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Introduction: Infrapatellar fat pad (IFP)-derived mesenchymal stem cells (MSCs) have high chondrogenic potential and are attractive cell sources for cartilage regeneration. During ceiling culture to acquire the characteristics of MSCs, mature adipocytes from fat tissue are known to undergo dedifferentiation, generating dedifferentiated fat (DFAT) cells. The purpose of the present study was to compare the yields and biological properties of IFP-derived MSCs and IFP-derived DFAT cells. Methods: IFPs were harvested from the knees of 8 osteoarthritis (OA) patients. DFAT cells were obtained using a ceiling culture of adipocytes isolated from the floating top layer of IFP digestion. MSCs were obtained by culturing precipitated stromal vascular fraction cells. We compared the P0 cell yields, surface antigen profile, colony formation ability, and multipotency of DFAT cells and MSCs. Results: The P0 cell yields per flask and the estimated total cell yields from 1 g of IFP were much greater for MSCs than for DFAT cells. Both MSCs and DFAT cells were positive for MSC markers. No obvious difference was observed in colony formation ability. In differentiation assays, DFAT cells produced greater amounts of lipid droplets, calcified tissue, and glycosaminoglycan than MSCs did. Adipogenic and chondrogenic gene expressions were upregulated in DFAT cells. Conclusions: IFP-derived DFAT cells showed higher adipogenic and chondrogenic potentials than IFP-derived MSCs, but they had a poor cell yield.

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Background/Objective: Rhabdomyolysis is a condition characterized by the destruction of skeletal muscle tissue that leads to systemic complications. We present a case of gender-affirming intramuscular (IM) testosterone therapy precipitating localized deltoid rhabdomyolysis. Case Report: A 34-year-old transgender man presented to the emergency department with dark-colored urine and pain in the left deltoid muscle where he had been injecting IM testosterone. He was found to have significant elevation in the level of creatinine kinase that was consistent with rhabdomyolysis and managed with intravenous fluids. He received trial therapy with IM testosterone again in the contralateral deltoid twice with recurrent rhabdomyolysis. He eventually transitioned to subcutaneous testosterone to achieve his masculinization goals without adverse effects. Discussion: Localized anabolic steroid use has been associated with rhabdomyolysis. However, to the best of our knowledge, this is the first case report of rhabdomyolysis attributed to gender-affirming testosterone therapy. Our patient had been administering testosterone intramuscularly into larger muscles (thigh and gluteus) for many years without any issues, whereas recurrent focal rhabdomyolysis developed only in association with deltoid injections. We theorize that a relative increase in dose and volume of testosterone per gram of muscle after switching to the deltoid site precipitated rhabdomyolysis. Subcutaneous testosterone is an acceptable alternative to IM testosterone for patients desiring an injectable delivery route with minimal adverse effects. Conclusion: This case report highlights the potential risk of rhabdomyolysis associated with IM testosterone administration in the deltoid region for gender-affirming care. Patients on IM testosterone should use the thigh or gluteal muscles rather than the deltoid.

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Skeletal-related events (SREs) are complications of bone metastases and carry a significant patient and economic burden. Denosumab is a receptor activator of nuclear factor-κB ligand (RANKL) inhibitor approved for SRE prevention in patients with multiple myeloma and patients with bone metastases from solid tumors. In phase 3 trials, denosumab showed superiority to the bisphosphonate zoledronate in reducing the risk of first on-study SRE by 17% (median time to first on-study SRE delayed by 8.2 months) and the risk of first and subsequent on-study SREs by 18% across multiple solid tumor types, including some patients with multiple myeloma. Denosumab also improved pain outcomes and reduced the need for strong opioids. Additionally, a phase 3 trial showed denosumab was noninferior to zoledronate in delaying time to first SRE in patients with newly diagnosed multiple myeloma. Denosumab has a convenient 120 mg every 4 weeks recommended dosing schedule with subcutaneous administration. Rare but serious toxicities associated with denosumab include osteonecrosis of the jaw, hypocalcemia, and atypical femoral fracture events, with multiple vertebral fractures reported following treatment discontinuation. After a decade of real-world clinical experience with denosumab, we are still learning about the optimal use and dosing for denosumab. Despite the emergence of novel and effective antitumor therapies, there remains a strong rationale for the clinical utility of antiresorptive therapy for SRE prevention. Ongoing studies aim to optimize clinical management of patients using denosumab for SRE prevention while maintaining safety and efficacy.

