Monoclonal antibodies (mAbs) are prone to post-translational modifications during manufacturing and storage, which can affect product quality. Modifications within the complementarity-determining regions (CDRs) impairing antigen interaction are considered as critical quality attributes. A modular workflow was developed using analytical antigen affinity chromatography to enable functional separation of mAb variants based on their antigen affinity. The workflow was optimized using trastuzumab and its antigen, human epidermal growth factor receptor-2 (Her2). A linear pH gradient (pH 7.4 - 2.5) was employed to separate binding-deficient variants, which were fractionated and characterized using surface plasmon resonance (SPR), size exclusion chromatography (SEC), ion exchange chromatography (IEC), reducing capillary gel electrophoresis (CGE), and peptide mapping. Critical modifications, such as HC-D102 isomerization and LCN30 succinimide (Asu) formation, were identified as contributors to binding loss, with HC-D102 isomerization showing the most significant impact. Binding was reduced by up to 6 % in deficient fractions. The workflow was applied to two additional mAbs, demonstrating its adaptability. For mAb1 variants, binding activity decreased 10 % with HCCDR isomerization and Asu formation being critical, while for mAb2, distinct variants with binding activity ranging from 2 % to 66 % were resolved. In mAb2, Asu formation in HCCDR and LCCDR was identified as the primary modification contributing to binding loss and was found to be irreversible under physiological-like conditions (37 °C, pH 7.4). This approach enables the targeted identification of critical modifications, supports early risk mitigation as well as control strategies during development, and ensures comprehensive characterization of mAb variants.

Monoclonal antibodies (mAbs) can mediate their therapeutic activity through antigen binding and by engaging Fc receptors (FcRs) or the complement system. FcRs are expressed on immune cells and can initiate effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis. The conserved Fc N-glycans of immunoglobulin G (IgG) at N297 modulate the strength and stability of Fc-FcγR interactions and the resulting effector responses. Conventional analysis typically considers individual glycans in isolation and neglects the fact that each Fc part carries two N-glycans, that may be paired symmetrically or asymmetrically. Recent studies with IgG Fc regions have demonstrated that glycan pairing can significantly shape Fc-FcR interactions. Here, the effect of glycan pairing was evaluated with full-length IgG using rituximab as a model. A set of pure, homogeneous, symmetrical, and asymmetrical Fc glyco-pair variants was generated by controlled chemoenzymatic remodeling and purification. Surface plasmon resonance binding studies across a comprehensive panel of human FcγRs revealed receptor- and allotype-specific effects: FcγRI binding was essentially unaffected, FcγRIIa showed allotype dependence, with FcγRIIa-H131 predominantly modulated by galactosylation and FcγRIIa-R131 by afucosylation, whereas FcγRIIIa required only a single afucosylation to reach maximal binding. Galactosylation exerted modest and receptor-dependent effects, most pronounced for FcγRIIIb. High-mannose species reduced binding to FcγRI and FcγRII, but displayed intermediate affinity to FcγRIIIa, which was lower than afucosylated complex types yet higher than fully fucosylated variants. To evaluate the biological relevance, cell-based ADCC reporter gene assays confirmed that single afucosylation was sufficient to drive the increase in ADCC potency, with galactosylation and high-mannose contributing only modest or context-dependent effects. Together, these findings establish glycan pairing as a critical quality attribute of therapeutic antibodies and provide a framework for precise evaluation of Fc N-glycan criticality and for tailoring mAbs with defined effector function profiles.

