is particularly relevant for IgG2s, which can have structurally distinct forms caused by different disulfide bond structures.

Consult Our Experts

Protein aggregation and degradation products, as well as heterogeneity in charge variants, are fundamental characteristics implicated in the stability, biological activity and immunogenicity of the therapeutic monoclonal antibody. These characteristics can be indicative of the nature of the clonal cell line, the manufacturing process, or the stability of the drug – information that is needed throughout the biological development cycle.

Example electropherogram for NIST mAb standard 

Capillary electrophoresis (CE) has become an effective replacement for manual slab gel electrophoresis due to its automation, quantitation, speed, and high efficiency. Sartorius’ CE-SDS technique provides information on the size variants of your protein. 

Capillary isoelectric focusing (cIEF) is a two-step technique that separates charge variants based on their isoelectric point (pI), The pI values can be determined through protein standards with known pI values. cIEF is an orthogonal technique done with IEX and run using the SCIEX 800 system. 

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Charge variants of a biological product can have implications on stability and biological function. Charge heterogeneity can be caused by sequence variants (e.g., C-terminal lysine clipping), chemical degradation products (e.g., deamidation) and some post-translational modification (e.g., glycans containing sialic acid). Variability can be inherently characteristic of the host cell line, or can be introduced by commercial manufacturing methods. Understanding and managing this heterogeneity is implicit in the requirements of ICH Q6b.

Using chromatographic methods, it is possible to separate proteins according to their overall net charge based on the isoelectric point (pI). Ion Exchange Chromatography (IEX) is a powerful technique to distinguish between molecules that have minor differences in net charge. Due to the amphoteric properties of several amino acid side chains, this characteristic is fundamental to understanding heterogeneity of the biologic. 

Capillary isoelectric focusing (cIEF) is a two-step technique that separates charge variants based on their isoelectric point (pI), The pI values can be determined through protein standards with known pI values. cIEF is an orthogonal technique done with IEX and run using the SCIEX 800 system. 

Reverse phase LC/MS profiling for IgG2 isoforms  

Reversed-phase liquid chromatography (LC) coupled to high-resolution mass spectrometry (MS) is a powerful analytical approach for the profiling of mAb therapeutics. The combination of high performance separations and accurate mass information can provide detailed insights into antibody heterogeneity and stability. However, to date, the highest resolution chromatographic separations of intact proteins have relied upon ion pairing reagents, which can compromise MS sensitivity.  

To facilitate the characterization of IgG2 antibodies, Sartorius developed a method that combines high resolution intact protein separations with high MS sensitivity. Leveraging recent advances in column technologies, this approach achieved excellent resolution of the different IgG2 isoforms, and a number of additional species were detected. In addition, the use of a novel ion pairing reagent improves the MS sensitivity, allowing achievement of a high quality mass spectra for each species.

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Example electropherogram for NIST mAb standard

Spectrophotometry is a well-established technique for this analysis. It involves passing light at a specified wavelength through the sample and measuring the absorbance. The wavelength commonly used for this process is 280 nm, due to the strong absorbance of aromatic amino acids at this region of the UV spectrum, which why it is often referred to as A280 analysis. The absorbance is related to the concentration using the Beer-Lambert law: 

A = εCL 

Where A = absorbance, ε = extinction coefficient, C = concentration and L = pathlength 
 

When using a fixed pathlength spectrophotometer, careful dilutions are often required in order to bring the samples concentration within the linear range of the instrument. This can be time consuming and any errors associated with the dilutions will lead to errors in the final concentration calculated for the sample. 

A solution to these problems is to use a variable pathlength instrument that alters the pathlength component of the Beer-Lambert law. By altering the pathlength instead of concentration, dilutions are usually not required. The assay developed by Sartorius is accurate and precise. A large number of samples can be analyzed in a short amount of time compared to a fixed path length spectrophotometer. Another benefit is that the sample is recoverable after analysis, which is especially useful when only a small amount of sample is available and a number of different tests are required.

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Related Downloads

Brochure: Physicochemical and Biological Analytical Comparability for Biosimilars

PDF | 739.5 KB

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