Evaluation of Neutralizing Antibodies against SARS-CoV-2 Variants after Infection and Vaccination Using a Multiplexed Surrogate Virus Neutralization Test.

BACKGROUND: The SARS-CoV-2 virus has mutated and evolved since the inception of the COVID-19 pandemic bringing into question the future effectiveness of current vaccines and antibody therapeutics. With evolution of the virus updated methods for the evaluation of the immune response in infected and vaccinated individuals are required to determine the durability of the immune response to SARS-CoV-2 variants. METHODS: We developed a multiplexed surrogate virus neutralization test (plex-sVNT) that simultaneously measures the ability of antibodies in serum to inhibit binding between angiotensin converting enzyme-2 (ACE2) and 7 SARS-CoV-2 trimeric spike protein variants, including wild type, B.1.1.7(α), B.1.351(ß), P.1(γ), B.1.617.2(δ), B.1.617.1(κ), and B.1.429(ε). The assay was validated against a plaque reduction neutralization test (PRNT).We evaluated 170 samples from 97 COVID-19 patients and 281 samples from 188 individuals that received the Pfizer-BioNTech or Moderna mRNA vaccines. RESULTS: The plex-sVNT demonstrated >96% concordance with PRNT. Antibody neutralization activity was significantly reduced for all SARS-CoV-2 variants compared to wild type in both the infected and vaccinated cohorts. There was a decline in overall antibody neutralization activity, within both cohorts, out to 5 months post infection or vaccination, with the rate of decline being more significant for the vaccinated. CONCLUSIONS: The plex-sVNT provides a correlative measure to PRNT and a convenient approach for evaluating antibody neutralization against SARS-CoV-2 variants. Neutralization of SARS-CoV-2 variants is reduced compared to wild type and declines over the ensuing months after exposure or vaccination within each cohort, however it is still unknown what degree of neutralizing capacity is protective.

A Fully Automated Multiplex Assay for Diagnosis of Lyme Disease with High Specificity and Improved Early Sensitivity.

Lyme borreliosis is a tick-borne disease caused by the Borrelia burgdorferisensu lato complex. Bio-Rad Laboratories has developed a fully automated multiplex bead-based assay for the detection of IgM and IgG antibodies to B. burgdorferi The BioPlex 2200 Lyme Total assay exhibits an improved rate of seropositivity in patients with early Lyme infection. Asymptomatic subjects from endemic and nonendemic origins demonstrated a seroreactivity rate of approximately 4% that was similar to other commercial assays evaluated in this study. Coupled to this result was the observation that the Lyme Total assay retained a high first-tier specificity of 96% while demonstrating a relatively high sensitivity of 91% among a well-characterized CDC Premarketing Lyme serum panel. The Lyme Total assay also performs well under a modified two-tier algorithm (sensitivity, 84.4 to 88.9%; specificity, 98.4 to 99.5%). Furthermore, the new assay is able to readily detect early Lyme infection in patient samples from outside North America.

Site-Specific Hydrolysis Reaction C-Terminal of Methionine in Met-His during Metal-Catalyzed Oxidation of IgG-1.

The metal-catalyzed oxidation by [Fe(II)(EDTA)](2-)/H2O2 of IgG-1 leads to the site-specific hydrolysis of peptide bonds in the Fc region. The major hydrolytic cleavage occurs between Met428 and His429, consistent with a mechanism reported for the site-specific hydrolysis of parathyroid hormone (1-34) between Met8 and His9 (Mozziconacci, O.; et al. Mol. Pharmaceutics 2013, 10 (2), 739-755). In IgG-1, to a lesser extent, we also observe hydrolysis reactions between Met252 and Ile253. After 2 h of oxidation (at pH 5.8, 37 °C) approximately 5% of the protein is cleaved between Met428 and His429. For comparison, after 2 h of oxidation, the amount of tryptic peptides containing a Met sulfoxide residue represents less than 0.1% of the protein. The effect of this site-specific hydrolysis on the conformational stability and aggregation propensity of the antibody was also examined. No noticeable differences in structural integrity and conformational stability were observed between control and oxidized IgG-1 samples as measured by circular dichroism (CD), fluorescence spectroscopy, and static light scattering (SLS). Small amounts of soluble and insoluble aggregates (3-6%) were, however, observed in the oxidized samples by UV-visible absorbance spectroscopy and size exclusion chromatography (SEC). Over the course of metal-catalyzed oxidation, increasing amounts of fragments were also observed by SEC. An increase in the concentration of subvisible particles was detected by microflow imaging (MFI).

