Evaluation Techniques for Physicochemical Properties in Biopharmaceutical Development: Candidate Selection

by Malvern Panalytical, Tuesday, 17th August 2021

This page guides you through the items to be evaluated at each stage of biopharmaceutical development and the Malvern Panalytical’s measurement techniques capable of evaluating them. On this page, we will detail “Candidate Selection”.

The start of new drug development begins with candidate selection. As the physicochemical properties of biopharmaceuticals largely depend on their constructs, it is necessary to select constructs with good physicochemical properties as early as possible. Malvern Panalytical’s measurement techniques support this assessment with particle size distribution, purity, thermal stability, and binding activity.

Evaluation of Particle Size Determination and Association, Aggregates Through Particle Size Distribution (DLS)

Measurement of Globin Size

It is important to screen the target sample conveniently and quickly from multiple candidates. Dynamic Light Scattering (DLS) can determine the size and dispersion state of sample particles within minutes and quickly identifies aggregates. The graph below shows the size (hydrodynamic radius) on the horizontal axis and light intensity distribution (Intensity (%)) on the vertical axis, depicting the particle size distribution of various globins. From the graph, it is clear that HbA (blue) and Hemocyanin (black) with scatter intensity standard values of 100% have a single peak. Meanwhile, Rice Hb1 (green) and Polytaur (gray) with lower values show a broad size distribution, indicating the presence of larger molecules.

By using DLS in this way, it is possible to determine the particle size and look at the peak area ratio to identify the proportion of monomers, dimers, and higher oligomers in the sample. Additionally, DLS can estimate molecular weight from particle size information and is effectively used to determine the oligomeric state and the proportion of aggregates present in the sample.

Globin Name	Subunit	Known Molecular Weight (kDa)	Peak Average (nm)	Scattering Intensity Standard (%)	Volume Standard (%)
Myoglobin	1	17.6	2.403	97.7	100
Rice Hb1	2	37	2.985	90.9	99.9
HbA	4	64.5	3.305	100	100
Polytaur	12	186	6.778	88.3	99.8
Hemocyanin	―	1720	12.36	100	100

→Formulation conditions can be selected based on the indicator of dispersion stability

Accurate Molecular Weight and Structural Information Evaluation Using Multiple Detectors (SEC-LS・Vis)

Evaluation of apo-RD and holo-RD

In traditional analysis methods based on retention capacity using standard substances, there is a risk of incorrect judgment in cases where the apparent volume increases due to structural changes in proteins, resulting in discrepancies in molecular weight. To obtain accurate molecular weight, it is effective to perform scattering light analysis by connecting a light scattering detector (LS) to Size Exclusion Chromatography (SEC). The figure below shows the results of measuring the Repeat in Toxin Domain (RD) protein that changes structure by binding with Ca ions with and without CaCl₂ (holo and apo respectively). Comparing the holo (solid line) and apo (dotted line) states, the faster elution time of apo indicates that, in the traditional method, apo is analyzed as aggregated with a molecular weight of about 600 kDa, while holo is 73 kDa. However, using light scattering analysis, it was confirmed that the molecular weights of both were around 73 kDa, as shown in the table. Moreover, comparing intrinsic viscosity obtained from the viscosity detector, it was noted that apo had 0.35 dL/g, while holo had 0.055 dL/g, indicating a significant difference. This suggests that the protein alters its structure in the presence of Ca ions.

Thus, using multiple detectors simultaneously allows for obtaining accurate molecular weight and structural information, which can help avoid misleading decisions during construct selection.

Parameter	Unit	apo-RD	holo-RD
Retention Volume	mL	10.4	13.8
Intrinsic Viscosity	dL/g	0.35	0.055
Molecular Weight	Kg/mol	73.6	73.2

→Avoid misleading during construct selection by accurate molecular weight and structural information

Particle Size Determination and Evaluation of Association, Aggregates Using Multiple Detectors (SEC-LS・Vis)

Assessment of Antibody Purity

Scattered light tends to be more intense as the molecular weight increases. By connecting a light scattering detector to SEC, it’s possible to clearly see the regions of aggregates that were difficult to identify with traditionally used RI or UV/VIS detectors. The figure below shows the result of measuring IgG using an RI detector connected to SEC and a light scattering detector. Monomers and dimers are confirmed by both the RI signal (red) and the light scattering signal (orange), but in the aggregate region, the light scattering signal is larger than the RI signal (as indicated by the red arrow). RI and UV/VIS detectors depend on concentration for signal changes, so even small amounts of aggregates tend to have smaller signals. Adding a light scattering detector allows for distinguishing whether there truly are aggregates or if it’s baseline fluctuation.

Therefore, using SEC-LS・Vis, it is possible to confirm the purity of the sample more accurately, enabling the selection of a construct with higher purity and desired characteristics.

→Construct selection based on purity indicators is possible

Construct Selection by Comparison of Thermal Stability (DSC)

Comparison of Thermal Stability of 12 Antibody Variants with Fab Mutations

It is known that protein mutations can change activity and stability, and improving stability is thought to affect activity. Differential Scanning Calorimetry (DSC) can evaluate protein thermal stability without labeling. The data below shows measurements of the wild type (WT) and 12 Fab mutants with tetanus toxoid antibody using DSC. The horizontal axis is temperature, and the vertical axis is the heat capacity required for denaturation. For WT Fab, the T_m (peak top temperature of main peak) was 78.7 ℃, while for the mutant V11, it was 92 ℃, confirming that stability improved by 13.3 ℃.

By using DSC, it is possible to compare how slight sequence differences in the variable region of an antibody affect thermal stability, allowing for narrowing down of more stable construct candidates.

→It is possible to select constructs with high thermal stability using T_m as an indicator

Construct Selection by Binding Activity (ITC)

Interaction of Histone Chaperone with Three Different Histone Peptides

In drug discovery development, it is important to efficiently select candidates with higher affinity for the target protein. Isothermal Titration Calorimetry (ITC) allows for simultaneous determination of affinity, specificity, and binding mechanisms of intermolecular interactions in a single measurement.

The figure below compares the interactions of the PHD domain (plant homodomain) of the histone chaperone TAF3 (TBP(TATA binding protein)-associated factor) with three different 10-mer histone peptides. The top row shows raw data, and the bottom row represents molar ratios on the horizontal axis and enthalpy changes (ΔH) from interactions on the vertical axis. These interactions yield a single sigmoidal curve indicative of typical 1:1 binding, suggesting that affinity varies between peptides through fitting analysis. Differences in affinity are reflected in the slope of the curve, indicating stronger affinity closer to vertical slopes and weaker affinity as the slope decreases.

In this way, ITC enables the selection of constructs with higher affinity.

→Select constructs with high affinity based on K_D as an indicator

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