Biophysical Characterisation of Biologics

Biophysical characterisation of biologics laboratory services – supporting protein development by applying UV, CD, NMR, FTIR, fluorescence, SEC-MALS, AUC, DLS and DSC

The biophysical characterisation of biologics plays an important role in the development of protein therapeutics such as or monoclonal antibodies or biosimilars. The biophysical behaviour of proteins can impact many aspects of biologic drug function, activity, and stability. Biophysical analytical methods can, for example, help to monitor or confirm conformational integrity, the nature of the folded state of the protein and how the peptides associate to form higher order structures. Understanding the interactions between drug substance and excipients – which may potentially alter protein structure and therefore product stability – is important during formulation development, stability studies, and comparability. Biophysical methods can also provide valuable information on protein degradation and aggregation of proteins in solution.

Techniques for Biophysical Characterisation
Our biologics biophysical characterisation experts apply a wide range of techniques to interrogate the biophysical behaviour of your molecule. We examine a product's higher-order structure (HOS), including secondary and tertiary structure, aggregation, oligomerization, and degradation of the drug substance. Moreover, our biophysical programs are phase specific. During pre-formulation, we study the proteins in the environment and behaviour in addition to protein forced degradation studies, and to support formulation development, we study proteins in the buffers and the presence of additives/excipients of interest. To support IND or NDA submissions, we conduct biophysical characterisation studies that assess spectroscopic, thermodynamic, and hydrodynamic properties.

Higher Order Structure Analysis
Our higher-order structure biophysical programs include high-resolution methods such as multi-dimensional nuclear magnetic resonance (NMR) and other spectroscopic techniques such as circular dichroism (CD), fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), second derivative ultraviolet absorbance spectroscopy (UV-vis), and intrinsic fluorescence.

Aggregation Biophysical Studies
Differential scanning calorimetry (DSC), analytical ultracentrifugation (AUC), and light scattering techniques (DLS, SEC-MALS) allows the study of non-covalent structural aspects of a protein, and also study of aggregates or oligomer formation in solution.

Spectroscopic Profiles for Protein Characterisation (ICH Q6B)
To establish spectroscopic profiles to support protein characterisation physico-chemical programs, we apply ultraviolet and visible absorption spectra, circular dichroism (CD), nuclear magnetic resonance (NMR), infrared (FTIR), or fluorescence spectroscopy.

Biophysical Characterisation Expertise
Our expertise in biophysical characterisation can support your product development process. With expertise across a wide range of product types including proteins, monoclonal antibodies, vaccines, PEGylated proteins, antibody drug conjugates, oligonucleotides, glycoproteins, and biosimilars, we can apply a wide range of biophysical technologies to deliver efficient and detailed information. As an industry leader in scientific and technical leadership, we can help you advance your biopharmaceutical development programs with expert project management and regulatory guidance. Bringing quality and safety to life, we apply our Total Quality Assurance expertise to help you to meet and exceed quality, safety, and regulatory standards for your biologic development programs.

Circular dichroism (CD) spectra of proteins provide insight into the secondary structures based on absorption by peptide bonds (far-UV CD) or the tertiary structure based on the absorption by either disulphide bonds or aromatic amino acids (near-UV CD) in the protein of interest. As part of our higher order structure analysis expertise, we use Far UV CD spectra to estimate the approximate secondary structural composition of a protein (α-helix, β-sheet, β-turns and random coil). Near UV CD depends upon the local folding environment around aromatic residues provides a unique fingerprint of the protein of interest.

Fourier Transform infrared spectroscopy (FTIR) involves the study of the stretching vibrational absorption bands of the Amide I (C=O) and the Amide II (C-N) groups of a protein background and allows quantification of different secondary structural elements. Compared to CD, FTIR is more sensitive to β –sheets. It is therefore, particularly useful for characterisation of monoclonal antibodies which have high β-sheet content.

Our team provide uniquely efficient high field 600 MHz NMR studies (1D and 2D NOESY (through space correlation) and TOCSY (through bond) correlation experiments) in comparison with reference materials which correlation map to be generated which facilitates detailed assessment of 3D structural differences or changes (perhaps as a result of process changes). This technique is not available commercially.

Intrinsic fluorescence assays use the fluorescence emission by aromatic amino acids tryptophan, tyrosine or phenylalanine to provide information about the tertiary structure of a protein. Tryptophan fluorescence, in particular, is sensitive to the local environment around Tryptophan and can be used to monitor changes in the tertiary structure of a protein making it a very useful method for formulation development, stability or comparability studies.

Ultraviolet absorbance spectroscopy can be used to monitor signals from the three aromatic amino acids: Phe, Tyr, and Trp in a protein. The second derivative of the spectrum (d2A/dλ2) allows reduction in the spectral overlap allowing the application of UV to detect conformational changes, for example in the presence of formulation matrix excipients or changes over a range of solution conditions.

Differential scanning calorimetry (DSC), gives information about the structural stability of the folded protein. It measures the heat change associated with thermal denaturation of a protein and gives the melting transition temperature (T_m) T_m of a protein is a temperature at which half the molecules are in their native conformation and half of the molecules are unfolded. Generally, the higher the T_m of a protein, the more stable it is considered to be. DSC is, therefore, a very useful method to assess effect of different excipients in formulation on protein stability and is a key technique in the development of a Biopharmaceutical product.

Sedimentation Velocity Analytical ultracentrifugation (SV-AUC) is a qualitative method that provides information about the level of homogeneity or heterogeneity of a protein solution and can detect low level of aggregates present. It also provides useful information about size and shape of a molecule and approximate molecular weight of the molecule being studied. It is a direct technique that allows analysis of a sample in its native formulation with minimal sample preparation in most cases. The range of molecular weights that can be detected in a single samples exceeds any other column based method. Different species present in solution can also be quantified with reasonable accuracy thus allowing AUC to be an ideal method for quantification of aggregate content in support of regulatory data submissions.

Dynamic light scattering (DLS) is used for qualitative characterization of particle size distribution of particles in solution. Unlike other orthogonal methods the sensitivity of DLS to detect very low levels of large aggregates make it a very powerful technique. DLS can detect particles between 1 nm and 10 microns and can be used during product development and formulation development. It is extremely useful as a tool in comparability studies and stability studies where it offers a robust method of observing and measuring the aggregates in solution.

SEC-MALS provides valuable insight into protein aggregation formation and aggregate levels in solution. When used in combination with orthogonal techniques that do not depend on chromatography, such as dynamic light scattering and SV-AUC, the data can support product development, stability and comparability studies.