Although therapeutic proteins are susceptible to visible light-induced photooxidation, the underlying mechanisms remain unclear because amino acid residues do not directly absorb light above 400 nm. To evaluate the role of light source characteristics, three monoclonal antibodies formulated in water were exposed to spectrally distinct fluorescent and light-emitting diode (LED) sources, eliminating excipient-related effects. Following exposure to visible light (400-800 nm), degradation was assessed by size-exclusion, Protein A, and ion-exchange chromatography, together with mass spectrometry. The LED spectrum displayed a pronounced emission at 451 nm and a broad band between 480 and 800 nm, whereas the fluorescent lamp exhibited prominent emissions at 436, 545, and 612 nm, with additional blue-light contributions overlapping antibody absorption. Both light sources induced dose- and antibody-dependent photodegradation, characterized by increased high molecular weight species, Fc-oxidation, acidic and basic charge variants, and methionine and tryptophan oxidation, without detectable fragmentation. Monomer content decreased by 1-4 %, Fc-oxidized variants increased by 5-20 %, and the ion-exchange main peak decreased by 2-8 %. LED-induced degradation was less pronounced than that induced by fluorescent light, reflecting reduced short-wavelength emission. These findings support photosensitizer-mediated oxidation at 400-450 nm and highlight the importance of controlling light exposure during bioprocessing.

The pharmacokinetics (PK) of therapeutic monoclonal antibodies (mAbs) are influenced by N-glycosylation, a critical quality attribute (CQA) that affects serum half-life and receptor interactions. High-mannose N-glycans are known to accelerate mAb clearance, likely via the mannose receptor (MR). However, the impact of high-mannose glycan pairing - whether symmetrical or asymmetrical - on this process remains poorly understood. MAbs enriched in high-mannose N-glycans were fractionated using mannose receptor—affinity chromatography to isolate symmetrical and asymmetrical high-mannose glyco-pairs. These fractions were characterized for physicochemical properties and labeled for a cell-based internalization assay using MR-expressing SUP-B15 cells to quantify internalization rates via flow cytometry. A PK study in rats was conducted using a high-mannose enriched mAb drug product, and glyco-pair-specific clearance was analyzed by mass spectrometry following immunocapture. Symmetrical high-mannose glyco-pair exhibited the highest internalization rate in vitro, followed by asymmetrical high-mannose and symmetrical complex glyco-pairs. In vivo, symmetrical high-mannose glyco-pair showed the fastest clearance, with a half-life of 2.4 days, compared to 7.2 days for asymmetrical high-mannose and 17.4 days for symmetrical complex glyco-pairs. The area under the curve was reduced to 73% and 38% for asymmetrical and symmetrical high-mannose glyco-pairs, respectively, relative to the symmetrical complex glyco-pair. These differences were attributed solely to glycan pairing, as other physicochemical properties remained consistent across fractions. Competitive inhibition with mannan confirmed MR-mediated uptake in vitro. The findings also suggest potential implications for immunogenicity, as increased internalization by antigen-presenting cells may enhance antigen presentation and anti-drug antibody formation. This study demonstrates that glycan pairing significantly influences the PK of mAbs by correlating with differential MR interactions, providing evidence for MR-mediated faster clearance of high-mannose containing mAbs. Symmetrical high-mannose glyco-pair are cleared more rapidly than asymmetrical counterparts, underscoring the need to consider glycan pairing as a distinct CQA. Current control strategies based solely on released N-glycans may overlook clinically relevant heterogeneity. Incorporating high-mannose glyco-pair analysis into manufacturing and quality control processes could improve therapeutic consistency and reduce immunogenicity risks.

The viscosity of high-concentration protein solutions is a critical parameter in biopharmaceutical formulation development. Conventionally, the viscosity is measured and optimized in labor-intensive experimental workflows that require a lot of material. While predicting the viscosity with atomistic molecular dynamics (MD) simulations is feasible, they are computationally prohibitively expensive due to the large system sizes and the long simulation times involved. Coarse-grained MD (CG-MD) simulations significantly reduce computational demands, but evaluating their accuracy and predictive power requires rigorous validation. Here, we assess the capability of the Martini 3 CG force field to predict the viscosity of high-concentration antibody solutions. We show that a refined Martini 3 force field, with optimized protein-protein interactions, can predict the elevated viscosities observed in concentrated solutions of F(ab')2 fragments of the therapeutic monoclonal antibody (mAb) omalizumab. Furthermore, we show that our previously developed Martini 3-exc model for arginine excipients successfully captures the trend of lowering viscosity, as observed in our rheology experiments. These findings open the way to physics-based computational prediction of the properties of dense biopharmaceutical solutions via large-scale MD simulations.

