Quantitative Laser Diffraction for Quantification of Protein Aggregates: Comparison With Resonant Mass Measurement, Nanoparticle Tracking Analysis, Flow Imaging, and Light Obscuration

One of the major product quality challenges for injectable biologics is controlling the amount of protein aggregates and particles present in the final drug product. This article focuses on particles in the submicron range (<2 mm). A cross-industry collaboration was undertaken to address some of the analytical gaps in measuring submicron particles (SMPs), developing best practices, and surveying the concentration of these particles present in 52 unique clinical and commercial protein therapeutics covering 62 dosage forms. Measured particle concentrations spanned a range of 4 orders of magnitude for nanoparticle tracking analysis and 3 orders of magnitude for resonant mass measurement. The particle concentrations determined by the 2 techniques differed significantly for both control and actual product. In addition, results suggest that these techniques exhibit higher variability compared to well-established subvisible particle characterization techniques (e.g., flow-imaging or light obscuration). Therefore, in their current states, nanoparticle tracking analysis and resonant mass measurementebased techniques can be used during product and process characterization, contributing information on the nature and propensity for formation of submicron particles and what is normal for the product, but may not be suitable for release or quality control testing. Evaluating the level of SMPs to which humans have been routinely exposed during the administration of several commercial and late-phase clinical products adds critical knowledge to our understanding of SMP levels that may be considered acceptable from a safety point of view. This article also discusses dependence of submicron particle size and concentration on the dosage form attributes such as physical state, primary packaging, dose strength, etc. To the best of our knowledge, this is the largest study ever conducted to characterize SMPs in late-phase and commercial products.

Section: Submicron Particle Analysis On Late-phase Clinical and Commesupporting

confidence: 88%

A Multicompany Assessment of Submicron Particle Levels by NTA and RMM in a Wide Range of Late-Phase Clinical and Commercial Biotechnology-Derived Protein Products

Hubert¹,

Yang

Kwok

et al. 2020

“…For the present study, we conducted a qLD analysis of mAb aggregates, which enabled estimation of the distribution of aggregates across a broad range of sizes, including submicron-sized aggregates. 24,42 Further studies using different types of samples and using other analytical methods such as resonance mass measurement and nanoparticle tracking analysis will be necessary to estimate the characteristics and amounts of submicron-sized particles sufficient to activate FcgRs and to confirm the risk of unintended immune cell activation caused by submicron-sized mAb aggregates. In addition, it should be considered that soluble oligomers and nanometer-sized particles could activate FcgRs.…”

Section: Discussionmentioning

confidence: 99%

Fcγ Receptor Activation by Human Monoclonal Antibody Aggregates

Tada

Aoyama

Ishii‐Watabe

2020

Protein aggregates are a potential risk factor for immunogenicity. The measurement, characterization, and control of protein aggregates in drug products are indispensable for the development of biopharmaceuticals, including therapeutic mAbs. In this study, Fcg receptor (FcgR)-expressing reporter cell lines were used to analyze the FcgR-activation properties of mAb aggregates. Comparison of aggregates of mAbs harboring different IgG subclasses revealed that the FcgR-activation profiles of the mAb aggregates were dependent on IgG subclass. In addition, aggregates of Fc-engineered mAb with enhanced FcgRactivation properties exhibited stronger activation of FcgRs than was observed in the wild-type aggregates, whereas aggregates of Fc-engineered mAb with decreased FcgR-activation properties showed reduced activation. These results suggest that FcgR activation by mAb aggregates depends greatly on the Fc functions of the native (nonaggregated) mAbs. We also showed that aggregates of mAbs smaller than 1 mm in size have the potential to directly activate FcgRs. Unintended immune cell activation can be induced on account of FcgR activation by mAb aggregates and such FcgR activation may contribute to immunogenicity, and therefore, analysis of the FcgR-activation properties of mAb aggregates using FcgR-expressing reporter cell lines is a promising approach for the characterization of mAb aggregates.

“…22 Quantitative laser diffraction (qLD) has been reported to measure a wide range of 0.3-20 µm sizes, which is wider than the size range measured by RMM and NTA. 25,30 Table 1 summarizes the submicron aggregate quantification methods RMM, NTA, qLD, and 3D-HLD. Compared to the other methods, NTA can quantify particles of smaller sizes (more than tens of nanometers), whereas qLD can quantify particles of larger sizes (0.3-20 µm).…”

Section: Discussionmentioning

confidence: 99%

“…The TIG aggregate stock solution was prepared as previously described. 25 Briefly, the TIG solution ejected from the Tetagam Ⓡ P syringe was diluted to 0.5 mg/mL with 20 mM sodium acetate buffer (pH 5.0) and 200 mM NaCl and was then stirred with a magnetic stirrer Magmixer MA300A…”

Section: Sample Preparation For Therapeutic Proteinsmentioning

confidence: 99%

Plate Reader-Based Analytical Method for the Size Distribution of Submicron-Sized Protein Aggregates Using Three-Dimensional Homodyne Light Detection

Fukuhara¹,

Anzai

Osawa

et al. 2021

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