Free-boundary Electrophoresis Using Electrophoretic Light Scattering (ELS) Detection

Purpose

Complementing traditional small-molecular drugs, therapeutic proteins hold a great promise for human therapy to treat hematological and solid tumors, autoimmune, inflammatory, infectious, and cardiovascular diseases. To ensure the safety, quality, integrity, and efficacy of the therapeutic protein, effective characterization techniques are needed to address their inevitably heterogeneity due to the biotechnological manufacture processes. In the present study, Laser Doppler Electrophoresis (LDE) is explored as an orthogonal and complementary free-boundary technique to the conventional capillary and gel electrophoresis

Methods

Measurement Techniques

Electrophoretic Light Scattering (ELS), a.k.a. LDE or LDV, evaluates motion of particulates under electric field. It is often quantified as the electrophoretic mobility (µe) of particulates.

μ= Ve / E

The optional frequency/phase shift due to electrophoretic motion can be evaluated via:

  • Spectral Analysis (ELS)
  • Phase Analysis (PALS)

Lyophilized proteins are commercially obtained (Sigma-Aldrich). Prior to light scattering tests, all the samples were dissolved in the designated buffers and filtered with a 0.02 µm filter. For LDE tests, the samples were also dialyzed overnight.

Results

1. Protein Electrophoretic Mobility
–Electric Field: 4 V, 2 Hz sinusoidal
–Duration: ~1 second

image of protein electrophoretic mobility graph
ProteinLyzozymeBSAApoferritinCasein
Buffer1xPBS1xPBS1xPBSPEG formula
M(103g/mol)1465450
µe [(µms l s -1) / V l cm-1]0.46-1.01-1.07-0.025

2. Effect of Buffer Conditions

image of effects of buffer conditions graphs

3. Protein Charge: Electrophoretic Effect
–Debye-Hükel-Henry Theory

“Electrical Double Layer (EDL)”

image of electrical double layer equation
image of electrical double layer table

4. Protein Size: Buffer Effect

image of protein buffer effect graph

5. Brownian Motion & Scatter Radius (Rh)
–Diffusive Motion (Fick’s Law: <ΔR(τ)2>τ = 6Dτ)

image of brownian motion and scatter radius graph
image of radius distribution graph

6. Protein Size: Scaling (Rh vs. Mw)

image of protein scaling graph

Conclusions

Light scattering detection delivers rapid (~ 1 second) and statistically reliable characterization of proteins electrophoretic motion. It provides a viable orthogonal and complementary free-boundary electrophoresis technique to the conventional gel and capillary ones. We demonstrate that the technique can be implemented with good sensitivity, precision and accuracy. It is shown that the important effects of solvation media can be assessed well within the measurement certainty. 

The electrophoretic effect of counter-ions is confirmed and analyzed with the Debye-Hückel-Henry theory. The interplay of protein size and charge can be tackled with back-to-back ELS and DLS measurements performed on the identical instrument.

Applications: Zeta
Instruments: NanoBrook Series