Understanding the structure and self-organisation of monoclonal antibodies (mAbs) at the air–water interface is crucial for the stability and efficacy of protein drug formulations. This paper investigates the competitive adsorption of mAb and two amphiphilic polymers, poloxamer 188 (P188) and polysorbate 20 (PS20), commonly used to stabilise mAb formulations. Our objective was twofold: to ascertain whether the surfactants in question are capable of preventing mAb adsorption; and to determine whether it is possible to desorb mAb molecules from the air–water interface by surfactant addition. Langmuir film balance measurements and drop shape tensiometry were used to obtain surface pressure and surface tension data. Infrared Reflection–Absorption Spectroscopy (IRRAS) provided information on the surface composition, including the amount of adsorbed molecules. The state adopted by P188 is contingent upon its surface concentration, which determines the self-assembled phases it adopts. We show that the phase state of P188 has a considerable influence on mAb adsorption. The presence of P188 in the brush phase (≥ 0.3 mg/L) consistently inhibits mAb adsorption, but addition of P188 subsequent to the formation of the mAb film does not result in mAb desorption. However, addition of PS20 results in the desorption of freshly-formed interfacial mAb layers of up to two hours’ age, whereas an aged mAb layer of 17 h was unable to be desorbed by PS20. Thus there is a time-dependent reorganisation of mAb at the air–water interface, increasing resistance to desorption, which we discuss in the context of potential intermolecular interactions within the interfacial film.

The high doses of drugs required for biotherapeutics, such as monoclonal antibodies (mAbs), and the small volumes that can be administered to patients by subcutaneous injections pose challenges due to high-concentration formulations. The addition of excipients, such as arginine and glutamate, to high-concentration protein formulations can increase solubility and reduce the tendency of protein particle formation. Molecular dynamics (MD) simulations can provide microscopic insights into the mode of action of excipients in mAb formulations but require large system sizes and long time scales that are currently beyond reach at the fully atomistic level. Computationally efficient coarse-grained models such as the Martini 3 force field can tackle this challenge but require careful parametrization, testing, and validation. This study extends the popular Martini 3 force field toward realistic protein-excipient interactions of arginine and glutamate excipients, using the Fab domains of the therapeutic mAbs trastuzumab and omalizumab as model systems. A novel all-atom to coarse-grained mapping of the amino acid excipients is introduced, which explicitly captures the zwitterionic character of the backbone. The Fab-excipient interactions of arginine and glutamate are characterized concerning molecular contacts with the Fabs at the single-residue level. The Martini 3 simulations are compared with results from all-atom simulations as a reference. Our findings reveal an overestimation of Fab-excipient contacts with the default interaction parameters of Martini 3, suggesting a too strong attraction between protein residues and excipients. Therefore, we reparametrized the protein-excipient interaction parameters in Martini 3 against all-atom simulations. The excipient interactions obtained with the new Martini 3 mapping and Lennard-Jones (LJ) interaction parameters, coined Martini 3-exc, agree closely with the all-atom reference data. This work presents an improved parameter set for mAb-arginine and mAb-glutamate interactions in the Martini 3 coarse-grained force field, a key step toward large-scale coarse-grained MD simulations of high-concentration mAb formulations and the stabilizing effects of excipients.

