Measuring Subvisible Particles in Protein Formulations Using a Modified Light Obscuration Sensor with Improved Detection Capabilities

Published on Jun 2, 2015in Analytical Chemistry6.785
· DOI :10.1021/ACS.ANALCHEM.5B00688
Anacelia Ríos Quiroz1
Estimated H-index: 1
(University of Basel),
Gabriela Québatte6
Estimated H-index: 6
(University of Basel)
+ 6 AuthorsMichael Adler11
Estimated H-index: 11
Although light obscuration is the “gold standard” for subvisible particle measurements in biopharmaceutical products, the current technology has limitations with respect to the detection of translucent proteinaceous particles and particles of sizes smaller and around 2 μm. Here, we describe the evaluation of a modified light obscuration sensor utilizing a novel measuring mode. Whereas standard light obscuration methodology monitors the height (amplitude) of the signal, the new approach monitors its length (width). Experimental evaluation demonstrated that this new detection mode leads to improved detection of subvisible particles of sizes smaller than 2 μm, reduction of artifacts during measurements especially of low concentrations of translucent protein particles, and higher counting accuracy as compared to flow imaging microscopy and standard light obscuration measurements.
📖 Papers frequently viewed together
22 Citations
4 Authors (Andrea Hawe, ..., John F. Carpenter)
5 Citations
23 Citations
#1Daniel Weinbuch (LEI: Leiden University)H-Index: 6
#2Wim Jiskoot (LEI: Leiden University)H-Index: 84
Last. Andrea HaweH-Index: 23
view all 3 authors...
Light obscuration (LO) is the current standard technique for subvisible particle analysis in the quality control of parenterally administered drugs, including therapeutic proteins. Some of those, however, exhibit high viscosities due to high protein concentrations, which can lead to false results by LO measurements. In this study, we show that elevated sample viscosities, from about 9 cP, lead to an underestimation of subvisible particle concentrations, which is easily overlooked when considerin...
13 CitationsSource
#1Tobias Werk (Hoffmann-La Roche)H-Index: 5
#2David B. Volkin (KU: University of Kansas)H-Index: 40
Last. Hanns-Christian Mahler (Hoffmann-La Roche)H-Index: 31
view all 3 authors...
Abstract Purpose Protein formulations may contain subvisible particle (SbVP) impurities that can vary (e.g., in number, size, shape, density, refractive index and transparency) depending on the formulation composition, environmental stresses and the type of protein. Additionally formulation solutions may differ in their physical properties including turbidity, color, viscosity, density and refractive index. This study examined the impact of these formulation matrix parameters on the ability to s...
43 CitationsSource
Micro-flow imaging (MFI™) is an increasingly important technique for the characterization of subvisible particles during the development of biopharmaceutical products. Protein particles are highly variable in size, appearance, and translucency posing challenges to optical detection techniques. We have developed a set of standard statistical tests applicable for routine evaluation of MFI™ particle dataset quality. The tests evaluate the spatial randomness of particles using nearest neighbor and q...
13 CitationsSource
#1Andrea HaweH-Index: 23
#2Frank SchaubhutH-Index: 3
Last. Gerhard Winter (LMU: Ludwig Maximilian University of Munich)H-Index: 59
view all 8 authors...
Abstract The draft for a new United States Pharmacopoeia (USP) monograph 〈787〉 “Sub-visible Particulate Matter in Therapeutic Protein Injections” describes the analysis of sub-visible particles by light obscuration at much lower sample volumes as so far required by the European Pharmacopoeia (Ph. Eur.) and the USP for parenterals in general. Our aim was to show the feasibility of minimizing the sample expenditure required for light obscuration similar to the new USP settings for standards and ph...
11 CitationsSource
#1Sarah Zölls (LMU: Ludwig Maximilian University of Munich)H-Index: 4
#2Manuel Gregoritza (LMU: Ludwig Maximilian University of Munich)H-Index: 9
Last. Andrea HaweH-Index: 23
view all 7 authors...
ABSTRACT: The aim of the present study was to quantitatively assess the relevance of transparency and refractive index (RI) on protein particle analysis by the light-based techniques light obscuration (LO) and Micro-Flow Imaging (MFI). A novel method for determining the RI of protein particles was developed and provided an RI of 1.41 for protein particles from two different proteins. An increased RI of the formulation by high protein concentration and/or sugars at pharmaceutically relevant level...
64 CitationsSource
#1Glenn A. WilsonH-Index: 2
#2Mark C. Manning (CSU: Colorado State University)H-Index: 60
The potential impact of subvisible particles (SVPs) in protein therapeutic products has received a great deal of attention recently. As a result, new analytical methods have emerged to characterize and quantify SVPs. Among these, flow imaging (also called flow microscopy) has been widely employed. As we have used both FlowCAM® and Micro-Flow Imaging™ in a variety of development projects, we wished to share our experiences and observations, with the intention of fostering a discussion that will l...
16 CitationsSource
#1Dean C. Ripple (NIST: National Institute of Standards and Technology)H-Index: 14
#2Mariana N. Dimitrova (MedImmune)H-Index: 5
All therapeutic protein products contain intrinsic particles formed by the aggregation of protein monomers. There is growing interest in understanding particles in biopharmaceutical products, fostered on one hand by significant advancements in particle analysis and on the other hand by concerns about potential impact of particles on product quality and safety. With currently available methods, particles in therapeutic proteins can be counted, sized, and characterized in a rudimentary way over a ...
