How Subvisible Particles Become Invisible—Relevance of the Refractive Index for Protein Particle Analysis

Zölls, Sarah; Gregoritza, Manuel; Tantipolphan, Ruedeeporn; Wiggenhorn, Michael; Winter, Gerhard; Frieß, Wolfgang; Hawe, Andrea

doi:10.1002/jps.23479

Cited by 95 publications

(97 citation statements)

References 34 publications

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“…An increased RI of the formulation, leading to a decreased RI difference between particles and formulation, can impede a correct detection of protein particles by light-based techniques. Compared to LO, Micro-Flow Imaging (MFI) was shown to be slightly more robust against such a decreased RI difference (13,26).…”

Section: Sarah Zölls and Daniel Weinbuch Equally Contributed To This mentioning

confidence: 99%

Flow Imaging Microscopy for Protein Particle Analysis—A Comparative Evaluation of Four Different Analytical Instruments

Zölls

Weinbuch

Wiggenhorn³

et al. 2013

AAPS J

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Abstract. Flow imaging microscopy was introduced as a technique for protein particle analysis a few years ago and has strongly gained in importance ever since. The aim of the present study was a comparative evaluation of four of the most relevant flow imaging microscopy systems for biopharmaceuticals on the market: Micro-Flow Imaging (MFI)4100, MFI5200, Flow Cytometer And Microscope (FlowCAM) VS1, and FlowCAM PV. Polystyrene standards, particles generated from therapeutic monoclonal antibodies, and silicone oil droplets were analyzed by all systems. The performance was critically assessed regarding quantification, characterization, image quality, differentiation of protein particles and silicone oil droplets, and handling of the systems. The FlowCAM systems, especially the FlowCAM VS1, showed high-resolution images. The FlowCAM PV system provided the most precise quantification of particles of therapeutic monoclonal antibodies, also under impaired optical conditions by an increased refractive index of the formulation. Furthermore, the most accurate differentiation of protein particles and silicone oil droplets could be achieved with this instrument. The MFI systems provided excellent size and count accuracy (evaluated with polystyrene standards) especially the MFI5200 system. This instrument also showed very good performance for protein particles, also in case of an increased refractive index of the formulation. Both MFI systems were easier to use and appeared more standardized regarding measurement and data analysis as compared to the FlowCAM systems. Our study shows that the selection of the appropriate flow imaging microscopy system depends strongly on the main output parameters of interest and it is recommended to decide based on the intended application.

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Section: Sarah Zölls and Daniel Weinbuch Equally Contributed To This mentioning

confidence: 99%

Flow Imaging Microscopy for Protein Particle Analysis—A Comparative Evaluation of Four Different Analytical Instruments

Zölls

Weinbuch

Wiggenhorn³

et al. 2013

AAPS J

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“…It is important to note that differentiation of protein and silicone-oil particles with MFI proved problematic; even with the use of the discriminant analysis described in the methods. This is a problem observed in previous papers due to optical similarities between protein and silicone-oil particles with a diameter less than 4m (Zölls et al, 2013;Zolls et al, 2012). Table 2 presents the (apparent) protein concentrations in buffer-only (i.e.…”

Section: Rmm and Mfi Characterisation Of Silicone-oil Droplets In Mabmentioning

confidence: 93%

“…Through the use of the RMM nano sensor and RICS, the smaller-sized aggregate population was analysed, detecting particles < 0.5 m in diameter. There are limited published reports on the use of RMM (Panchal et al, 2014;Weinbuch et al, 2013b;Zölls et al, 2013), and to our knowledge, this is the first study reporting the use of both RMM sensors on the same solutions. A careful observation of size ranges detected by RMM micro and nano sensors (SI, Figure S1) revealed uncertainties regarding the use of both detectors.…”

Section: Considerations Regarding the Different Techniquesmentioning

confidence: 97%

“…For example, in the case of HIAC and MFI, both of which are optical-based particle counting techniques, the techniques are influenced by the refractive index difference between protein particles and the formulation. As highlighted by Ripple and Hu (Ripple and Hu, 2015) and Zölls et al (Zölls et al, 2013), the change in refractive index at higher concentrations has led to the underestimation of particle concentrations. Hence, although novel and emerging techniques may not be appropriate for quality control in their current state, they are capable of monitoring the early stages of aggregation, require minimal sample volume and are therefore directly relevant to early stages of formulation development.…”