Monoclonal antibodies' Fc N-glycans play a crucial role in their therapeutic efficacy, as they influence effector functions through Fc receptor binding. However, the impact of asymmetrical Fc glyco-pairs is often overlooked in assessing Fc receptor binding and effector functions. This study addresses this gap by generating pure asymmetrical Fc glyco-pairs and evaluating their Fc receptor binding properties, thereby providing a comprehensive understanding of the impact of Fc N-glycans. Utilizing redox pairing and affinity chromatography, homogeneously asymmetrical Fc glyco-pairs were generated, and their interaction properties toward Fcγ receptors IIIa, IIa, IIb, and I were determined by surface plasmon resonance. The results underscore the importance of considering the apparent glycan distribution of Fc N-glycans as glycan pairing was found to individually influence Fc receptor binding. Notably, single afucosylation significantly increased the affinity for FcγRIIIa, while the effect of galactosylation was detectable but less pronounced. Galactosylation, however, played a crucial role in FcγRIIa binding, with asymmetrical galactosylation being sufficient for the whole effect. In contrast, for FcγRIIb, afucosylation was more important, while galactosylation played a minor role. Furthermore, glycosylation-dependent Fc-FcγRI complex stability differences could be resolved, challenging the commonly held belief that this interaction is glycosylation independent.

During the development process of therapeutic monoclonal antibodies (mAbs), it is crucial to control (critical) quality attributes such as N-glycosylation influencing pharmacokinetics (PK) and Fc effector functions. Previous reports have shown that mAbs containing high-mannose N-glycans are cleared faster from blood circulation, leading to reduced half-lives. The high-mannose N-glycan content of mAbs can be influenced during the cell culture process by factors such as cell lines, process conditions, and media. Furthermore, mAbs have either one high mannose N-glycan (asymmetrical high-mannose glyco-pair) or two high mannose N-glycans (symmetrical high-mannose glyco-pair). The hypothesis that the mannose receptor (MR, CD206) accelerates clearance by facilitating their internalization and subsequent lysosomal degradation is widespread. However, the interaction between MR and mAbs has not been explicitly demonstrated. This study aimed to investigate this interaction, providing the first systematic demonstration of MR binding to the Fc region of mAbs with high-mannose N-glycans. Two novel analytical methods, MR surface plasmon resonance and MR affinity chromatography, were developed and applied to investigate the MR-mAb interaction. The interaction is found to be dependent on high-mannose content, but is independent of the mAb format or sequence. However, different glyco-pairs exhibited varying binding affinities to the MR, with the symmetrical high-mannose glyco-pair showing the strongest binding properties. These findings strengthen the hypothesis for the MR-mediated mAb interaction and contribute to a deeper understanding of the MR-mAb interaction, which could affect the criticality of high-mannose containing mAbs development strategies of IgG-based molecules and improve their PK profiles.

Protein formulations may form proteinaceous particles that vary in size from nanometers to millimeters. Monitoring the kinetics of protein particle formation, e.g., through accelerated degradation studies, is an attempt to understand and assess the rate and progression of particle populations. Little is known about whether the initial level of high molecular weight (HMW) species, or initial HMW level (IHL), of a protein solution influences the propagation of protein particle formation, and thus affects the storage stability of proteins.In this study, we have established a method to generate protein solutions of different IHLs by thermal stress. We have evaluated a 16-week thermal stability study at 40 °C of two monoclonal antibodies (mAb-A and mAb-B) at different IHLs using size exclusion chromatography (SEC) and sub-visible particle analysis. We have performed an isothermal stress study with guanidinium hydrochloride (GuaHCl) at room temperature for 300-min to evaluate the formation of HMWs analysed by SEC. The application of the Finke-Watzky (F-W) two-step nucleation model allowed us to mathematically describe the kinetics of HMW formation and to extract kinetic parameters of this process.For mAb-A, the IHLs had a marginal influence on the loss of monomer rate; instead, mAb-A exhibited fragmentation at 40 °C, which was independent of the IHL. Nevertheless, above a threshold of ≥ 7 % IHL, existing trimers/tetramers undergo conversion into higher-order oligomers at 40 °C, which is not observed at lower IHLs. In contrast, mAb-B exhibited an increased HMW formation rate above a threshold of ≥ 4 % IHL, which was reflected in the monomer decay rates at 40 °C and the F-W kinetic parameters of the chemical stress study.This case study shows that the initial level of HMWs exerts a differential influence on the progression of HMW formation. In one instance, there is a discernible acceleration in the formation of HMWs with rising IHLs. Conversely, in another example, the IHL exerts only a slight influence on HMW formation. Moreover, the results of our short-term chemical stress study are in accordance with those of a classical storage stability study conducted at 40 °C, which evaluated different IHLs. The analysis of HMW formation kinetics will enhance our understanding of the protein particle formation process and facilitate the formulation development of biotherapeutics.