A case report of parotid gland epithelioid hemangioendothelioma.

Epithelioid hemangioendothelioma (EHE) is a rare low-grade malignant vascular tumor. It mainly occurs in the liver, lungs, bones, and other parts of the body. Reports of epithelioid hemangioendothelioma in the parotid gland are rare in both domestic and international literature. Here, we present a case report of a parotid gland epithelioid hemangioendothelioma, including its complete clinical course and imaging findings, to improve the diagnosis and treatment of this unusual disease. Case presentation: The patient, a 75-year-old female, presented with a swelling around the right ear for 2 months and pain for 20 days. Enhanced MRI of the parotid gland revealed a well-defined, round mass with homogeneous signal intensity. The mass showed low signal intensity on T1-weighted imaging, high signal intensity on T2-weighted imaging, nodular low signal intensity within, significant high signal intensity on DWI sequence, low signal intensity on ADC sequence, and heterogeneous enhancement in the arterial phase after intravenous injection of Gd-DTPA. Nodular non-enhancing low signal intensity was observed internally, and slight clearance was seen in the venous phase. The initial diagnosis before surgery was a benign lesion, but after histopathological and immunohistochemical examination, it was confirmed as epithelioid hemangioendothelioma. Intervention: Complete tumor resection was performed. Results: The patient experienced a favorable recovery, with meticulous follow-up conducted for up to 1 year revealing no signs of recurrence or metastasis. Continued patient surveillance is ongoing to substantiate and validate the long-term efficacy of the treatment. Conclusion: Due to the extreme rarity of parotid gland epithelioid hemangioendothelioma, it often leads to a high misdiagnosis rate. The most common misdiagnosis is salivary gland lymphoma, followed by epithelioid hemangiosarcoma. When the lesion is multifocal, fusiform, with internal necrosis, and shows punctate low signal intensity on T2-weighted imaging, significant enhancement in the arterial phase, particularly with more pronounced peripheral enhancement, and persistent enhancement in the venous and delayed phases, epithelioid hemangioendothelioma should be considered. However, the current clinical diagnosis of epithelioid hemangioendothelioma still primarily relies on immunohistochemical methods.

Fluorogenic tagging methodology applied to characterize oxidized tyrosine and phenylalanine in an immunoglobulin monoclonal antibody.

PURPOSE: Metal-catalyzed oxidation (MCO) of proteins is of primary concern in the development of biotherapeutics as it represents a prominent degradation pathway with potential undesired biological and biotherapeutic consequences. METHODS: We developed a fluorogenic derivatization methodology to study the MCO of IgG1 using a model oxidation system, CuCl2/L-ascorbic acid. RESULTS: Besides the oxidation of Met, Trp and His residues, we detected significant oxidation of Phe and Tyr in IgG1. CONCLUSION: The fluorogenic derivatization method provides an alternative approach for the rapid detection of oxidized Tyr and Phe as their benzoxazole derivatives by fluorescence spectrometry and size exclusion chromatography coupled to fluorescence detection.

The photolysis of disulfide bonds in IgG1 and IgG2 leads to selective intramolecular hydrogen transfer reactions of cysteine Thiyl radicals, probed by covalent H/D exchange and RPLC-MS/MS analysis.

PURPOSE: The evaluation of photo-instability of biotherapeutic products is mandated by regulatory agencies. Photo-irradiation can induce oxidative modifications in proteins, which may lead to undesired biological and therapeutic consequences. Among the modifications, epimerization of amino acid residues can occur upon photo-irradiation of IgGs. METHODS: We show here, that UV irradiation (λ = 253.7 nm) of IgG1 and IgG2 leads to the formation of intermediary carbon-centered radicals, validated by covalent incorporation of deuterium into the protein primary sequence. RESULTS: By MS/MS analysis we identified the sites of deuterium incorporation, such as the sequence QD [303:304, HC], present in the peptide of VVSVLTVVHQDWLNGK [294:309, HC] in both IgG1 and IgG2, and V [111, LC] and K [116, LC], present in the peptide VTVLGQPK [109:116, LC] in IgG2. Both peptides are in the proximity of intrachain disulfide bonds. CONCLUSIONS: The exposure of IgG1 and IgG2 to UV-light (λ = 253.7 nm) generates specific carbon-centered radicals. The latter were evidenced by a covalent H-D exchange reaction that likely occurred through a hydrogen atom transfer reaction between cysteine thiyl radical and C-H bond.

