Shawn Cao

A Critical Review of Analytical Methods for Subvisible and Visible Particles

The subvisible and visible particles present in a solution are often classified based on size, and are quantified by the actual number of particles present rather than by weight or molar amounts. The analysis of these particles in protein therapeutics are governed by compendial methods and the regulatory agencies, and the methods available to measure them originally evolved focusing on potential safety issues, including capillary occlusion and immunogenicity, that might arise from their presence. Ultracentrifugation, size exclusion chromatography, etc., discussed in previous articles, can be used to analyze aggregates of less than 0.10 microns. This article will focus on methods for analyzing and quantitating sub visible particles (SbVP) of 2 microns or larger. At the present time there is no routine method for quantitating sub visible particles (SbVP) between 0.1 microns and 2 microns. The most common technique for quantitating the amount of subvisible particles between 2 and 100 microns is the light obscuration method. This technique can determine size and amount of particles, but cannot differentiate between the types of particles, such as protein particles, foreign material, micro bubbles or silicone oil droplets, that can be present in protein solutions. The difficulties in adapting this method, originally developed for small molecule drugs for IV administration, to protein therapeutics delivered subcutaneously is discussed. The flow imaging techniques can determine morphology and optical characteristics of the particles, but still not identify the chemical composition. Other methods that can also be used, but are applicable for characterization purposes only, are discussed. The primary method for quantitating visible particles is visual inspection, a method that can be subjective and relies on adequate training of the human inspectors. Automated methods for visible particle determination are being developed. Identification of the chemical composition of isolated particles greater than about 50 microns is possible using several micro-spectroscopic methods, and these will also be discussed.

Separation and characterization of protein aggregates and particles by field flow fractionation.

Field flow fractionation (FFF) is a technique that holds great promise for the analysis and characterization of protein aggregates and particles, due to its wide dynamic range and matrix-free separation mechanism. FFF can be routinely used to achieve good monomer-oligomer separation and quantification for a variety of protein types, and is a reasonable choice for an orthogonal method for size exclusion chromatography and analytical ultracentrifugation. Quantifying sub-micrometer particles in protein therapeutics is a potential of the FFF technique that is yet to be realized, due to the lack of detection with sufficient sensitivity. In this article the effect of several important parameters on the optimization of FFF analyses are explored, and the strengths, weaknesses, and potential new applications of the technique are discussed.

Ion-specific modulation of protein interactions: Anion-induced, reversible oligomerization of a fusion protein

Ions can significantly modulate the solution interactions of proteins. We aim to demonstrate that the salt‐dependent reversible heptamerization of a fusion protein called peptibody A or PbA is governed by anion‐specific interactions with key arginyl and lysyl residues on its peptide arms. Peptibody A, an E. coli expressed, basic (pI = 8.8), homodimer (65.2 kDa), consisted of an IgG1‐Fc with two, C‐terminal peptide arms linked via penta‐glycine linkers. Each peptide arm was composed of two, tandem, active sequences (SEYQGLPPQGWK) separated by a spacer (GSGSATGGSGGGASSGSGSATG). PbA was monomeric in 10 mM acetate, pH 5.0 but exhibited reversible self‐association upon salt addition. The sedimentation coefficient (sw) and hydrodynamic diameter (DH) versus PbA concentration isotherms in the presence of 140 mM NaCl (A5N) displayed sharp increases in sw and DH, reaching plateau values of 9 s and 16 nm by 10 mg/mL PbA. The DH and sedimentation equilibrium data in the plateau region (>12 mg/mL) indicated the oligomeric ensemble to be monodisperse (PdI = 0.05) with a z‐average molecular weight (Mz) of 433 kDa (stoichiometry = 7). There was no evidence of reversible self‐association for an IgG1‐Fc molecule in A5N by itself or in a mixture containing fluorescently labeled IgG1‐Fc and PbA, indicative of PbA self‐assembly being mediated through its peptide arms. Self‐association increased with pH, NaCl concentration, and anion size (I− > Br− > Cl− > F−) but could be inhibited using soluble Trp‐, Phe‐, and Leu‐amide salts (Trp > Phe > Leu). We propose that in the presence of salt (i) anion binding renders PbA self‐association competent by neutralizing the peptidyl arginyl and lysyl amines, (ii) self‐association occurs via aromatic and hydrophobic interactions between the ..xx..xxx..xx.. motifs, and (iii) at >10 mg/mL, PbA predominantly exists as heptameric clusters.