N-glycosylation of the Fc part is a (critical) quality attribute of therapeutic antibodies and Fc-containing biotherapeutics, that impacts their stability, immunogenicity, pharmacokinetics, and effector functions. Current glycosylation analysis methods focus on the absolute amounts of glycans, neglecting the apparent glycan distribution over the entirety of proteins. The combination of the two Fc N-glycans, herein referred to as glyco-pair, therefore remains unknown, which is a major drawback for N-glycan impact assessment. This study presents a comprehensive workflow for the analysis and characterization of Fc N-glycan pairing in biotherapeutics, addressing the limitations of current glycosylation analysis methods. The applicability of the method across various biotherapeutic proteins including antibodies, bispecific antibody formats, and a Fc-Fusion protein is demonstrated, and the impact of method conditions on glycan pairing analysis is highlighted. Moreover, the influence of the molecular format, Fc backbone, production process, and cell line on glycan pairing pattern was investigated. The results underscore the significance of comprehensive glycan pairing analysis to accurately assess the impact of N-glycans on important product quality attributes of therapeutic antibodies and Fccontaining biotherapeutics.

Silicone oil droplets in biopharmaceutical products can originate from sources such as siliconized surfaces of primary packaging materials, potentially triggering the formation of protein–silicone oil particles. To better understand this phenomenon, there is a need for particle detection devices that cannot only distinguish between protein particles and silicone oil droplets but also determine particle sizes ranging from nanometers to micrometers. In this study, we conducted a systematic assessment of imaging flow cytometry (IFC) using the FlowSight® instrument. Our first step was to investigate specific instrument settings using protein particle samples spiked with silicone oil for particle classification. Based on these findings, we established suitable, harmonized working templates. Next, we evaluated the instrument’s accuracy and precision for particle sizes within the range of 0.5 to 100 µm and their respective concentrations. Finally, we investigated any constraints in particle concentration within this size range. This study demonstrates that IFC can effectively distinguish protein particles from silicone oil droplets when the latter is labeled with a specific fluorescent dye. Our findings suggest that fluorescently labeled particles ≥ 0.5 µm can be reliably detected. Through our research, we determined the particle concentration limits for each particle size in the range of 0.5 to 10 µm, with a precision deviation of less than 15

Protein formulation development relies on the selection of excipients that inhibit protein-protein interactions preventing aggregation. Empirical strategies involve screening many excipient and buffer combinations by physicochemical characterization using forced degradation or temperature-induced stress, mostly under accelerated conditions. Such methods do not readily provide information on the inter- and intramolecular interactions responsible for the effects of excipients. Here, we describe a combined experimental and computational approach for investigating the effect of protein-excipient interactions on formulation stability, which allows the identification of preferential interaction sites and thus can aid in the selection of excipients to be experimentally screened. Model systems composed of two marketed therapeutic IgG1 monoclonal antibodies with identical Fc domain sequences, trastuzumab and omalizumab, were investigated with commonly used excipients arginine, glutamate, and equimolar arginine/glutamate mixtures. Protein-excipient interactions were studied using all-atom molecular dynamics (MD) simulations, which show accumulation of the excipients at specific antibody regions. Preferential excipient-interaction sites were particularly found for charged and aromatic residues and in the complementary-determining regions, with more pronounced arginine contacts for omalizumab than trastuzumab. These computational findings are in line with the more pronounced stabilizing effects of arginine observed in the long-term storage stability study. Furthermore, the aggregation and solubility propensity predicted by commonly used in silico tools do not align with the preferential excipient-interaction sites identified by the MD simulations, suggesting that different physicochemical mechanisms are at play.