Malignant proliferative ependymoma of the neck with lymph node metastasis: A case report.

BACKGROUND: Malignant proliferating trichilemmal tumor (MPTT) is an infrequent malignant neoplasm originating from cutaneous appendages, with only a handful of documented cases. This report delineates a unique instance of MPTT situated in the neck, accompanied by lymph node metastasis. A comprehensive exposition of its clinical trajectory and imaging manifestation is presented, aiming to enhance comprehension and management of this atypical ailment. CASE SUMMARY: Patient concerns: A 79-year-old male presented with a longstanding right neck mass persisting for over six decades, exhibiting recent enlargement over the past year. Diagnoses: Enhanced magnetic resonance imaging of the neck unveiled an elliptical mass on the right neck side, characterized by an ill-defined border and a heterogeneous signal pattern. The mass exhibited subdued signal intensity on T1-weighted imaging (T1WI) and a heterogeneous high signal on T2-weighted imaging (T2WI), interspersed with a lengthy T1 and T2 cystic signal motif. Close anatomical association with the submandibular gland joint was noted, and intravenous gadolinium diethylene triamine pentaacetic acid administration facilitated conspicuous enhancement. Substantial enhancement of the solid segment prompted an initial preoperative diagnosis of malignant nerve sheath tumor. However, post-surgery histopathological and immunohistochemical analysis conclusively confirmed the diagnosis as malignant hyperplastic external hair root sheath tumor. Intervention: Complete excision of the tumor was successfully executed. Outcomes: The patient experienced a favorable postoperative recovery. CONCLUSION: Malignant proliferative trichilemmal tumor external hair root sheath tumor is a cystic-solid lesion, appearing as low signal on T1WI images or high signal on T2WI with enhancement of the solid component. Suspicions of malignancy are heightened when the tumor border is indistinct, tissue planes are breached, or when linear or patchy high signals are observed in the subcutaneous tissue on T1 liver acquisition with volume acceleration enhanced images along with intermediate signal on T2WI and restricted diffusion on diffusion-weighted imaging images. Strong consideration for malignancy should arise if there are signs of compromised adjacent tissue relationships or direct invasion evident on imaging. We have incorporated the above-mentioned content into the entire manuscript.

Biotherapeutic formulation factors affecting metal leachables from stainless steel studied by design of experiments.

Trace amounts of metals are inevitably present in biotherapeutic products. They can arise from various sources. The impact of common formulation factors such as protein concentration, antioxidant, metal chelator concentration and type, surfactant, pH, and contact time with stainless steel on metal leachables was investigated by a design of experiments approach. Three major metal leachables, iron, chromium, and nickel were monitored by inductively coupled plasma-mass spectrometry. It was observed that among all the tested factors, contact time, metal chelator concentration, and protein concentration were statistically significant factors with higher temperature resulting in higher levels of leached metals. Within a pH range of 5.5-6.5, solution pH played a minor role for chromium leaching at 25°C. No statistically significant difference was observed due to type of chelator, presence of antioxidant, or surfactant. In order to optimize a biotherapeutic formulation to achieve a target drug product shelf life with acceptable quality, each formulation component must be evaluated for its impact.

Transient expression of antinuclear RNP-A antibodies in patients with acute COVID-19 infection.

Introduction: Viral infections have been implicated in the initiation of the autoimmune diseases. Recent reports suggest that a proportion of patients with COVID-19 develop severe disease with multiple organ injuries. We evaluated the relationship between COVID-19 severity, prevalence and persistence of antinuclear and other systemic and organ specific autoantibodies as well as SARS-CoV-2 infection specific anti-nucleocapsid (N) IgG antibodies and protective neutralizing antibody (Nab) levels. Methods: Samples from 119 COVID-19 patients categorized based on their level of care and 284 healthy subjects were tested for the presence and persistence of antinuclear and other systemic and organ specific autoantibodies as well as SARS-CoV-2 and neutralizing antibody levels. Results: The data shows significantly increased levels of anti RNP-A, anti-nucleocapsid and neutralizing antibody among patients receiving ICU care compared to non-ICU care. Furthermore, subjects receiving ICU care demonstrated significantly higher nucleocapsid IgG levels among the RNP-A positive cohort compared to RNP-A negative cohort. Notably, the expression of anti RNP-A antibodies is transient that reverts to non-reactive status between 20 and 60 days post symptom onset. Conclusions: COVID-19 patients in ICU care exhibit significantly higher levels of transient RNP-A autoantibodies, anti-nucleocapsid, and SARS-CoV-2 neutralizing antibodies compared to patients in non-ICU care.