Subvisible (2–100 μm) particle analysis during biotherapeutic drug product development: Part 2, experience with the application of subvisible particle analysis

Measurement and characterization of subvisible particles (including proteinaceous and non-proteinaceous particulate matter) is an important aspect of the pharmaceutical development process for biotherapeutics. Health authorities have increased expectations for subvisible particle data beyond criteria specified in the pharmacopeia and covering a wider size range. In addition, subvisible particle data is being requested for samples exposed to various stress conditions and to support process/product changes. Consequently, subvisible particle analysis has expanded beyond routine testing of finished dosage forms using traditional compendial methods. Over the past decade, advances have been made in the detection and understanding of subvisible particle formation. This article presents industry case studies to illustrate the implementation of strategies for subvisible particle analysis as a characterization tool to assess the nature of the particulate matter and applications in drug product development, stability studies and post-marketing changes.

Classification of Glass Particles in Parenteral Product Vials by Visual, Microscopic, and Spectroscopic Methods

Glass vials have been used as primary containers for parenteral drugs including biopharmaceuticals. Different types of glass-related particles, although in low occurrence rate, may be adventitiously introduced in these parenterals. Proper classification and investigations of these glass-related particles may help to understand their formation, improve process control, reduce glass-related particles, and deliver safe parenteral drugs to patients. In this article, we introduced a classification scheme, and identification procedures and methods, for the glass-related particles. We propose to classify them as glass chip, glass lamella/flake, and silica gel. Eight characteristics for each glass particle type have been identified and described for the visual inspection method. The limitations of the visual method and the need to correlate visual results with forensic analysis are discussed. Using representative examples from each type of glass particle, this study summarized their forensic differentiations based on microscopic methods of optical microscopy, scanning electron microscopy, micro-flow imaging, and spectroscopic methods of dnergy-dispersive spectroscopy and Fourier transform infrared spectroscopy. The mechanisms of glass particle formation are listed as references for drug development scientists to investigate the root causes and improve process control on visible glass particles in parenteral vials. LAY ABSTRACT: Glass vials have been used as primary containers for parenteral drugs including biopharmaceuticals. Different types of glass-related particles, although in low occurrence rate, may be adventitiously introduced in these parenterals. Proper classification and investigations of these glass-related particles may help to understand their formation, improve process control, reduce glass-related particles, and deliver safe parenteral drugs to patients. In this article, we introduced a classification scheme, and identification procedures and methods, for the glass-related particles. We propose to classify them as glass chip, glass lamella/flake, and silica gel. Using representative examples from each type of glass particle, this study summarized their forensic differentiations based on microscopic and spectroscopic methods. The mechanisms of glass particle formation are listed as references for drug development scientists to investigate the root causes and improve process control on visible glass particles in parenteral vials.