Nanoparticle tracking analysis (NTA) is an emerging technique for the analysis of particles in the submicron range of 50–1000 nm. It tracks the Brownian motion of individual particles and calculates the diffusion coefficient and subsequently the hydrodynamic diameter based on the Stokes-Einstein equation. In this study, we provide guidance on the capabilities and limitations using NTA for particle analysis. We have used polystyrene (PS) particle size standards to evaluate various experimental parameters such as the influence of particle concentration, measurement temperature, and neutral density (ND) filter on sizing and counting. We have also used bimodal samples in different ratios to assess the resolution power of NTA as well as trimodal samples to evaluate two different analysis algorithms. Within the working range of 106–109 particles/mL, lower particle concentrations of monomodal samples lead to an increase in the detected particle size but allow for more accurate particle concentration measurements. The measurement temperature in the range of 21 °C to 29 °C causes a trend of increasing particle size up to 8

Bispecific antibodies (BsAbs) capable of recognizing two distinct epitopes or antigens offer promising therapeutic options for various diseases by targeting multiple pathways. The favorable pharmacokinetic (PK) properties of monoclonal antibodies (mAbs) are crucial, as they directly influence patient safety and therapeutic efficacy. For numerous mAb therapeutics, optimization of neonatal Fc receptor (FcRn) interactions and elimination of unfavorable molecular properties have led to improved PK properties. However, many BsAbs exhibit unfavorable PK, which has precluded their development as drugs. In this report, we present studies on the molecular determinants underlying the distinct PK profiles of three IgG1-scFv BsAbs. Our study indicated that high levels of nonspecific interactions, elevated isoelectric point (pI), and increased number of positively charged patches contributed to the fast clearance of IgG1-scFv. FcRn chromatography results revealed specific scFv-FcRn interactions that are unique to the IgG1-scFv, which was further supported by molecular dynamics (MD) simulation. These interactions likely stabilize the BsAb FcRn interaction at physiological pH, which in turn could disrupt FcRn-mediated BsAb recycling. In addition to the empirical observations, we also evaluated the impact of in silico properties, including pI differential between the Fab and scFv and the ratio of dipole moment to hydrophobic moment (RM) and their correlation with the observed clearance. These findings highlight that the PK properties of BsAbs may be governed by novel determinants, owing to their increased structural complexity compared to immunoglobulin G (IgG) 1 antibodies.

Biological drugs intended for multi-dose application require the presence of antimicrobial preservatives to avoid microbial growth. As the presence of certain preservatives has been reported to increase protein and peptide particle formation, it is essential to choose a preservative compatible with the active pharmaceutical ingredient in addition to its preservation function. Thus, this review describes the current status of the use of antimicrobial preservatives in biologic formulations considering (i) appropriate preservatives for protein and peptide formulations, (ii) their physico-chemical properties, (iii) their in-/compatibilities with other excipients or packaging material, and (iv) their interactions with the biological compound. Further, (v) we present an overview of licensed protein and peptide formulations.

The computational prediction of the viscosity of dense protein solutions is highly desirable, for example, in the early development phase of high-concentration biopharmaceutical formulations where the material needed for experimental determination is typically limited. Here, we use large-scale atomistic molecular dynamics (MD) simulations with explicit solvation to de novo predict the dynamic viscosities of solutions of a monoclonal IgG1 antibody (mAb) from the pressure fluctuations using a Green-Kubo approach. The viscosities at simulated mAb concentrations of 200 and 250 mg/mL are compared to the experimental values, which we measured with rotational rheometry. The computational viscosity of 24 mPa·s at the mAb concentration of 250 mg/mL matches the experimental value of 23 mPa·s obtained at a concentration of 213 mg/mL, indicating slightly different effective concentrations (or activities) in the MD simulations and in the experiments. This difference is assigned to a slight underestimation of the effective mAb-mAb interactions in the simulations, leading to a too loose dynamic mAb network that governs the viscosity. Taken together, this study demonstrates the feasibility of all-atom MD simulations for predicting the properties of dense mAb solutions and provides detailed microscopic insights into the underlying molecular interactions. At the same time, it also shows that there is room for further improvements and highlights challenges, such as the massive sampling required for computing collective properties of dense biomolecular solutions in the high-viscosity regime with reasonable statistical precision.

