Product Characterization

Figure 1: Immunoglobulin G (IgG) structure consists of four polypeptide chains, including two identical light chains (~25 kDa) and two identical heavy chains (~50 kDa). Each light chain consists of one constant domain (CL) and one variable domain (VL), and each heavy chain consists of three constant domains (CH1, CH2, and CH3) and one variable domain (VH). The Fab is the antigenbinding region containing hypervariable or complementarity-determining regions (CDRs), whereas the Fc region is highly conserved across molecular species. The hinge region contains two disulfide bonds and glycosylation (shown as red dots) at CH2 domain of Fc fragment.

Biosimilar Therapeutic Monoclonal Antibodies: Gaps in Science Limit Development of an Industry Standard for Their Regulatory Approval, Part 2

Last month, Part 1 of this discussion briefly described the regulatory landscape for developing biosimilar therapeutic monoclonal antibodies (TMAbs). We identified certain specific structural components of TMAb drug substances that warrant particular attention because alterations to them are likely to affect therapeutic safety and effectiveness. Now we conclude by considering whether studies of reference materials can further the development of analytical industry standards to ensure comparability of putative biosimilar TMAbs with innovator TMAbs. We suggest that the time is right…

Figure 2: Intact ESI-MS spectra of both nonsuccinylated and succinylated proteins; upper figures represent the full charge envelope of the two proteins, and lower figures illustrate a narrower m/z charge range. Each m/z charge state of the nonsuccinylated protein was observed to be a primary species, whereas significant heterogeneity was seen for the charge states of the succinylated protein.

Mass Spectrometric Conjugate Characterization: Process Qualification of Recombinant Protein–Hapten Conjugation

Conjugated protein biotherapeutics such as PEGylated proteins (with polyethylene glycol), antibody–drug conjugates (ADCs), and protein–haptens often present unique analytical challenges related to characterizing the conjugation aspect of their manufacturing processes. Analytical characterization of this class of proteins requires knowledge of the sites of conjugation, the degree of conjugation, and the drug-to-protein ratio. Here we present case studies in development of reliable methods based on mass spectrometry (MS) to characterize a protein–hapten drug substance during late-phase process validation. This protein is…

Figure 1: Absorbance Fourier-transform infrared (FT-IR) spectra of different test articles made of polypropylene

Investigation of Foreign-Particle Contamination: Practical Application of FT-IR, Raman, and SEM-EDS Technologies

The presence of visible foreign particulate matter is considered a critical defect in parenteral products and one of the main reasons they can be recalled (1). Foreign particles present during any stage of manufacturing are considered to be contaminants and can impose a risk to the control of the manufacturing processes (2). For those reasons, particle contamination arising in any manufacturing step initiates a nonconformance or out-of-specification observation. That requires an investigation to identify root cause so as to mitigate…

JUPITER IMAGES (WWW.THINKSTOCKPHOTOS.COM)

Science, Risks, and Regulations: Current Perspectives on Host Cell Protein Analysis and Control

State-of-the-art analytics guide process development by providing companies with thorough understanding, effective removal, suitable control, and comparability assessment after process changes of host cell proteins (HCPs) in recombinant biotechnology products. An array of analytical techniques and approaches can be used to establish control strategies for host cell proteins. Techniques used for HCP characterization and comparability include two-dimensional (2D) gel electrophoresis with a range of stains, 2D immunoblotting, 2D high-performance liquid chromatography (HPLC), 2D difference gel electrophoresis (DIGE), and increasingly mass…

BRISTOL-MYERS SQUIBB (WWW.BMS.COM)

Osmolality Measurements for High-Concentration Protein–Polymer Solutions: Variation Based on Working Principles of Osmometers

Osmolality is a critical attribute for injectable formulations. It is desirable to have products match physiological osmotic conditions. Furthermore, osmolality provides confirmation of soluble content in solution. Preventing injection of hypo- or hyperosmotic solutions is a key element of parenteral formulation development. Additionally, some investigators have explored correlations between injection pain and formulation osmolality, although no significant correlation has yet been observed (1–4). Osmolality is a valuable in-process test also because it provides a reliable and repeatable value that reflects…

The Zetasizer Helix system combines Raman spectroscopy with dynamic light scattering.  MALVERN INSTRUMENTS (WWW.MALVERN.COM)

Enhanced Biosimilar Product Characterization: A Case Study Using Raman Spectroscopy Combined with Dynamic Light Scattering

Biophysical characterization has drawn great attention from the biopharmaceutical industry and regulatory agencies across the globe, especially for use in biosimilar drug product development. Currently available biophysical characterization tools can help in screening and optimizing better (more stable) formulations for such products. However, most tools cannot be used for head-to-head comparison of the biophysical properties of an optimized biosimilar formulation with those of an innovator product at higher concentrations. We developed and optimized a formulation for monoclonal antibody MAb B…

liposomes_vironova

Ask the Expert Liposome and Viral Vector Characterization: Use of Electron Microscopy and Image Analysis

with Dr. Josefina Nilsson For this webcast, Josefina Nilsson (EM Services business unit head) discussed Vironova’s work, including case studies. She focused on characterization of drug and gene delivery platforms with electron microscopy and image analysis, specifically for systems that use viral vectors or liposomes. Along with two colleagues — Gustaf Kylberg (image analysis expert) and Mathieu Colomb-Delsuc (electron microscopist) — she then answered questions from the audience. Nilsson’s Presentation Structural characterization provides important insights into the quality of development and…

Schematic_ADCC

Fucosylation of a Therapeutic Antibody: Effects on Antibody-Dependent, Cell-Mediated Cytotoxicity (ADCC) Potency and Efficacy

Product quality attributes are critical for the functionality and manufacturability of therapeutic antibodies. They can be significantly influenced by a number of production process parameters, such as cell culture media. The composition of growth and feed media can influence antibody glycosylation, including the concentration of ammonia, glutamine, glucose, and metal ions (1, 2). Thus, it is critical during media development and optimization to monitor and consider a culture medium’s impact on glycosylation. For therapeutic antibodies whose mechanism of action includes…

WWW.PHOTOS.COM

Benefits of Using Tetradetector SEC to Characterize Membrane Proteins

About 60% of all drug targets are membrane proteins. So understanding their structure and function as well as their interactions with drug candidates is critical to discovery and development of new therapeutic agents. Solubilization of these proteins is an essential precursor to in vitro studies of receptor function, structure, and activity. Purification and crystallization are important aspects. However, solubilization is complicated by the hydrophobic nature of a protein’s membrane-spanning part. Surfactant micelles and other stabilizing systems thus are used to…

Bridging Analytical Methods for Release and Stability Testing: Technical, Quality and Regulatory Considerations

To monitor the control and consistency of products derived from biological systems, a broad array of analytical methods are used for biopharmaceutical release and stability testing. These methods include both classical and state-of-the-art technologies as well as new technologies as they emerge over time.During the life cycle of a product, several reasons can arise for making changes in existing analytical methods: e.g., improved sensitivity, specificity, or accuracy; increased operational robustness; streamlined workflows; shortened testing times; and lowered cost of testing.…