Protein Higher-Order Structural Characterization Methods
When developing biologics, more specifically therapeutic protein products, unwanted immune responses can not only be harmful to patients, but also inhibit the efficacy of a product. Maintaining correct higher-order structure (HOS) is critical to ensuring proper functionality, activity, and stability of a biopharmaceutical product. A well-developed panel of methods for HOS characterization is an essential component of a complete product characterization program. All of our biophysical characterization assays can be qualified and validated for use in your product’s routine lot release or stability studies.
Analytical Ultracentrifugation (AUC)
Analytical ultracentrifugation (AUC) separates protein species directly in solution, without the use of a stationary phase such as in size-exclusion chromatography (SEC). The sedimentation rate of the molecule(s) is induced by the centrifugal force, and is monitored continuously by UV absorbance, fluorescence, or interferometry to produce a size distribution profile of the species present within the test sample. Sedimentation velocity analytical ultracentrifugation (SV-AUC) generates sedimentation coefficient values and reports the relative percentages of monomer, multimer, and aggregate species. Information on molecular weight (MW) and hydrodynamic shape are also obtained.
Differential Scanning Calorimetry (DSC)
Differential scanning calorimetry (DSC) is conducted by heating the molecule at a constant rate and the detectable changes in heat capacity associated with thermal denaturation are recorded. A single DSC experiment can determine the transition midpoint, Tm, and the enthalpy (ΔH) and heat capacity change (ΔCp) associated with unfolding. DSC is a very useful biophysical characterization technique for comparing lots of the same product to ensure lot-to-lot consistency, comparability of product upon manufacturing changes, and to establish biosimilarity.
Circular Dichroism (CD)
Circular dichroism (CD) measures differences in the absorption of circularly polarized light arising from structural asymmetry. Far UV scan, 195-250 nm, is dependent on peptide bond alignment/positioning. Ellipticity data in the far UV is de-convoluted to estimate secondary structures such as alpha-helix, beta-sheet, or random coil. Near UV scan, 250-350 nm, measures absorbance of the chromophores of aromatic residues and disulfide bonds. The CD signal within this spectral range provides a fingerprint of the overall tertiary structure of the protein.
Dynamic Light Scattering (DLS)
Dynamic light scattering (DLS) provides information on the hydrodynamic size and size distribution of particle emulsions and molecules dispersed or dissolved in a liquid. Population size information is expressed as a distribution curve. Dynamic light scattering allows for the measurement of a protein’s diffusion coefficient, along with the hydrodynamic size, and an estimation of molecular weight.
Intrinsic Tryptophan Fluorescence (ITF)
Intrinsic tryptophan fluorescence (ITF) assesses the conformational state of a protein. The intrinsic fluorescence of a folded protein is due primarily to tryptophan residue emission, with some small contributions from tyrosine and phenylalanine residue. Tryptophan has a wavelength of maximum absorption of 280 nm and an emission peak that is solvatochromic, ranging from 300 to 350 nm depending on the polarity of the local environment. Hence, ITF is used as a diagnostic for structural characterization of a protein.
Fluorescence Polarization Anisotropy (FPA)
Fluorescence polarization anisotropy (FPA) determines binding constants from the interaction of relatively small fluorescently emitting ligands with larger receptor molecules. This biophysical characterization technique is based on the use of polarized light to excite a fluorescent molecule, which in turn will emit polarized light. However, the degree of polarization and thus anisotropy of the emitted light is directly dependent on the rotational diffusion of the fluorescent molecule. The measurement of anisotropy, therefore, can be used to generate binding curves upon varying the concentration of ligand with respect to its receptor. Affinity constants can then be calculated from the respective binding curves.
Surface Plasmon Resonance (SPR)
Surface plasmon resonance (SPR) protein binding studies commonly use biacore instrumentation and take on various configurations and formats, including both Protein:Protein and Protein:Drug binding studies. Rank-order screening methods for lead selection as well as optimization of running conditions to determine Kon, Koff, and KD values for IND/CMC programs can be developed as part of the biophysical characterization of the product.