Biologics formulation factors affecting metal leachables from stainless steel.

An area of increasing concern and scientific scrutiny is the potential contamination of drug products by leachables entering the product during manufacturing and storage. These contaminants may either have a direct safety impact on the patients or act indirectly through the alteration of the physicochemical properties of the product. In the case of biotherapeutics, trace amounts of metal contaminants can arise from various sources, but mainly from contact with stainless steel (ss). The effect of the various factors, buffer species, solution fill volume per unit contact surface area, metal chelators, and pH, on metal leachables from contact with ss over time were investigated individually. Three major metal leachables, iron, chromium, and nickel, were monitored by inductively coupled plasma-mass spectrometry because they are the major components of 316L ss. Iron was primarily used to evaluate the effect of each factor since it is the most abundant. It was observed that each studied factor exhibited its own effect on metal leachables from contact with ss. The effect of buffer species and pH exhibited temperature dependence over the studied temperature range. The metal leachables decreased with the increased fill volume (mL) per unit contact ss surface area (cm(2)) but a plateau was achieved at approximately 3 mL/cm(2). Metal chelators produced the strongest effect in facilitating metal leaching. In order to minimize the metal leachables and optimize biological product stability, each formulation factor must be evaluated for its impact, to balance its risk and benefit in achieving the target drug product shelf life.

An Industry Perspective on Compatibility Assessment of Closed System Drug-Transfer Devices for Biologics.

The Formulation Workstream of the BioPhorum Development Group (BPDG), an industry-wide consortium, has identified the increased use of closed system drug-transfer devices (CSTDs) with biologics, without an associated compatibility assessment, to be of significant concern. The use of CSTDs has increased significantly in recent years due to the recommendations by NIOSH and USP that they be used during preparation and administration of hazardous drugs. While CSTDs are valuable in the healthcare setting to reduce occupational exposure to hazardous compounds, these devices may present particular risks that must be adequately assessed prior to use to ensure their compatibility with specific types of drug products, such as biologic drugs, which may be sensitive. The responsibility of ensuring quality of biologic products through preparation and administration to the patient lies with the drug product sponsor. Due to the significant number of marketed CSTD systems, and the large variety of components offered for each system, a strategic, risk-based approach to assessing compatibility is recommended herein. In addition to traditional material compatibility, assessment of CSTD compatibility with biologics should consider additional parameters to address specific CSTD-related risks. The BPDG Formulation Workstream has proposed a systematic risk-based evaluation approach as well as a mitigation strategy for establishing suitability of CSTDs for use.

Dependency of γ-secretase complex activity on the structural integrity of the bilayer.

γ-secretase is a membrane protein complex associated with the production of Aß peptides that are pathogenic in Alzheimer's disease. We have characterized the activity of γ-secretase complexes under a variety of detergent solubilization and reconstitution conditions, and the structural state of proteoliposomes by electron microscopy. We found that γ-secretase activity is highly dependent on the physical state or integrity of the membrane bilayer--partial solubilization may increase activity while complete solubilization will abolish it. The activity of well-solubilized γ-secretase can be restored to near native levels when properly reconstituted into a lipid bilayer environment.

An ultrastructural study on indirect injury of dental pulp caused by high-speed missile projectile to mandible in dogs.

The aim of this study was to evaluate the characteristics of indirect injury of dental pulp caused by high-speed missile projectile to mandible in dogs. Eighteen dogs aged 12-13 months were divided equally into six groups (n = 3 in each group) with random allocation, then a high-speed missile projectile (a ball bearing of stainless steel, phi6.0 mm, 0.88 g) was shot at right mandible body (the wound tract was below the fourth premolar, 1 cm or so to the root tips) of each dog, but the teeth were not wounded directly. The dogs were killed 6 h (n = 3), 24 h (n = 3), 3 days (n = 3), 7 days (n = 3), 2 weeks (n = 3) and 4 weeks (n = 3) after the wound, respectively; then ultrastructural change of dental pulp of the fourth premolar and the second premolar of right mandible, and the second premolar of left mandible was observed through transmission electron microscope. The results showed that mean initial velocity of projectiles was 778.0 +/- 33.2 m s(-1) and mean projection energy was 266.1 +/- 19.1 J, which were in conformity with parameters of gunshot wound. On the wound side, dental pulp of the fourth mandibular premolar was injured seriously and irreversible necrosis happened in the end; yet, dental pulp of the second mandibular premolar was injured less seriously, reversibly; on the opposite side, dental pulp of the second mandibular premolar was injured slightly and temporarily. It may be concluded that there are several characteristics in indirect injury of dental pulp caused by high-speed missile projectile to dogs' mandible: the injured area is relatively extensive; traumatic degree decreases progressively and sharply with the distance to the wound tract increasing; ultrastructural change of nerval damage takes place in early stage after wound, etc.