Analytical and functional similarity of Amgen biosimilar ABP 215 to bevacizumab

ABSTRACT ABP 215 is a biosimilar product to bevacizumab. Bevacizumab acts by binding to vascular endothelial growth factor A, inhibiting endothelial cell proliferation and new blood vessel formation, thereby leading to tumor vasculature normalization. The ABP 215 analytical similarity assessment was designed to assess the structural and functional similarity of ABP 215 and bevacizumab sourced from both the United States (US) and the European Union (EU). Similarity assessment was also made between the US- and EU-sourced bevacizumab to assess the similarity between the two products. The physicochemical properties and structural similarity of ABP 215 and bevacizumab were characterized using sensitive state-of-the-art analytical techniques capable of detecting small differences in product attributes. ABP 215 has the same amino acid sequence and exhibits similar post-translational modification profiles compared to bevacizumab. The functional similarity assessment employed orthogonal assays designed to interrogate all expected biological activities, including those known to affect the mechanisms of action for ABP 215 and bevacizumab. More than 20 batches of bevacizumab (US) and bevacizumab (EU), and 13 batches of ABP 215 representing unique drug substance lots were assessed for similarity. The large dataset allows meaningful comparisons and garners confidence in the overall conclusion for the analytical similarity assessment of ABP 215 to both US- and EU-sourced bevacizumab. The structural and purity attributes, and biological properties of ABP 215 are demonstrated to be highly similar to those of bevacizumab.

Characterizing Reversible Protein Association at Moderately High Concentration Via Composition-Gradient Static Light Scattering

Analysis of weakly self-associating macromolecules at concentrations beyond a few g/L is challenging on account of the confounding effect of thermodynamic nonideality on the association signal. When the reversible association comprises only 1 or 2 oligomeric species in equilibrium with the monomer, the nonideality may be accounted for in a relatively rigorous manner, but if more association states are involved, the analysis becomes quite complex. We show that under reasonable assumptions, the nonideality in a composition-gradient static light scattering measurement may be accounted for in a simple fashion. The correction is applied to determining the stoichiometry and binding affinity of a protein previously characterized via sedimentation equilibrium and dynamic light scattering. The results of the new analysis are remarkably self-consistent and in line with the expectations for the form of self-association predicted previously from analysis of the surface residuals, establishing composition-gradient multi-angle static light scattering with nonideality corrections as a critical technology for characterizing associative interactions in concentrated solutions.

Field-Flow Fractionation in Therapeutic Protein Development

The development lifecycle for pharmaceutical proteins begins with target identification and demonstration of the biological relevance of a particular protein or protein property, continues to identification of which lead product candidate and cell line to advance, then proceeds through process and formulation development and characterization, clinical trials and commercialization. The launch of a product represents the beginning of a different kind of product support, which includes lot release, exploration of different delivery devices, comparability, and support for investigations. The past several decades have seen demonstration and documentation of the utility of asymmetrical flow field-flow fractionation (AF4) in biotechnology applicable to each of these protein drug development phases, but as yet, with limited industrial or routine implementation. This chapter seeks to provide a survey of such applications and potential opportunities for inspiration and exploitation of the distinct characteristics of AF4 throughout the long, winding and multifaceted drug development process.

Field-Flow Fractionation for Assessing Biomolecular Interactions in Solution

Many biological systems are primarily governed by protein-protein interactions. It is important to develop sensitive analytical techniques to identify and characterize these bimolecular interactions in order to understand their fundamental roles in biological processes and in disease. In this book chapter, we summarize three case studies that applied asymmetrical flow field-flow fractionation (AF4) to access the protein-protein interactions of therapeutic proteins with their counterparts. These new applications of AF4 provide a unique and innovative tool that extends the bioanalytical capability to study protein complexes beyond micro-molar affinity.

Visible and Subvisible Protein Particle Inspection Within a QbD-Based Strategy

In spite of significant progress in many analytical technologies, nondestructive particle characterization remains a challenge. Guidance on foreign particle matter in conventional therapeutics dates back many years in contrast to protein aggregate particles present in the relatively recent biotechnology protein therapeutic products. In this chapter the focus will be on protein aggregates that manifest themselves as both visible and subvisible particles. Protein aggregation is undesired and when unavoidable must be controlled. This requires the ability to characterize this attribute, ideally by size and number and its distribution amongst a manufactured drug product lot as a function of time. Once quantitation is available, in-process controls can be established to ensure that those aggregation levels observed in the manufacture of clinical materials are not exceeded in the commercial product during its shelf life.