The presence of visible and sub-visible particles is a critical quality attribute of biotherapeutics that needs to be monitored closely. Backgrounded membrane imaging (BMI) is an alternative technique to determine the size and concentration of particles in liquid formulations. The particles are imaged in a dry state on a membrane, as the liquid phase is removed during the preparation of BMI samples. Therefore, the refractive index difference (ΔRI) of the particle to the background is drastically increased and the detection of translucent particles improved. In this study, we compared BMI to established methods such as microflow imaging (MFI) and light obscuration (LO). We have evaluated several experimental parameters, such as the sample volume and the washing routine, to determine a suitable handling procedure for optimizing the accuracy and reproducibility of the measurements. Particle concentrations of polystyrene and protein samples were compared using BMI, MFI, and LO. Furthermore, the influence of various additives, as well as hard- and software parameters on BMI measurements, was evaluated. BMI is a high-throughput microscopic method that requires a sample volume of only 25 µl to monitor particles as small as 2 µm. This enables particle detection in case of limited sample availability, but the final particle concentrations are calculated by data extrapolation to particle counts in 1 ml. During the verification of the system’s performance, we have observed a clustering of the hydrophobic polystyrene standard beads on the hydrophilic membrane. This can be avoided by using negatively charged polystyrene particle standard beads. For stressed monoclonal antibody (mAb) samples, the particle concentrations determined by BMI were about one to two orders of magnitude increased in comparison to MFI and LO. Furthermore, the influence of various additives such as sucrose, polysorbate 20, silicone oil, and glycerol (to mimic high viscous samples) was evaluated. Critical hard- and software parameters such as the camera focus, the particle detection algorithm, and the sample efficiency, which have impaired the measurements, as well as accuracy of BMI measurements were discussed. We have addressed several new aspects to improve the experimental procedure and accuracy of BMI measurements. In general, BMI is an alternative method to determine particle size and concentration in pharmaceutical products, especially if the sample volume is limited.

Sub-visible particles (SVPs) in pharmaceutical products are a critical quality attribute, and therefore should be monitored during development. Although light obscuration (LO) and microscopic particle count tests are the primary pharmacopeial methods used to quantify SVPs, flow imaging methods like Micro-Flow Imaging (MFITM) appear to overcome shortcomings of LO such as limited sensitivity concerning smaller translucent SVPs in the size range < 10 mu m. Nowadays, MFITM is routinely utilized during development of biologicals. Oftentimes multiple devices are distributed across several laboratories and departments. This poses challenges in data interpretation and consistency as well as in the use of multiple devices for one purpose. In this study, we sys-tematically evaluated seven MFITM instruments concerning their counting and size precision and accuracy, using an inter-comparable approach to mimic daily working routine. Therefore, we investigated three different types of particles (i) NIST certified counting standards, (ii) protein-coated particles, and (iii) stress-induced particles from a monoclonal antibody. We compared the results to alternative particle detection methods: LO and Back -grounded Membrane Imaging (BMI). Our results showed that the precision and accuracy of particle count and size, as well as the comparability of instruments, depended on the particle source and its material properties. The various MFITM instruments investigated showed high precision (<15 %) and data generated on different in-struments were of the same order of magnitude within pharmacopeial relevant size ranges for NIST certified counting standards. However, we found limitations in the upper and lower detection limits, contrary to the limits claimed by the manufacturer. In addition, proteinaceous and protein-containing particles showed statistically significant differences in particle counts, while the measured particle diameters of all sizes were quite consistent.