Colonic adenomatous polyp with florid presence of monoclonal lambda Russell bodies: Case report and etiopathogenic hypothesis / Pólipo adenomatoso colónico con presencia florida de cuerpos de Russell monoclonales lambda: comunicación de un caso e hipótesis etiopatogénica

Russell bodies (RBs) are round eosinophilic intracytoplasmic inclusions formed by condensed immunoglobulins in mature plasma cells, which are called Mott cells. These cells are rarely found in the gastric tract, with even less cases reported in the colorectal region. There are still many questions about this event, as it is still unknown the relationship between the agents reported of increasing the probability of appearance of these cells and the generation of RBs. In this case report we describe the fifth patient presenting an infiltration of Mott cells in a colorectal polyp, being the second case with a monoclonal origin without a neoplastic cause, and the first one monoclonal for lambda. A comparison with previously similar reported cases is also done, and a possible etiopathogenic hypothesis proposed. (AU)

Los cuerpos de Russell (RB) son inclusiones intracitoplasmáticas eosinofílicas redondas formadas por inmunoglobulinas condensadas en las células plasmáticas maduras, que se denominan células de Mott. Estas células rara vez se encuentran en el tracto gástrico, y son aún más infrecuentes en la región colorrectal. Actualmente hay muchas dudas sobre este evento, ya que se desconoce la relación entre los agentes causantes de aumentar la probabilidad de aparición tanto de estas células como de la de RB. En este caso describimos al quinto paciente con un pólipo colorrectal, localizado en el tracto colorrectal e infiltrado por células de Mott, siendo el segundo caso de origen monoclonal sin causa neoplásica y el primero monoclonal para lambda. También se hace una comparación con casos similares previamente reportados y se propone una hipótesis etiopatogénica. (AU)

Identification of Acyl-Protein Thioesterase-1 as a Polysorbate-degrading Host Cell Protein in a Monoclonal Antibody Formulation Using Activity-based Protein Profiling.

Polysorbate (PS) degradation in monoclonal antibody (mAb) formulations poses a significant challenge in the biopharmaceutical industry. PS maintains protein stability during drug product's shelf life but is vulnerable to breakdown by low-abundance residual host cell proteins (HCPs), particularly hydrolytic enzymes such as lipases and esterases. In this study, we used activity-based protein profiling (ABPP) coupled with mass spectrometry to identify acyl-protein thioesterase-1 (APT-1) as a polysorbate-degrading HCP in one case of mAb formulation with stability problems. We validated the role of APT1 by matching the polysorbate degradation fingerprint in the mAb formulation with that of a recombinant APT1 protein. Furthermore, we found an agreement between APT1 levels and PS degradation rates in the mAb formulation, and we successfully halted PS degradation using APT1-specific inhibitors ML348 and ML211. APT1 was found to co-purify with a specific mAb via hitchhiking mechanism. Our work provides a streamlined approach to identifying critical HCPs in PS degradation, supporting quality-by-design principles in pharmaceutical development.

The Logic and History of Passive Immunity and Antibody Therapies.

This volume takes a broad overview of antibody-based therapies prior to and during the COVID pandemic and examines their potential use in future pandemics. Passive antibody therapy was the first effective antimicrobial treatment and its development in the early twentieth century helped catalyze immunological and microbiological research. During the era of serum therapy (1890-1940) antibody-based therapies were developed against both viral and bacterial diseases. Effective treatment required an understanding of how to quantify antibodies, how to develop serotype-specific sera and recognition of the need to treat early in disease. Thus, although the era of serum therapy essentially ended with the development of small molecule antimicrobial therapy in the 1940s, antibody-based therapies led to important new scientific understanding, while remaining in use for some toxin and venom-caused diseases and in the prevention of outbreaks of viral hepatitis. A renewed interest in antibody-based therapies was seen in the widespread deployment of convalescent plasma and monoclonal antibodies during the COVID-19 pandemic. Convalescent plasma will likely be the first specific therapy during outbreaks with new pathogens for which there is no other therapy. For all forms of antibody-based therapies, effectiveness relies on the key principles of antibody therapy, namely, treatment early in disease with preparations containing sufficient antibody specific to the microbe in question.

Concurrent Crystalline Light-Chain Proximal Tubulopathy and Membranous Nephropathy: A Case Report and Literature Review.

Light-chain proximal tubulopathy (LCPT) is typically characterized by the intracytoplasmic deposition of light chains within the proximal tubular epithelial cells, which is usually classified into crystalline and noncrystalline subgroups. Membranous nephropathy (MN) is a common glomerular disease characterized by diffused subepithelial electron-dense deposits along the capillary loop accompanied by the effacement and microvillus transformation of the foot process. Here, we report a biopsy-confirmed case of a concurrence of LCPT with crystals (κ light chains restricted) and antigen-undetermined MN in a male patient. The patient presented with low-molecular-weight proteinuria, increased serum creatinine levels, and incomplete Fanconi syndrome. To our knowledge, this is the first report of a concurrence of LCPT and independent MN of unknown target antigens, which may enrich our recognition of monoclonal gammopathy of renal significance with synchronous MN.

The Future of Atopic Dermatitis Treatment.

This chapter thoroughly examines recent breakthroughs in atopic dermatitis (AD) treatment, with a primary focus on the medications in the development pipeline. Biologics agents targeting new interleukin receptors like interleukin-31, interleukin-22, and interleukin-2 are discussed along with the novel pathway looking at the OX40-OX40L interaction. Oral agents and small molecule therapies like Janus kinase inhibitors, sphingosine-1-phosphate modulators, and Bruton's tyrosine kinase inhibitors are also discussed along with the various new topical medications. Newly approved topicals like phosphodiesterase-4 and JAK inhibitors are highlighted while also discussing the potential of tapinarof and emerging microbiome-targeted therapies. Beyond conventional approaches, the chapter touches upon unconventional therapies currently being studied. The goal of this chapter is to discuss new advances in AD treatment from medications in the initial stages of development to those nearing FDA approval.