67 CitationsSource
#1Wim Jiskoot (LEI: Leiden University)H-Index: 84
#2Theodore W. Randolph (CU: University of Colorado Boulder)H-Index: 86
Last. John F. Carpenter (University of Colorado Denver)H-Index: 106
view all 9 authors...
Protein instability and immunogenicity are two main roadblocks to the clini- cal success of novel protein drug delivery systems. In this commentary, we discuss the need for more extensive analytical characterization in relation to concerns about protein instability in injectable drug delivery systems for sustained release. We then will briefly address im- munogenicity concerns and outline current best practices for using state-of-the-art analytical assays to monitor protein stability for both co...
182 CitationsSource
#2Wim JiskootH-Index: 84
15 CitationsSource
#1James G. Barnard (University of Colorado Denver)H-Index: 5
#2Matthew N. Rhyner (Beckman Coulter)H-Index: 2
Last. John F. Carpenter (University of Colorado Denver)H-Index: 106
view all 3 authors...
ABSTRACT: In this paper, we evaluate the suitability of Coulter method for detecting and quantifying subvisible particles in protein solutions and compare results with other particle-counting technologies. The effects of key instrument and operational parameters such as aperture diameter, solution conductivity, and cleaning procedures are demonstrated. Degraded and nondegraded intravenous immunoglobulin and human serum albumin were chosen as model proteins and sample types for this evaluation. M...
29 CitationsSource
Cited By6
#1Xinyi Hua (Hunan University of Science and Technology)H-Index: 2
#2Zhichao Yang (Hunan University of Science and Technology)
Last. Haowen Huang (Hunan University of Science and Technology)H-Index: 23
view all 8 authors...
Abstract Modification of proteins is widespread throughout nature responsible for the vast biodiversity. In this study, we developed a facile method to rapid and selectively modify heme of hemoglobin (Hb) with tartaric acid to form complex (TA-Hb), which exhibits strong peroxidase-like activity comparable to the native horseradish peroxidase (HRP). The structural change of Hb accompanied by the formation of TA-Hb complex was validated by UV-vis absorption, fluorescence, fluorescence lifetime and...
#1Stephanie K. Vargas (BioMarin Pharmaceutical)H-Index: 1
#2Aydin Eskafi (BioMarin Pharmaceutical)H-Index: 1
Last. Natalie Ciaccio (BioMarin Pharmaceutical)H-Index: 1
view all 4 authors...
Abstract A recently developed high-throughput background membrane imaging (BMI) technique, the HORIZON, was assessed for its ability to quantify subvisible particles (SVP) generated during protein therapeutic development. The HORIZON platform method was optimized and compared to three well-characterized SVP counting techniques: light obscuration, micro-flow imaging (MFI), and FlowCam®. A head-to-head comparison was performed for precision, linearity, SVP concentration, and morphological output o...
3 CitationsSource
#1Anja MatterH-Index: 3
#2Atanas KoulovH-Index: 13
Last. Roman MathaesH-Index: 9
view all 8 authors...
Abstract Subvisible particles (SVPs) are an obligatory critical quality attribute of the product, and yet, they are found in all biopharmaceutical products intended for infusion or injection. Light obscuration (LO) is the primary pharmacopeial method used to quantify SVPs. However, the method may not be equally sensitive toward all particles that can possibly occur. Calibration of LO instruments is usually performed using polystyrene beads suspended in water. In this study, the dependence of the...
3 CitationsSource
#1Wei Wang (Bayer)H-Index: 11
#2Christopher J. Roberts (UD: University of Delaware)H-Index: 42
Abstract Protein aggregation has been recognized as one of the major challenges in the development and commercialization of successful protein-based drug products because of the inherent and high aggregation tendency during various manufacturing processes, storage, and administration. Protein aggregates may impact significantly the product quality, safety and/or efficacy. This review is intended to summarize four major aspects of protein aggregation – (1) aggregation mechanisms, (2) aggregation-...
61 CitationsSource
This minireview focuses on novel degradation pathways of proteins in solution via intermediary tryptophan (Trp) radical cations, which are generated via photo-induced electron transfer to suitable acceptors such as disulfide bonds.Gas-phase mass spectrometry studies had indicated the potential for Trp radical cations to fragment via release of 3-methylene-3H-indol-1-ium from the side chain. HPLC-MS/MS analysis demonstrates that analogous fragmentation reactions occur during the exposure of pepti...
14 CitationsSource
#1Anacelia Ríos Quiroz (University of Basel)H-Index: 3
#2Christof Finkler (Hoffmann-La Roche)H-Index: 11
Last. Atanas Koulov (Hoffmann-La Roche)H-Index: 13
view all 6 authors...
Abstract A number of new techniques for subvisible particle characterization in biotechnological products have emerged in the last decade. Although the pharmaceutical community is actively using them, the current knowledge about the analytical performance of some of these tools is still inadequate to support their routine use in the development of biopharmaceuticals (especially in the case of submicron methods). With the aim of increasing this knowledge and our understanding of the most prominen...
14 CitationsSource