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INTRODUCTION: Mature adipocyte-derived dedifferentiated fat cells (DFATs) are mesenchymal stem cell (MSC)-like cells with high proliferative ability and multilineage differentiation potential. In this study, we first examined whether DFATs can be prepared from infrapatellar fat pad (IFP) and then compared phenotypic and functional properties of IFP-derived DFATs (IFP-DFATs) with those of subcutaneous adipose tissue (SC)-derived DFATs (SC-DFATs). METHODS: Mature adipocytes isolated from IFP and SC in osteoarthritis patients (n = 7) were cultured by ceiling culture method to generate DFATs. Obtained IFP-DFATs and SC-DFATs were subjected to flow cytometric and microarray analysis to compare their immunophenotypes and gene expression profiles. Cell proliferation assay and adipogenic, osteogenic, and chondrogenic differentiation assays were performed to evaluate their functional properties. RESULTS: DFATs could be prepared from IFP and SC with similar efficiency. IFP-DFATs and SC-DFATs exhibited similar immunophenotypes (CD73+, CD90+, CD105+, CD31-, CD45-, HLA-DR-) and tri-lineage (adipogenic, osteogenic, and chondrogenic) differentiation potential, consistent with the minimal criteria for defining MSCs. Microarray analysis revealed that the gene expression profiles in IFP-DFATs were very similar to those in SC-DFATs, although there were certain number of genes that showed different levels of expression. The proliferative activity in IFP-DFATs was significantly (p < 0.05) higher than that in the SC-DFATs. IFP-DFATs showed higher chondrogenic differentiation potential than SC-DFATs in regard to production of soluble galactosaminogalactan and gene expression of type II collagen. CONCLUSIONS: IFP-DFATs showed higher cellular proliferative potential and higher chondrogenic differentiation capacity than SC-DFATs. IFP-DFAT cells may be an attractive cell source for chondrogenic regeneration.

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INTRODUCTION: Several drugs of abuse (DOA) are capable of modulating neurohypophysial hormones, such as oxytocin (OT) and vasopressin (VP), potentially resulting in the development of psychological abnormalities, such as cognitive dysfunction, psychoses, and affective disorders. Efavirenz (EFV), widely used in Africa and globally to treat HIV, induces diverse neuropsychiatric side effects while its abuse has become a global concern. The actions of EFV may involve neurohypophysial system (NS) disruption like that of known DOA. This study investigated whether sub-chronic EFV exposure, at a previously-determined rewarding dose, alters peripheral OT and VP levels versus that of a control, ∆9-tetrahydrocannabinol (∆9-THC), methamphetamine (MA) and cocaine. MATERIALS AND METHODS: To simulate the conditions under which reward-driven behavior had previously been established for EFV, male Sprague Dawley rats (n = 16/exposure) received intraperitoneal vehicle (control) or drug administration across an alternating sixteen-day dosing protocol. Control administration (saline/olive oil; 0.2 ml) occurred on odd-numbered and drug administration (EFV: 5 mg/kg, ∆9-THC: 0.75 mg/kg, MA: 1 mg/kg, or cocaine: 20 mg/kg) on even-numbered days followed by euthanasia, trunk blood collection and plasma extraction for neuropeptide assay. Effect of drug exposure on peripheral OT and VP levels was assessed versus controls and quantified using specific ELISA kits. Statistical significance was determined by Kruskal-Wallis ANOVA, with p < 0.05. Ethics approval: NWU-00291-17-A5. RESULTS: Delta-9-THC reduced OT and VP plasma levels (p < 0.0001, p = 0.0141; respectively), cocaine reduced plasma OT (p = 0.0023), while MA reduced plasma VP levels (p = 0.0001), all versus control. EFV reduced OT and VP plasma levels (p < 0.0001; OT and VP) versus control, and similar to ∆9-THC. CONCLUSION: EFV markedly affects the NS in significantly reducing both plasma OT and VP equivalent to DOA. Importantly, EFV has distinct effects on peripheral OT and VP levels when assessed within the context of drug dependence. The data highlights a possible new mechanism underlying previously documented EFV-induced effects in rats, and whereby EFV may induce neuropsychiatric adverse effects clinically; also providing a deeper understanding of the suggested abuse-potential of EFV.