The discovery and role of CD147 as a subunit of gamma-secretase complex.

Gamma-secretase is a membrane protein complex with unusual aspartyl protease activity that cleaves a variety of type I transmembrane proteins, such as APP, Notch and E-cadherin, within their transmembranous regions. Gamma-secretase was first recognized because of its role in the production of Abeta peptides that are pathogenic in Alzheimer's disease. There is overwhelming evidence demonstrating that four components, presenilin, nicastrin, APH-1 and PEN-2, are necessary and sufficient for gamma-secretase activity. However, based on the findings of studies conducted on cells overexpressing these four components, the existence of regulatory components of the gamma-secretase complex has been postulated. Recently, an additional subunit of the gamma-secretase complex, membrane protein CD147, has been identified through the purification and characterization of endogenous complexes from HeLa cell membranes. Removal of CD147 from gamma-secretase complexes increases the production of Abeta-peptides. Elucidating the molecular mechanism by which CD147 exerts its effect on the activity of the gamma-secretase complex will help us to further understand the pathogenesis of Alzheimer's disease, and may allow for the development of novel therapeutics.

Locally applied nerve growth factor enhances bone consolidation in a rabbit model of mandibular distraction osteogenesis.

Distraction osteogenesis is widely used in treating deformities, defects, and fractures of both long bones and craniofacial bones. Demands for acceleration of bone consolidation are increased in distraction osteogenesis. Nerve growth factor (NGF) can enhance innervation and bone regeneration in a fracture model and stimulate differentiation of osteoblastic cells. In this study, we tested the ability of locally applied NGF to enhance bone regeneration in a rabbit model of mandibular distraction osteogenesis. Twenty rabbits underwent bilateral distraction osteogenesis with a rate of 0.5 mm per 12 h. Two times 0.04 mg human NGFbeta (hNGFbeta) in buffer was injected into the callus after distraction. The contralateral side received placebo injections. Rabbits were euthanized at consolidation times of 14 and 28 days. Specimens were subjected to radiography, callus dimensions measurement, mechanical testing, and bone histological and histomorphometric analysis. The maximum load, bone volume/total volume, mineral apposition rate of the 1st to 11th day, and mineralized bone percentage were significantly higher in the hNGFbeta side at 14 and 28 days (p<0.05). The data indicate that locally applied hNGFbeta can accelerate callus maturation and may be an option to shorten the consolidation period in distraction osteogenesis.

An ultrastructural study on indirect trauma of dental pulp caused by maxillofacial impact injury in dogs.

BACKGROUND: Indirect injuries of adjacent tissues and organs usually accompany maxillofacial impact injuries. However, studies on indirect dental pulp injury are rare. This study was designed to determine the characteristics of indirect dental pulp injury caused by impact injury of mandible in dogs. METHODS: Eighteen dogs were divided equally into six groups with random allocation. Right mandible of each dog was impacted but teeth were not injured directly. Then, the animals were killed at appointed time points and ultrastructural changes in dental pulp of assigned teeth of each dog were investigated with transmission electron microscope. RESULTS: Dental pulp of the fourth premolar of right mandible was injured very severely, but irreversible necrosis did not occur in the end. Dental pulp of the second premolar of right mandible was injured less severely and reversibly. Dental pulp of the second premolar of left mandible was injured mildly and temporarily. CONCLUSION: In the indirect injury of dental pulp caused by maxillofacial impact injury, the injured area is relatively extensive. The effect of the trauma decreases progressively and sharply as the distance to the impact site increases. Ultrastructural changes in the damaged nerves take place early.

The Use of Poly-L-Lysine as a Capture Agent to Enhance the Detection of Antinuclear Antibodies by ELISA.