Section: Considerations Regarding the Different Techniquesmentioning

confidence: 97%

“…The discriminant analysis used in this study is based on certain particle parameters relating to apparent optical properties and circularity (aspect ratio, intensity mean, intensity minimum and intensity standard deviation), devised by Weinbuch et al (Weinbuch et al, 2013b). However, due to some optical similarities between mAb and silicone-oil, the reliability of this analysis has previously been questioned for particles < 4m (Weinbuch et al, 2013b;Zölls et al, 2013;Zolls et al, 2012).…”

Section: Considerations Regarding the Different Techniquesmentioning

confidence: 99%

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Evaluation of aggregate and silicone-oil counts in pre-filled siliconized syringes: An orthogonal study characterising the entire subvisible size range

Shah

Rattray

Day³

et al. 2017

International Journal of Pharmaceutics

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Characterisation of particulates in therapeutic monoclonal antibody (mAb) formulations is routinely extended to the sub-visible size-range (0.1-, with the increased use of prefilled syringes (PFS), particle differentiation is required between proteinaceous and non-proteinaceous particles such as silicone-oil droplets. Here, three orthogonal techniques: Raster Image Correlation Spectroscopy (RICS), Resonance Mass Measurements (RMM) and Micro-Flow Imaging (MFI), were evaluated with respect to their sub-visible particle measurement and characterisation capabilities. Particle formation in mAb PFS solutions was evaluated with increasing polysorbate-20 (PS-20) concentrations. All three techniques provided complementary but distinct information on protein aggregate and silicone-oil droplet presence. PS-20 limited the generation of mAb aggregates during agitation, while increasing the number of silicone-oil droplets (PS-20 concentration dependent). MFI and RMM revealed PS-20 lead to the formation of larger micron-sized droplets, with RICS revealing an increase in smaller submicron droplets. Subtle differences in data sets complicate the apparent correlation between silicone-oil sloughing and mAb aggregates' generation. RICS (though the use of a specific dye) demonstrates an improved selectivity for mAb aggregates, a broader measurement size-range and smaller sample volume requirement. Thus, RICS is proposed to add value to the currently available particle measurement techniques and enable informed decisions during mAb formulation development. ABSTRACTCharacterisation of particulates in therapeutic monoclonal antibody (mAb) formulations is routinely extended to the sub-visible size-range (0.1-10 m). Additionally, with the increased use of pre-filled syringes (PFS), particle differentiation is required between proteinaceous and non-proteinaceous particles such as silicone-oil droplets. Here, three orthogonal techniques: Raster Image Correlation Spectroscopy (RICS), Resonance Mass Measurements (RMM) and Micro-Flow Imaging (MFI), were evaluated with respect to their sub-visible particle measurement and characterisation capabilities. Particle formation in mAb PFS solutions was evaluated with increasing polysorbate-20 (PS-20) concentrations. All three techniques provided complementary but distinct information on protein aggregate and silicone-oil droplet presence. PS-20 limited the generation of mAb aggregates during agitation, while increasing the number of silicone-oil droplets (PS-20 concentration dependant). MFI and RMM revealed PS-20 lead to the formation of larger micron-sized droplets, with RICS revealing an increase in smaller sub-micron droplets. Subtle differences in data sets complicate the apparent correlation between silicone-oil sloughing and mAb aggregates' generation. RICS (though the use of a specific dye) demonstrates an improved selectivity for mAb aggregates, a broader measurement size-range and smaller sample volume requirement. Thus, RICS is proposed to add value to the currently available pa...

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Trends in Formulation and Drug Delivery for Antibodies

Mahler

Mathäs

2017

Process Scale Purification of Antibodies

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How Subvisible Particles Become Invisible—Relevance of the Refractive Index for Protein Particle Analysis

Cited by 95 publications

References 34 publications

Flow Imaging Microscopy for Protein Particle Analysis—A Comparative Evaluation of Four Different Analytical Instruments

Flow Imaging Microscopy for Protein Particle Analysis—A Comparative Evaluation of Four Different Analytical Instruments

Evaluation of aggregate and silicone-oil counts in pre-filled siliconized syringes: An orthogonal study characterising the entire subvisible size range

Trends in Formulation and Drug Delivery for Antibodies

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