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The need for long-term treatments of chronic diseases has motivated the widespread development of long-acting parenteral formulations (LAPFs) with the aim of improving drug pharmacokinetics and therapeutic efficacy. LAPFs have been proven to extend the half-life of therapeutics, as well as to improve patient adherence; consequently, this enhances the outcome of therapy positively. Over past decades, considerable progress has been made in designing effective LAPFs in both preclinical and clinical settings. Here we review the latest advances of LAPFs in preclinical and clinical stages, focusing on the strategies and underlying mechanisms for achieving long acting. Existing strategies are classified into manipulation of in vivo clearance and manipulation of drug release from delivery systems, respectively. And the current challenges and prospects of each strategy are discussed. In addition, we also briefly discuss the design principles of LAPFs and provide future perspectives of the rational design of more effective LAPFs for their further clinical translation.

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Proteins and peptides (PPs) have gradually become more attractive therapeutic molecules than small molecular drugs due to their high selectivity and efficacy, but fewer side effects. Owing to the poor stability and limited permeability through gastrointestinal (GI) tract and epithelia, the therapeutic PPs are usually administered by parenteral route. Given the big demand for oral administration in clinical use, a variety of researches focused on developing new technologies to overcome GI barriers of PPs, such as enteric coating, enzyme inhibitors, permeation enhancers, nanoparticles, as well as intestinal microdevices. Some new technologies have been developed under clinical trials and even on the market. This review summarizes the history, the physiological barriers and the overcoming approaches, current clinical and preclinical technologies, and future prospects of oral delivery of PPs.

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Rheumatoid Arthritis (RA) is more common and severe in women compared to men. Both women and men with RA express autoantibodies to post-translationally modified antigens, including citrullinated and homocitrullinated proteins or peptides. These autoantibodies are strongly linked with the HLA-DR4 gene. The objective of this study was to determine sex differences in immune responses to homocitrullinated antigens. We used a humanized animal model of RA, DR4-transgenic mice and immunized them with a homocitrullinated peptide called HomoCitJED. Immune responses in these mice were measured for splenocyte proliferation by tritiated thymidine incorporation, serum autoantibody production by ELISA and cytokine levels by multiplex. We found that T cell and antibody responses to homocitrullinated antigens were similar in male and female mice. However, we found sex differences in serum cytokine profiles with female mice having higher ratio of IL-1α to IL-5, suggesting imbalances in immune regulation. This is the first study to report that immune responses to homocitrullinated antigens can be differentiated by sex.

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Managing patients with severe asthma during the coronavirus pandemic and COVID-19 is a challenge. Authorities and physicians are still learning how COVID-19 affects people with underlying diseases, and severe asthma is not an exception. Unless relevant data emerge that change our understanding of the relative safety of medications indicated in patients with asthma during this pandemic, clinicians must follow the recommendations of current evidence-based guidelines for preventing loss of control and exacerbations. Also, with the absence of data that would indicate any potential harm, current advice is to continue the administration of biological therapies during the COVID-19 pandemic in patients with asthma for whom such therapies are clearly indicated and have been effective. For patients with severe asthma infected by SARS-CoV-2, the decision to maintain or postpone biological therapy until the patient recovers should be a case-by-case based decision supported by a multidisciplinary team. A registry of cases of COVID-19 in patients with severe asthma, including those treated with biologics, will help to address a clinical challenge in which we have more questions than answers.