Apolar Fluorescent Dye 8-Anilinonaphthalene-1-Sulfonic Acid (ANS) Method
The apolar fluorescent dye 8-Anilinonaphthalene-1-sulfonic acid (ANS) method evaluates the surface hydrophobicity of a protein in its native state. This method is a relatively rapid, non-destructive, and simple means of quantitatively assessing the apolar or hydrophobic nature of a protein. The number and relative size of hydrophobic sites on a protein’s surface may be influenced by conditions of pH, temperature, and ionic strength. As the molecule unfolds, more hydrophobic regions are exposed. Thus, this method is very sensitive to the conformational state of the protein.
Size-Exclusion Chromatography with Multi-Angle Laser Light Scattering (SEC-MALLS)
Size-exclusion chromatography with multi-angle laser light scattering (SEC-MALLS) detection is used to separate proteins based on size and to determine the molar mass of the separated proteins. The angular dependence for large species (>200 kDa) may also be used to estimate size and shape factors, such as the protein radius of gyration.
Fourier Transform Infrared (FT-IR) Spectroscopy
Fourier transform infrared (FT-IR) spectroscopy is a well-established experimental technique for the analysis of secondary structure of polypeptides and protein. Analysis of polypeptide and proteins by FT-IR yields a series of characteristic IR absorption bands. Amide I and II bands are the two most significant vibrational bands of the protein backbone. Furthermore, the more sensitive of the two amide bands is the amide I band (1700−1600 cm−1), which corresponds to the C=O stretch vibrations of the peptide linkages. The frequencies of the amide I band components are well correlated with the secondary structural characteristics of proteins.
Aggregation & Sizing
One type of biophysical characterization that should be completed is aggregation and sizing. Protein aggregates are defined as any self-associated protein species and can be classified based on five characteristics: size, reversibility/dissociation, conformation, chemical modification, and morphology. Here are some of our methods of protein aggregation analysis:
- Size-exclusion chromatography with multi angle laser light scattering (SEC-MALLS)
- Analytical ultracentrifugation (AUC)
- Dynamic light scattering (DLS)
- Electrophoresis via SDS PAGE and CE-SDS
- Circular dichroism (CD) spectropolarimetry
- Fourier transform infrared spectroscopy (FT-IR)
- Intrinsic tryptophan fluorescence (ITF) spectroscopy
- Extrinsic fluorescence ANS dye binding
- Disulfide linkage mapping via peptide mapping LC-MS/MS
- CD thermal denaturation study
- Differential scanning calorimetry (DSC)
- Intrinsic tryptophan fluorescence (ITF) spectroscopy
- Extrinsic fluorescence ANS dye binding
Protein Binding Kinetics
- Surface plasmon resonance (SPR) via biacore
- Fluorescence polarization anisotropy (FPA)
Frequently Asked Questions (FAQs) About Biophysical Characterization
What are the sample requirements for doing AUC on AAV samples?
For routine AUC analysis of an AAV sample we would usually require about 0.200 mL at around 1e13 vg/mL. A validation can consume up to 5 mL or more at 1e13 vg/mL. Although AUC sample requirements are not small, we are currently working on methods to reduce them.
In AUC assays, how do you control for misalignment?
We have a number of mechanisms to ensure alignment including the common ways: visual alignment with marks on the cell and rotor and use of an alignment tool. We are experimenting with a quantitative method of alignment that makes use of image analysis.
How consistently is AUC used in clinical lot release and stability?
AUC is becoming more and more useful in lot release for rAAV products. It is difficult to say precisely how many filings are now including AUC, but we believe this will be on the increase, and will be included in future FDA guidance documents.
Is it possible to validate an AUC method for AAV empty/full when the sample is heterogeneous?
A sample that is heterogeneous can present a number of problems. In particular, if the main species i.e. the "full" capsid is low in abundance, or on-par with the abundance levels of impurities like partially packaged variants. If a sample is very heterogeneous, a recommendation would be to explore purification strategies prior to advancing very far with a non-ideal product batch.
Is 20,000 rpm optimized to have the better separation for AAV particle characterization?
Centrifugal velocities in the range 15,000 - 20,000 rpm are the most common and probably optimal for empty/full resolution of AAV samples, although as low as 12,000 rpm may be used at times. If the speed is too slow then broadening of the profiles from diffusion can lead to poor resolution, while faster speeds can risk sedimenting larger species too fast which can cause them to be under-represented in fitting.