Antibodies to nuclear antigens (antinuclear antibodies or ANAs) are the serological hallmark of systemic lupus erythematosus (SLE). These antibodies bind diverse nuclear antigens that include DNA, histones and non-histone proteins as well as complexes of proteins with DNA and RNA. Because of the frequency of ANA expression in SLE, testing is an important component of clinical evaluation as well as determination of eligibility for clinical trials or utilization of certain therapies. Immunofluorescence assays have been commonly used for this purpose although this approach can be limited by issues of throughput, variability and difficulty in determining positivity. ELISA and multiplex assays are also useful approaches although these assays may give an incomplete picture of antibodies present. To develop a sensitive and quantitative ANA assay, we have explored an ELISA platform in which plates are pre-coated with a positively charged nucleic acid binding polymer (NABP) to increase adherence of antigens containing DNA or RNA. As a source of antigens, we have used supernatants of Jurkat cells undergoing apoptosis in vitro. As results presented show, a poly-L-lysine (PLL) pre-coat significantly enhances detection of antibodies to DNA as well as antigens such as histones, SSA, SSB and RNP. Comparison of the ELISA assay with the PLL pre-coat with a multiplex assay using the BioPlex® 2200 system indicated good agreement in results for a panel of lupus sera. Together, these studies indicate that a pre-coat with a positively charged polymer can increase the sensitivity of an ANA ELISA using as antigens molecules released from dead and dying cells. This assay platform may facilitate ANA testing by providing an ensemble of antigens more similar in composition and structure with antigens present in vivo, with a NABP promoting adherence via charge-charge interactions.

T-cadherin negatively regulates the proliferation of cutaneous squamous carcinoma cells.

T-cadherin is a unique member of the cadherin superfamily that lacks the transmembrane and cytoplasmic domains, and is instead linked to the cell membrane via a glycosyl-phosphatidylinositol anchor. We previously reported that T-cadherin was specifically expressed on the basal keratinocytes of the epidermis, and the expression of T-cadherin was significantly reduced in invasive cutaneous squamous cell carcinoma (SCC) and in the lesional skin of psoriasis vulgaris. In this study, to obtain an insight into the role of T-cadherin in keratinocytes, we used transfection methods and examined the effect of overexpression or knockdown of T-cadherin in immortalized keratinocyte cell lines derived from SCC. T-cadherin overexpressed cells showed clearly reduced cell proliferation, but the influence of cell-cell adhesiveness and cell mobility was not detected. Using a tetracycline-regulated expression system, we also confirmed that the suppression of cell proliferation was dependent on the expression level of T-cadherin. Cell cycle analysis demonstrated that over expression of T-cadherin induced a delay in the G(2)/M phase. Our findings suggest that T-cadherin acts as an endogenous negative regulator of keratinocyte proliferation and its inactivation is the cause for keratinocyte hyperproliferation in SCC or in psoriasis vulgaris.

Three-dimensional sutural expansion osteogenesis to expand zygomatic bone: an experimental study.

Sutural expansion osteogenesis was used to extend zygomatic bone three-dimensionally by the formation of new bone in eight goats. A 3 cm incision without osteomy was made in the infraorbital part of the zygomatic bone. The external expansion appliance was then placed on the zygomatic bone. The expansion appliance was activated 10 days after the operation at a rate of 0.9 mm/day for 10 days. The direction could be changed through turning the nuts and moving the brace boards. All the zygomatic bones were expanded anterosuperolaterally and inferolaterally by a mean of 9 mm (range 7.8-11.5mm). The regenerating bone tissues in the distracted sutures were recorded radiographically, clinically, and histologically. The experiment succeeded in expanding the zygoma quickly. The three-dimensional external expansion appliance was simple to operate. The result of sutural expansion osteogenesis is stable and credible, and the method is feasible.

Calculating the mass of subvisible protein particles with improved accuracy using microflow imaging data.

Although formation of subvisible particles (1-100 µm) during manufacturing and/or storage is a major stability concern with protein therapeutics, particle numbers are often too low to permit for direct experimental measurement of their protein content (mass). The objective of this work was to develop a novel, accurate, and easy-to-implement method to calculate the mass of subvisible protein particles using particle number, size, and morphology data obtained from microflow imaging (MFI) measurements. The method was evaluated using (1) spherical and nonspherical polystyrene standards and (2) shake and stir-stressed IgG1 mAb solutions. For extensively stressed mAb samples, in which protein mass loss after particle removal could be measured experimentally, calculated results were in good agreement and showed improvements in accuracy and precision compared with other methods. Improved estimates of protein mass in particles were made possible by using morphological data to better model particle volume, and by using literature-based values for protein density and particle composition. This method improves estimations of protein particle mass when total amounts are too low to be measured experimentally and also facilitates a better understanding of protein particle formation by accounting for particle mass as well as number.