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A hybrid biological-biomaterial antigen delivery vector comprised of a polymeric shell encapsulating an Escherichia coli core was previously developed for in situ antigen production and subsequent delivery. Due to the engineering capacity of the bacterial core, the hybrid vector provides unique opportunities for immunogenicity optimization through varying cellular localization (cytoplasm, periplasm, cellular surface) and type (protein or DNA) of antigen. In this work, three protein-based hybrid vector formats were compared in which the pneumococcal surface protein A (PspA) was localized to the cytoplasm, surface, and periplasmic space of the bacterial core for vaccination against pneumococcal disease. Furthermore, we tested the hybrid vector's capacity as a DNA vaccine against Streptococcus pneumoniae by introducing a plasmid into the bacterial core to facilitate PspA expression in antigen presenting cells (APCs). Through testing these various formulations, we determined that cytoplasmic accumulation of PspA elicited the strongest immune response (antibody production and protection against bacterial challenge) and enabled complete protection at substantially lower doses when compared to vaccination with PspA + adjuvant. We also improved the storage stability of the hybrid vector to retain complete activity after 1 month at 4 °C using an approach in which hybrid vectors suspended in a microbial freeze drying buffer were desiccated. These results demonstrate the flexibility and robustness of the hybrid vector formulation, which has the potential to be a potent vaccine against S. pneumoniae.

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Impaired cardiorenal response to acute saline volume expansion in preclinical systolic dysfunction (PSD) may lead to symptomatic heart failure. The objective was to determine if combination phosphodiesterase-V inhibition and exogenous B-type natriuretic peptide (BNP) administration may enhance cardiorenal response. A randomized double-blinded, placebo-controlled study was conducted in 21 subjects with PSD and renal dysfunction. Pre-treatment with tadalafil and subcutaneous BNP resulted in improved cardiac function, as evidenced by improvement in ejection fraction, left atrial volume index, and left ventricular end-diastolic volume. However, there was reduced renal response with reduction in renal plasma flow, glomerular filtration rate, and urine flow. (Tadalafil and Nesiritide as Therapy in Pre-clinical Heart Failure; NCT01544998).

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The synthetic cathinones methylone, butylone, and pentylone differ from each other through the one carbon lengthening of the α-alkyl chain: methylone (-CH3), butylone (-CH2CH3), and pentylone (-CH2CH2CH3) while 3,4-methylenedioxymethamphetamine (MDMA) differs from methylone by a single oxygen atom. Studies with MDMA, suggests that there may be male and female pharmacokinetic and pharmacodynamic differences. In the present study, we present the plasma pharmacokinetic data relative to a 20 mg/kg, subcutaneous doses of methylone, butylone and pentylone in female Sprague-Dawley rats. Briefly, plasma samples were collected via a jugular vein cannula, purified, and analyzed using a HPLC system. While we have previously reported on the consistent relationship between structure and pharmacokinetics of these synthetic cathinones in male, Sprague-Dawley rats (Grecco and Sprague, 2016), this data set suggests that there is no consistent relationship of chemical structure and pharmacokinetics of methylone, butylone and pentylone in female Sprague-Dawley rats. The findings from the present study further emphasize the need for the inclusion of female subjects in the pharmacokinetic studies of synthetic cathinones as it is very possible male-female differences may exist in rodent models.

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INTRODUCTION: The objective of this study is to evaluate the insulin secretion of mixed aggregates of insulinoma cells (INS-1) and gelatin hydrogel microspheres after their subcutaneous transplantation. METHODS: Gelatin hydrogel microspheres were prepared by the conventional w/o emulsion method. Cell aggregates mixed with or without the hydrogel microspheres were encapsulated into a pouched-device of polytetrafluoroethylene membrane. An agarose hydrogel or MedGel™ incorporating basic fibroblast growth factor (bFGF) was subcutaneously implanted to induce vascularization. After the vascularization induction, cell aggregates encapsulated in the pouched-device was transplanted. RESULTS: The vascularization had the potential to enable transplanted cell aggregates to enhance the level of insulin secretion compared with those of no vascularization induction. In addition, the insulin secretion of cell aggregates was significantly promoted by the mixing of gelatin hydrogel microspheres even in the pouched-device encapsulated state. CONCLUSION: It is possible that the microspheres mixing gives cells in aggregates better survival condition, resulting in promoted insulin secretion.

