Physicochemical Evaluation Techniques in Biopharmaceutical Development: Quality Control

by Malvern Panalytical, Tuesday, 17th August 2021

We introduce the items to be evaluated at each stage of biopharmaceutical development and the measurement technologies of Malvern Panalytical capable of evaluating them. This page details “Quality Control”.

Formulated products need to maintain the same quality consistently. Malvern Panalytical’s measurement technology supports the evaluation and management of equivalency between batches and sites using multiple parameters as indicators.

Multifaceted Size Evaluation Using Multiple Detectors (SEC-LS・Vis)

Absolute Molecular Weight and Intrinsic Viscosity Evaluation of β-Amylase

By combining light scattering detectors (LS) with Size Exclusion Chromatography (SEC), it is possible to determine the absolute molecular weight. This molecular weight correlates with the weight of the molecule, where heavier molecules scatter more light and lighter molecules scatter less. The figure below shows the measurement results of β-Amylase with SEC-LS. Using the molecular weight determined from the calibration curve, the two peaks are judged as an aggregate in the first half and a monomer in the latter. However, analysis with a light scattering detector shows that the molecular weights of these two peaks are almost the same, suggesting different molecular structures due to different intrinsic viscosity values. Different structures indicate different molecular volumes, which explains the appearance of two peaks.

Thus, comprehensive evaluation using SEC with multiple parameters allows for detailed and accurate size comparison of formulated proteins.

	Peak1	Peak2
Elution Volume (mL)	14.60	15.97
Molecular Weight (g/mol)	209,200	214,200
Intrinsic Viscosity (dL/g)	0.1572	0.05705

→Accurate size and molecular weight evaluation of formulated protein formulations is possible

T_m and T_1/2 as Indicators for Instability Prediction (DSC)

Impact of Forced Oxidation on Thermal Stability of Antibodies

Differential Scanning Calorimetry (DSC) allows for comparison and examination of effects such as oxidation during post-formulation storage and transport, using thermal stability as an indicator. The figure below shows DSC measurements before and after forced oxidation of antibodies. The onset temperature of denaturation (T_onset) for T_m1 after oxidation is lower compared to before, with the peak becoming broader. Meanwhile, the main peak T_m2 remains largely unaffected, suggesting that the Fc-CH2 domain of this antibody is destabilized by oxidation.

DSC thus enables evaluation of which domains in antibody drugs are affected by oxidation.

→Evaluation of the impact of oxidation on stability for protein formulations is possible

Confirmation of Binding Activity Using Enthalpy Change as an Indicator (DSC)

Correlation Between Thermal Stability of gp120 and Binding Activity with CD4

The data obtained by DSC peaks-identically overlaps if the high-order structure of the sample is maintained at the same concentration and buffer conditions. Figure (a) below shows DSC data for different batches of gp120 protein. The peak tops are around 61°C, but the peak sizes differ. In figure (b), the enthalpy change obtained in (a) (equivalent to the peak area) is plotted on the horizontal axis, while the vertical axis plots the binding activity (%) with CD4 that binds to gp120. The linear correlation between enthalpy change and binding activity implies that reduced enthalpy change indicates batches containing inactive molecules.

Thus, DSC allows for quality evaluation post-manufacturing.

→Comparison of binding activity between batches and sites is possible using ΔH and T_m

Equivalency Assessment Using Peak Shape Similarity as an Indicator (DSC)

Equivalency Evaluation Among Antibody Lots

The thermograms obtained by DSC indicate that if the high-order structure of the sample is intact, the same shape (fingerprint) is obtained under equivalent concentration and buffer conditions. A difference in the obtained shape suggests the inclusion of structurally unstable samples. Similar shape but reduced peak area could indicate already denatured samples in the solution.

The figure below evaluates the equivalency between batches of post-formulation antibodies. The good match of thermograms suggests maintained equivalency across batches.

Thus, DSC enables equivalency evaluation of protein formulations between lots and sites.

→Equivalency assessment using thermograms as fingerprints between batches and sites is possible

Equivalency Assessment Using Affinity and Binding Ratio as Indicators (ITC)

Binding Activity Evaluation of Proteins from Different Lots

The binding ratio (n) obtained from Isothermal Titration Calorimetry (ITC) is represented by the value of “syringe sample concentration/cell sample concentration” on the data’s horizontal axis. Generally, protein is often set in the cell, allowing evaluation of protein binding activity by comparing binding ratios. The figure below compares the binding activity of a protein between different batches using ITC. The syringe is filled with peptidyl positive control. The obtained sigmoidal curve’s slopes are nearly identical, and looking at affinity, lot 1 is 97.1 nM while lot 2 is 135 nM, indicating similar results. However, the binding ratio shows lot 1 at 1.05 sites, while lot 2 is at 0.235 sites. A theoretically 1 binding ratio indicates 100% binding activity, and lot 2 suggests that about 24% of the protein maintains binding activity.

Thus, ITC allows for evaluation of retained binding activity of protein formulations between lots and sites.

→Evaluation of equivalency between lots and sites is possible using K_D and binding ratio

Size and Particle Size Distribution Evaluation Using Particle Size and PdI as Indicators (DLS)

Colloidal Stability Comparison of Antibodies Under Different Storage Conditions

Storage conditions of antibodies hugely impact their quality. Measurement of particle size using Dynamic Light Scattering (DLS) allows for assessing whether the conditions used are optimal.

The figure below shows particle size measurement results of IgG under different storage conditions. Under 4°C storage for 35 days (green), there are no aggregates, and the PdI (Polydispersity Index) is small. In contrast, under conditions where freeze/thaw cycles were repeated five times (red), aggregates occur and the PdI is very large. For samples stored at 25°C for 31 days (blue), while there are not many aggregates, the PdI is slightly larger. Therefore, for detailed evaluation of storage impact, it is effective to compare and evaluate using both particle size and PdI.

Thus, using DLS, the size and particle size distribution of formulated proteins under different storage conditions can be evaluated over time, optimizing storage conditions.

→Quality assessment under different long-term storage conditions using particle size and PdI is possible

Temporal Evaluation of SVP by Scattered Light (NTA)

Temporal Observation of SVP in Antibodies Exposed to Harsh Conditions

In quality control, accelerated testing of pharmaceuticals is required. Adding particle concentration by Nanoparticle Tracking Analysis (NTA) to the parameters allows for more detailed evaluations beyond just particle size. The figure below evaluates IgG exposed to harsher conditions than regular accelerated testing using NTA. Analyzing SVP formation in IgG adjusted to 1 mg/mL and heated at 50°C clearly shows SVP concentration increasing over time and larger aggregates forming.

Thus, quantitative evaluation of SVP is possible using NTA.