Qualitative High-Throughput Analysis of Subvisible Particles in Biological Formulations Using Backgrounded Membrane Imaging

Murphy, Markela Ibo; Bruque, Maria; Hanford, Alexis; Trayton, Isabelle; Handali, Melody; Leissa, Jesse A.; Hasige, Sathish; Day, Katie; Patel, Sajal M.

doi:10.1016/j.xphs.2022.03.010

Cited by 8 publications

(3 citation statements)

References 21 publications

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“…Subvisible particulate matter was analyzed by a backgrounded membrane imaging (BMI) technique using the Horizon equipment from Halo Labs (Burlingame, CA). This high-throughput technique enables particle characterization and quantification using sample volumes as low as 25 μL and presents high sensitivity due to the large difference in the refractive indexes of proteins and air when compared with methods that rely on the analysis of protein in solution, which presents a lower difference in the refractive indexes . After protein adsorption to the air–water interface for 120 min, samples were carefully aspirated from the interface with a syringe and a 31-gauge needle.…”

Section: Methodsmentioning

confidence: 99%

“…This high-throughput technique enables particle characterization and quantification using sample volumes as low as 25 μL and presents high sensitivity due to the large difference in the refractive indexes of proteins and air when compared with methods that rely on the analysis of protein in solution, which presents a lower difference in the refractive indexes. 55 After protein adsorption to the air−water interface for 120 min, samples were carefully aspirated from the interface with a syringe and a 31-gauge needle. At the beginning of each run, an unused 96-well membrane was imaged before sample loading.…”

Section: Materials the National Institute Of Standards Andmentioning

confidence: 99%

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Using Passive Microrheology to Measure the Evolution of the Rheological Properties of NIST mAb Formulations during Adsorption to the Air–Water Interface

Escobar,

Vaclaw,

Lozenski

et al. 2024

Langmuir

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The development of novel protein-based therapeutics, such as monoclonal antibodies (mAbs), is often limited due to challenges associated with maintaining the stability of these formulations during manufacturing, storage, and clinical administration. An undesirable consequence of the instability of protein therapeutics is the formation of protein particles. MAbs can adsorb to interfaces and have the potential to undergo partial unfolding as well as to form viscoelastic gels. Further, the viscoelastic properties may be correlated with their aggregation potential. In this work, a passive microrheology technique was used to correlate the evolution of surface adsorption with the evolution of surface rheology of the National Institute of Standards and Technology (NIST) mAb reference material (NIST mAb) and interface-induced subvisible protein particle formation. The evolution of the surface adsorption and interfacial shear rheological properties of the NIST mAb was recorded in four formulation conditions: two different buffers (histidine vs phosphate-buffered saline) and two different pHs (6.0 and 7.6). Our results together demonstrate the existence of multiple stages for both surface adsorption and surface rheology, characterized by an induction period that appears to be purely viscous, followed by a sharp increase in protein molecules at the interface when the film rheology is viscoelastic and ultimately a slowdown in the surface adsorption that corresponds to the formation of solid-like or glassy films at the interface. When the transitions between the different stages occurred, they were dependent on the buffer/pH of the formulations. The onset of these transitions can also be correlated to the number of protein particles formed at the interface. Finally, the addition of polysorbate 80, an FDA-approved surfactant used to mitigate protein particle formation, led to the interface being surfactant-dominated, and the resulting interface remained purely viscous.

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Section: Methodsmentioning

confidence: 99%

Section: Materials the National Institute Of Standards Andmentioning

confidence: 99%

Using Passive Microrheology to Measure the Evolution of the Rheological Properties of NIST mAb Formulations during Adsorption to the Air–Water Interface

Escobar,

Vaclaw,

Lozenski

et al. 2024

Langmuir

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show abstract

“…However, sizing and counting is less reliable as compared to flow-imaging microscopy because the boundaries of low-contrast particles might not be detected correctly. 118 , 119 Moreover, low analyzed volumes result in low particle counts and high extrapolation factors, which is a statistical challenge for any particle analysis method. 120 Nevertheless, a relative comparison or ranking by using backgrounded membrane imaging or other low-volume methods to rule out bad candidates is often sufficient for early screenings.…”

Section: Orthogonal Approaches For the Selection Of Drug-like Antibodiesmentioning

confidence: 99%

Approaches to expand the conventional toolbox for discovery and selection of antibodies with drug-like physicochemical properties

Svilenov

Arosio

Menzen³

et al. 2023

mAbs

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Antibody drugs should exhibit not only high-binding affinity for their target antigens but also favorable physicochemical drug-like properties. Such drug-like biophysical properties are essential for the successful development of antibody drug products. The traditional approaches used in antibody drug development require significant experimentation to produce, optimize, and characterize many candidates. Therefore, it is attractive to integrate new methods that can optimize the process of selecting antibodies with both desired target-binding and drug-like biophysical properties. Here, we summarize a selection of techniques that can complement the conventional toolbox used to de-risk antibody drug development. These techniques can be integrated at different stages of the antibody development process to reduce the frequency of physicochemical liabilities in antibody libraries during initial discovery and to co-optimize multiple antibody features during early-stage antibody engineering and affinity maturation. Moreover, we highlight biophysical and computational approaches that can be used to predict physical degradation pathways relevant for long-term storage and in-use stability to reduce the need for extensive experimentation.

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Backgrounded Membrane Imaging—A Valuable Alternative for Particle Detection of Biotherapeutics?

et al. 2023

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Qualitative High-Throughput Analysis of Subvisible Particles in Biological Formulations Using Backgrounded Membrane Imaging

Cited by 8 publications

References 21 publications

Using Passive Microrheology to Measure the Evolution of the Rheological Properties of NIST mAb Formulations during Adsorption to the Air–Water Interface

Using Passive Microrheology to Measure the Evolution of the Rheological Properties of NIST mAb Formulations during Adsorption to the Air–Water Interface

Approaches to expand the conventional toolbox for discovery and selection of antibodies with drug-like physicochemical properties

Backgrounded Membrane Imaging—A Valuable Alternative for Particle Detection of Biotherapeutics?

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