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Advancements in in silico techniques of lead molecule selection have resulted in the failure of around 70% of new chemical entities (NCEs). Some of these molecules are getting rejected at final developmental stage resulting in wastage of money and resources. Unfavourable physicochemical properties affect ADME profile of any efficacious and potent molecule, which may ultimately lead to killing of NCE at final stage. Numerous techniques are being explored including nanocrystals for solubility enhancement purposes. Nanocrystals are the most successful and the ones which had a shorter gap between invention and subsequent commercialization of the first marketed product. Several nanocrystal-based products are commercially available and there is a paradigm shift in using approach from simply being solubility enhancement technique to more novel and specific applications. Some other aspects in relation to parenteral nanosuspensions are concentrations of surfactant to be used, scalability and in vivo fate. At present, there exists a wide gap due to poor understanding of these critical factors, which we have tried to address in this review. This review will focus on parenteral nanosuspensions, covering varied aspects especially stabilizers used, GRAS (Generally Recognized as Safe) status of stabilizers, scalability challenges, issues of physical and chemical stability, solidification techniques to combat stability problems and in vivo fate.

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Parenteral diuretics form the cornerstone of decongestion in heart failure. However, parenteral therapy routinely requires emergency room or inpatient care. A novel buffered furosemide formulation with neutral pH was developed to offer "hospital-strength" diuresis for outpatient use, including self-administration at home. Subcutaneous infusion using a biphasic delivery profile resulted in complete bioavailability (99.65%) and equivalent diuresis when compared with intravenous administration. Subcutaneous administration of buffered furosemide was well tolerated with no evidence of any drug-induced skin reactions. Subcutaneous infusion of buffered furosemide in the outpatient setting or home may help to reduce the burden of heart failure.

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OBJECTIVE: A potential strategy to treat obesity - and the associated metabolic consequences - is to increase energy expenditure. This could be achieved by stimulating thermogenesis through activation of brown adipose tissue (BAT) and/or the induction of browning of white adipose tissue (WAT). Over the last years, it has become clear that several metalloproteinases play an important role in adipocyte biology. Here, we investigated the potential role of ADAMTS5. METHODS: Mice deficient in ADAMTS5 (Adamts5-/-) and wild-type (Adamts5+/+) littermates were kept on a standard of Western-type diet for 15 weeks. Energy expenditure and heat production was followed by indirect calorimetry. To activate thermogenesis, mice were treated with the ß3-adrenergic receptor (ß3-AR) agonist CL-316,243 or alternatively, exposed to cold for 2 weeks. RESULTS: Compared to Adamts5+/+ mice, Adamts5-/- mice have significantly more interscapular BAT and marked browning of their subcutaneous (SC) WAT. Thermogenic pathway analysis indicated, in the absence of ADAMTS5, enhanced ß3-AR signaling via activation of the cAMP response element-binding protein (CREB). Additional ß3-AR stimulation with CL-316,243 promoted browning of WAT in Adamts5+/+ mice but had no additive effect in Adamts5-/- mice. However, cold exposure induced more pronounced browning of WAT in Adamts5-/- mice. CONCLUSIONS: These data indicate that ADAMTS5 plays a functional role in development of BAT and browning of WAT. Hence, selective targeting of ADAMTS5 could provide a novel therapeutic strategy for treatment/prevention of obesity and metabolic diseases.

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OBJECTIVE: To investigate the role played by leptin in thermoregulation, we studied the effects of physiological leptin replacement in leptin-deficient ob/ob mice on determinants of energy balance, thermogenesis and heat retention under 3 different ambient temperatures. METHODS: The effects of housing at 14 °C, 22 °C or 30 °C on core temperature (telemetry), energy expenditure (respirometry), thermal conductance, body composition, energy intake, and locomotor activity (beam breaks) were measured in ob/ob mice implanted subcutaneously with osmotic minipumps at a dose designed to deliver a physiological replacement dose of leptin or its vehicle-control. RESULTS: As expected, the hypothermic phenotype of ob/ob mice was partially rescued by administration of leptin at a dose that restores plasma levels into the physiological range. This effect of leptin was not due to increased energy expenditure, as cold exposure markedly and equivalently stimulated energy expenditure and induced activation of brown adipose tissue irrespective of leptin treatment. Instead, the effect of physiological leptin replacement to raise core body temperature of cold-exposed ob/ob mice was associated with reduced thermal conductance, implying a physiological role for leptin in heat conservation. Finally, both leptin- and vehicle-treated ob/ob mice failed to match energy intake to expenditure during cold exposure, resulting in weight loss. CONCLUSIONS: The physiological effect of leptin to reduce thermal conductance contributes to maintenance of core body temperature under sub-thermoneutral conditions.

