Characterization Techniques in Biopharmaceutical Development: Formulation Studies
In this page, we introduce the items to be evaluated at each stage of biopharmaceutical development, along with the characterization techniques of Malvern Panalytical that enable these evaluations. Here, we introduce details on “formulation studies”.
Evaluation of Dispersion Stability Using the Diffusion Coefficient (DLS)
Comparison of diffusion coefficients under different formulation conditions from the concentration dependency of antibodies
In biopharmaceuticals, by plotting the diffusion coefficient obtained by Dynamic Light Scattering (DLS) against concentration, it is possible to evaluate dispersion stability. The diagram below shows the results of measuring the change in particle size with concentration changes for IgG antibodies dispersed in different buffer solutions using DLS.
In Buffer Solution 1 (upper left in the diagram), it can be confirmed that as the concentration increases, the particle size becomes larger, and the diffusion coefficient becomes smaller. Conversely, in Buffer Solution 2 (upper right in the diagram), it is confirmed that the particle size becomes smaller and the diffusion coefficient becomes larger. This is influenced by the repulsive forces of intermolecular interactions, causing the apparent diffusion coefficient to be larger in Buffer Solution 2. The lower diagram plots the diffusion coefficient against concentration, showing that if the slope is negative (blue), dispersion stability is low, and if the slope is positive (red), dispersion stability is high and there are fewer aggregates.
In this way, using DLS allows the evaluation of the dispersion stability of samples under different formulation conditions.
Evaluation of Dispersion Stability Using the Second Virial Coefficient (SLS)
Comparison of the Second Virial Coefficient under Different Formulation Conditions from Sample Concentration Dependency
Static Light Scattering (SLS), a type of light scattering method, can determine the molecular weight and second virial coefficient (B22) of a sample, allowing dispersion stability to be suggested using these parameters. The upper section illustrates the dispersion stability of lysozyme in a buffer containing different concentrations of NaCl, evaluated by SLS. By modifying protein concentration for each NaCl concentration and plotting KC/Rθ (Debye plot), B22 can be determined from the slope. This parameter indicates the relationship between solute and solvent, and generally, if B22 is positive, it suggests that the protein’s dispersion is stable. This indicated that lysozyme has high dispersion stability under buffer conditions containing 0% NaCl.
The lower section compares B22 when the same antibody is placed in different buffer solutions. This result shows that dispersion stability is higher in Buffer Solution 2.
As such, using SLS enables the evaluation of protein taking into account dispersion stability under different formulation conditions.
Evaluation of Dispersion Stability Using Electrophoretic Light Scattering (ELS)
Comparison of Zeta Potential of Antibodies under Different Formulation Conditions
Zeta potential, an indicator of dispersion, is measured by Electrophoretic Light Scattering (ELS), another type of light scattering method, and it is determined by the molecule and its surrounding environment (types of buffer solutions). The figure below shows the comparison of zeta potential for IgG in different buffer solutions. Since ELS tends to be unstable due to high salt concentrations, multiple measurements were taken to confirm reproducibility. In Buffer Solution 1 (blue), the zeta potential is low, and since the charge calculated from electrophoretic mobility is also small at 0.8 (data not shown), dispersion stability is low, suggesting dipolar interaction. In Buffer Solution 2 (red), given that the zeta potential and charge are large at 6.5, high electrostatic stability is presumed.
In this manner, ELS allows the evaluation of differences in dispersion stability due to molecular surface interactions under different formulation conditions.
Tm and Tagg Used as Indicators for Thermal Stability Evaluation (DLS)
Comparison of Recombumin Thermal Stability Across 10 Different Formulation Conditions
In the development of the Active Pharmaceutical Ingredient (API), it is important to design a formulation that provides sufficient stability for long-term storage under recommended handling conditions.
In DLS, the aggregation onset temperature (Tagg) is obtained from changes in particle size under heating conditions, allowing evaluations of thermal stability under various solvent conditions. Conditions with high thermal stability are thought to be also stable in long-term storage. The figure below shows the results of heating measurements conducted with recombumin dispersed in buffers adjusted at various pHs using DLS. Most of the samples prepared at pH 3-10 showed similar Tm (67-68 °C). Two samples formulated at pH 6 (red frames) showed the greatest thermal stability as aggregation did not initiate until Tm exceeded 74 °C. Contrastingly, the two samples formulated at pH 3 (green) and 4 (yellow) are already considered to be in a denatured state since their particle size at the start of heating is large.
Thus, conducting heating measurements using DLS allows for the evaluation of thermal stability under different formulation conditions.
