Zeta Potential BSA

Unlike the small organic molecules that have traditionally been used in drugs, proteins have chemical and physical properties that can cause instability and product variability. Instability occurs when protein therapeutics undergo chemical and physical changes during manufacturing, shipping, storage and administration. Physical instability such as aggregation or precipitation can negatively affect drug dosage, efficacy, and safety. Monitoring protein stability is therefore essential when manufacturing and storing pharmaceutical proteins.

 

Dynamic light scattering (DLS) and electrophoretic light scattering (ELS) offer the user the ability to monitor both the size and zeta potential of protein particles. Protein aggregates can vary in size. They can range from submicron soluble aggregates to much larger insoluble precipitates. In addition, zeta potential and isoelectric point of proteins can be used for predicting steric stabilization.

DLS also known as photon correlation spectroscopy (PCS) , is commonly used for sizing submicron particles. In solution, particles undergo Brownian motion and scatter light with time-dependent fluctuations in scattering intensity. By looking at the changes in the amplitude of scattered light with time and determining the coefficient of diffusion, the particle radius can be calculated using the Stokes-Einstein equation. As particle size decreases the fluctuations in time will become more rapid. Nicomp 380 ZLS instruments have the unique ability to calculate particle size using both Gaussian and a proprietary (Nicomp) deconvolution algorithm which allows for unimodal, skewed unimodal, and bimodal size distribution analysis with high resolution.

One of the benefits to using a DLS instrument is the ability to couple DLS techniques with ELS methods to obtain the zeta potential of a dispersed system of particles. The measurement is made by immersing two electrodes in a cuvette containing the sample and applying a suitable electric field. The particles in the electric field, if charged, will move with a certain velocity along the field lines toward the oppositely charged electrode. Zeta potential measurements give the potential at the shear plane of a particle.

From the zeta potential the isoelectric point (IEP) can be determined. The IEP is the pH at which a particle, colloid, or molecule carries no net electrical charge at the shear plane. At this pH value, a colloidal particle remains stationary in the applied electric field. To determine the IEP, a titration is performed through a range of pHs. The IEP is found at the pH where the zeta potential is zero. At and around this pH particles are no longer feel the effects of electrostatic stabilization and agglomeration occurs.

Figure 1 shows the volume weighted particle size distribution of bovine serum albumin (BSA) purchased from Sigma Aldrich and diluted 1:100 with deionized water. The primary particle size is ~5nm with aggregates of ~25nm. The histogram shows a fairly “clean” sample with only 0.5% of the sample volume being made up of aggregates.

The diluted BSA solution was then titrated with 0.01M HCl and 0.01M KOH. Zeta potential measurements were taken over a range of pHs. The results are shown in Figure 2. The IEP was then determined to be at pH 5.07. With the Nicomp 380 ZLS it is possible to not only size submicron proteins and their aggregates but it also provides a tool for predicting stability via zeta potential measurements.