Ziping Wei

Improved Particle Counting and Size Distribution Determination of Aggregated Virus Populations by Asymmetric Flow Field-Flow Fractionation and Multiangle Light Scattering Techniques

A method using a combination of asymmetric flow field‐flow fractionation (AFFFF) and multiangle light scattering (MALS) techniques has been shown to improve the estimation of virus particle counts and the amount of aggregated virus in laboratory samples. The method is based on the spherical particle counting approach given by Wyatt and Weida in 2004, with additional modifications. The new method was tested by analyzing polystyrene beads and adenovirus samples, both having a well‐characterized particle size and concentration. Influenza virus samples were analyzed by the new AFFFF‐MALS technique, and particle size and aggregate state were compared with results from atomic force microscopy analysis. The limitations and source of possible errors for the new AFFFF‐MALS analysis are discussed.

Evaluation of a synthetic peptide as a replacement for the recombinant fusion protein of respiratory syncytial virus in a potency ELISA

This report describes the development of a potency ELISA using a peptide derived from the motavizumab binding epitope of respiratory syncytial virus (RSV) F-protein. Motavizumab is an antibody therapeutic studied for the prevention of RSV disease. It binds to the RSV glycoprotein F (F-protein), blocking the ability of RSV to fuse with target cells. This binding is the basis for a potency ELISA, however, due to inefficient F-protein production, development of an alternative ligand for the potency ELISA was investigated. A series of synthetic peptides spanning the motavizumab epitope on F-protein were evaluated for motavizumab binding activity. A 26-mer peptide was identified with desirable motavizumab binding kinetics, as shown by ELISA and surface plasmon resonance. The peptide corresponds to a portion of the motavizumab binding domain on the F-protein, and is referred to as F-peptide. The binding of motavizumab to the F-peptide is used in a new motavizumab potency ELISA, which was shown to be robust and statistically comparable to the F-protein ELISA. In addition, based on a qualitative observation, this new ELISA may be able to detect motavizumab degradation with greater sensitivity compared to the F-protein ELISA.

Biophysical Techniques for Protein Size Distribution Analysis

This chapter discusses the biophysical methods for size distribution analysis of biopharmaceutical protein products. Protein aggregates, conformational variants, reversible self-association, fragments, and glycosylation are common contributors to size heterogeneity in protein pharmaceuticals. Size distribution analysis is essential to product understanding and for ensuring product quality during development. The choice of specific techniques for determination of size heterogeneity may not be straightforward due to a broad range of possible sizes from protein fragments to visible particles. We discuss commonly used biophysical methods for the determination of protein size distribution including size-exclusion chromatography, field-flow fractionation, sedimentation, light scattering, microscopy, light obscuration, electrical zone sensing, and mass spectrometry. The advantages and limitations of each method are also presented. A combination of several methods is often necessary to obtain a comprehensive view of size heterogeneity.

A Statistical Approach to Determining Criticality of Residual Host Cell DNA

We propose a method for determining the criticality of residual host cell DNA, which is characterized through two attributes, namely the size and amount of residual DNA in biopharmaceutical product. By applying a mechanistic modeling approach to the problem, we establish the linkage between residual DNA and product safety measured in terms of immunogenicity, oncogenicity, and infectivity. Such a link makes it possible to establish acceptable ranges of residual DNA size and amount. Application of the method is illustrated through two real-life examples related to a vaccine manufactured in Madin Darby Canine Kidney cell line and a monoclonal antibody using Chinese hamster ovary (CHO) cell line as host cells.