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Nerve agents with low volatility such as VX are primarily absorbed through the skin when released during combat or a terrorist attack. The barrier function of the stratum corneum may be compromised during certain stages of development, allowing VX to more easily penetrate through the skin. However, age-related differences in the lethal potency of VX have yet to be evaluated using the percutaneous (pc) route of exposure. Thus, we estimated the 24 and 48 h median lethal dose for pc exposure to VX in male and female rats during puberty and early adulthood. Pubescent, female rats were less susceptible than both their male and adult counterparts to the lethal effects associated with pc exposure to VX possibly because of hormonal changes during that stage of development. This study emphasizes the need to control for both age and sex when evaluating the toxicological effects associated with nerve agent exposure in the rat model.

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Vaccines containing multiple antigens may induce broader immune responses and provide better protection against Mycobacterium tuberculosis (Mtb) infection as compared to a single antigen. However, strategies for incorporating multiple antigens into a single vector and the immunization routes may affect their immunogenicity. In this study, we utilized recombinant adenovirus type 5 (rAd5) as a model vaccine vector, and Ag85A (Rv3804c) and Mtb32 (Rv0125) as model antigens, to comparatively evaluate the influence of codon usage optimization, signal sequence, fusion linkers, and immunization routes on the immunogenicity of tuberculosis (TB) vaccine containing multiple antigens in C57BL/6 mice. We showed that codon-optimized Ag85A and Mtb32 fused with a GSG linker induced the strongest systemic and pulmonary cell-mediated immune (CMI) responses. Strong CMI responses were characterized by the generation of a robust IFN-γ ELISPOT response as well as antigen-specific CD4(+) T and CD8(+) T cells, which secreted mono-, dual-, or multiple cytokines. We also found that subcutaneous (SC) and intranasal (IN)/oral immunization with this candidate vaccine exhibited the strongest boosting effects for Mycobacterium bovis bacille Calmette-Guérin (BCG)-primed systemic and pulmonary CMI responses, respectively. Our results supported that codon optimized Ag85A and Mtb32 fused with a proper linker and immunized through SC and IN/oral routes can generate the strongest systemic and pulmonary CMI responses in BCG-primed mice, which may be particularly important for the design of TB vaccines containing multiple antigens.

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There is a need for new analytical approaches to better characterize the nature of the concentration-dependent, reversible self-association (RSA) of monoclonal antibodies (mAbs) directly, and with high resolution, when these proteins are formulated as highly concentrated solutions. In the work reported here, hydrogen exchange mass spectrometry (HX-MS) was used to define the concentration-dependent RSA interface, and to characterize the effects of association on the backbone dynamics of an IgG1 mAb (mAb-C). Dynamic light scattering, chemical cross-linking, and solution viscosity measurements were used to determine conditions that caused the RSA of mAb-C. A novel HX-MS experimental approach was then applied to directly monitor differences in local flexibility of mAb-C due to RSA at different protein concentrations in deuterated buffers. First, a stable formulation containing lyoprotectants that permitted freeze-drying of mAb-C at both 5 and 60 mg/mL was identified. Upon reconstitution with RSA-promoting deuterated solutions, the low vs. high protein concentration samples displayed different levels of solution viscosity (i.e., approx. 1 to 75 mPa.s). The reconstituted mAb-C samples were then analyzed by HX-MS. Two specific sequences covering complementarity-determining regions CDR2H and CDR2L (in the variable heavy and light chains, respectively) showed significant protection against deuterium uptake (i.e., decreased hydrogen exchange). These results define the major protein-protein interfaces associated with the concentration-dependent RSA of mAb-C. Surprisingly, certain peptide segments in the VH domain, the constant domain (CH2), and the hinge region (CH1-CH2 interface) concomitantly showed significant increases in local flexibility at high vs. low protein concentrations. These results indicate the presence of longer-range, distant dynamic coupling effects within mAb-C occurring upon RSA.

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