Tm and T1/2 Used as Indicators for Thermal Stability Evaluation (DSC)
Comparison of Antibody Thermal Stability Across 19 Different Formulation Conditions
Formulation conditions for biopharmaceuticals (types of buffer solutions, pH, additives, etc.) can be narrowed down by comparing the thermal stability of samples in various solutions using Differential Scanning Calorimetry (DSC). The top data compares DSC data of the same antibody in three solution conditions with different pH levels. At pH 3.5, the main peak position shifts to the lower temperature side compared to pH 5.5 and 6.2, and the denaturation onset temperature is also lower, indicating low thermal stability conditions. The middle graph compares the denaturation temperatures (Tm) of the main peak for an antibody measured under 19 different buffer and pH conditions using DSC. Twelve conditions from Acetate 5.0 to Tris 7.5 show almost identical Tm (middle red arrows) and were considered formulation condition candidates. Typically, formulation conditions are narrowed down using this method, but additionally, the graph in the bottom section compares the antibodies immediately after preparation (blue) with those after one week (yellow) under the same conditions, using the half-width temperature of the main peak height (T1/2). The narrower the temperature width of T1/2, the more compact and stable the structure is assumed to be, leading to more refined conditions (bottom red arrow), compared to just using Tm for comparison.
In this way, multiple parameters obtained from DSC allow comparison of the sample’s thermal stability under different buffer conditions, enabling the determination of better formulation conditions.
Quantification of Protein Aggregates by Scattered Light (NTA)
In drug development, it is important to detect and quantify Sub Visible Particles (SVP).
Nano Tracking Analysis (NTA) can directly irradiate lasers onto particles in liquid to detect submicron and nanosized particles from as small as 10 nm* by capturing scattered light with a camera. It also tracks Brownian motion in liquid by image processing to calculate particle size from its speed. Protein aggregates of 50 nm or more can be detected, bridging the analytical scope of microscopes and chromatography with flow cytometry.
The figure below detects aggregates of proteins in buffer solutions that become strong enough to be detected by scattered light. The horizontal axis represents size, and the vertical axis represents the number of particles.
In this way, using NTA allows for visual confirmation and detection of aggregates that conventional methods struggled to detect, supporting better formulation condition decisions. *Dependent on particle material and density
Quantitative Evaluation of SVP Under Different Formulation Conditions Using Scattered Light (NTA)
SVP Detection in Insulin with Four Different Antioxidants Added
NTA can quantify SVP particle concentration since the measurement area is known. The figure below shows results from analyzing SVP in 1 mg/mL insulin with various antioxidants (100 μM) added, stored for 72 hours at 4°C. The horizontal axis represents size, and the vertical axis denotes the number of particles. Insulin in PBS without antioxidants (A) shows low SVP concentration detected. Differences were observed depending on the added antioxidant, with some causing more aggregated particles (B, D, E) and some not (C).
Thus, using NTA, quantitative evaluations of SVP content under different formulation conditions are possible, supporting better formulation condition decisions.
Evaluation of Thermal Stability By Changes in Particle Size Distribution (DLS)
Comparison of Size Changes in Accelerated Tests with Four Different Sugars Added to ConA
In DLS, evaluation of thermal stability is possible when additives are added to protein formulations.
The figure below shows the results of heating measurements conducted on Concanavalin A (Con A) at 1 mg/mL with various sugars (10 mM) added, to evaluate how the binding with these sugars affects thermal stability using DLS. The horizontal axis represents size, and the vertical axis represents intensity distribution (Intensity(%)). In the top row at 35 °C, it is confirmed that a clean monodisperse state exists, and that the peak tops also overlap. Meanwhile, in the lower row at 47 °C, it is confirmed that aggregation states (size, quantity) differ depending on the type of sugar.
From the state near the main peak at approximately 10 nm, it is suggested that No Sugar (light purple), and Galactose (green) show significant size shifts and aggregation, while Glucose (blue), Fructose (black), and Mannose (red) bind with Con A and enhance thermal stability.
Thus, using DLS, the impact of different additives on thermal stability can be evaluated through size changes, supporting better formulation condition decisions.
Size Change as an Indicator for Stability Evaluation (SEC-LS)
Verification of Antibody Association, Aggregation, and Fragmentation Through Accelerated Testing
Size Exclusion Chromatography (SEC) is often used for stability evaluation through accelerated testing of protein formulations.
The figure below shows the results of measuring samples of IgG antibody set in vials and subjected to specified heat using a water bath, analyzed by SEC. Comparing immediately after preparation (red) and those held for 135 minutes at 60°C (purple), the latter shows a higher monomer ratio and a smaller dimer ratio. In samples held for 60 hours at 60°C (green), aggregation accelerated with larger aggregates (red arrow: around RV 6 mL) was confirmed. Also, a peak appeared at the back of the monomer peak (yellow arrow: around RV 10 mL), indicating fragmentation.
As such, using SEC enables the evaluation of aggregation and fragmentation due to stability in samples through accelerated testing, supporting better formulation condition decisions.
| Molecular Weight (Da) (Content (%)) | ||||
| Aggregates | Dimers | Monomers | Unknown | |
| Start | 654,500 (4.5) | 294,500 (14.3) | 147,100 (81.2) | ― |
| 135 min at 60°C | 547,100 (1.8) | 293,500 (5.2) | 145,900 (93.0) | ― |
| 60 hrs at 60°C | 3,081,000 (1.2) | 588,400 (9.6) | 156,800 (75.0) | 133,400 (17.2) |
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