IMpower210: A phase III study of second-line atezolizumab vs. docetaxel in East Asian patients with non-small cell lung cancer.

Objective: IMpower210 (NCT02813785) explored the efficacy and safety of single-agent atezolizumab vs. docetaxel as second-line treatment for advanced non-small cell lung cancer (NSCLC) in East Asian patients. Methods: Key eligibility criteria for this phase III, open-label, randomized study included age ≥18 years; histologically documented advanced NSCLC per the Union for International Cancer Control/American Joint Committee on Cancer staging system (7th edition); Eastern Cooperative Oncology Group performance status of 0 or 1; and disease progression following platinum-based chemotherapy for advanced or metastatic NSCLC. Patients were randomized 2:1 to receive either atezolizumab (1,200 mg) or docetaxel (75 mg/m2). The primary study endpoint was overall survival (OS) in the intention-to-treat (ITT) population with wild-type epidermal growth factor receptor expression (ITT EGFR-WT) and in the overall ITT population. Results: Median OS in the ITT EGFR-WT population (n=467) was 12.3 [95% confidence interval (95% CI), 10.3-13.8] months in the atezolizumab arm (n=312) and 9.9 (95% CI, 7.8-13.9) months in the docetaxel arm [n=155; stratified hazard ratio (HR), 0.82; 95% CI, 0.66-1.03]. Median OS in the overall ITT population was 12.5 (95% CI, 10.8-13.8) months with atezolizumab treatment and 11.1 (95% CI, 8.4-14.2) months (n=377) with docetaxel treatment (n=188; stratified HR, 0.87; 95% CI, 0.71-1.08). Grade 3/4 treatment-related adverse events (TRAEs) occurred in 18.4% of patients in the atezolizumab arm and 50.0% of patients in the docetaxel arm. Conclusions: IMpower210 did not meet its primary efficacy endpoint of OS in the ITT EGFR-WT or overall ITT populations. Atezolizumab was comparatively more tolerable than docetaxel, with a lower incidence of grade 3/4 TRAEs.

Preclinical development of humanized monoclonal antibodies against CD169 as a broad antiviral therapeutic strategy.

New therapies to treat or prevent viral infections are essential, as recently observed during the COVID-19 pandemic. Here, we propose a therapeutic strategy based on monoclonal antibodies that block the specific interaction between the host receptor Siglec-1/CD169 and gangliosides embedded in the viral envelope. Antibodies are an excellent option for treating infectious diseases based on their high specificity, strong targeting affinity, and relatively low toxicity. Through a process of humanization, we optimized monoclonal antibodies to eliminate sequence liabilities and performed biophysical characterization. We demonstrated that they maintain their ability to block viral entry into myeloid cells. These molecular improvements during the discovery stage are key if we are to maximize efforts to develop new therapeutic strategies. Humanized monoclonal antibodies targeting CD169 provide new opportunities in the treatment of infections caused by ganglioside-containing enveloped viruses, which pose a constant threat to human health. In contrast with current neutralizing antibodies that bind antigens on the infectious particle, our antibodies can prevent several types of enveloped viruses interacting with host cells because they target the host CD169 protein, thus becoming a potential pan-antiviral therapy.

Therapeutics Pipeline.

Children have unique physiologic, developmental, and psychosocial needs and unique vulnerabilities, making them a challenging population for which to develop therapeutics. This is particularly apparent in the urgent and chaotic environment of a pandemic or outbreak. Advances in the development of medical countermeasures (MCMs) for pediatric populations have grown substantially over the last decade, and the coronavirus disease 2019 pandemic forced advancements in how we approach pediatric MCM development. Consequently, a MCMs pipeline targeting the pediatric population is essential. This article addresses the challenges inherent in these differences that must be taken into account.

Antibody Isolation in C. neoformans.

The importance of humoral immunity to fungal infections remains to be elucidated. In cryptococcosis, patients that fail to generate antibodies against antigens of the fungus Cryptococcus neoformans are more susceptible to the disease, demonstrating the importance of these molecules to the antifungal immune response. Historically, antibodies against C. neoformans have been applied in diagnosis, therapeutics, and as important research tools to elucidate fungal biology. Throughout the process of generating monoclonal antibodies (mAbs) from a single B-cell clone and targeting a single epitope, several immunization steps might be required for the detection of responsive antibodies to the antigen of interest in the serum. This complex mixture of antibodies comprises the polyclonal antibodies. To obtain mAbs, B-lymphocytes are harvested (from spleen or peripheral blood) and fused with tumor myeloma cells, to generate hybridomas that are individually cloned and specifically screened for mAb production. In this chapter, we describe all the necessary steps, from the immunization to polyclonal antibody harvesting, hybridoma generation, and mAb production and purification. Additionally, we discuss new cutting-edge approaches for generating interspecies mAbs, such as humanized mAbs, or for similar species in distinct host backgrounds.

Switching CGRP(r) MoAbs in migraine: what evidence?

INTRODUCTION: Approximately 50% of patients that receive a CGRP(r) MoAb for the preventative treatment of migraine are expected to discontinue therapy. For patients that discontinue CGRP(r) MoAb therapy, few clinical options are available. One potential option is to switch CGRP(r) MoAbs, however, data concerning the efficacy of this intervention is scarce. AREAS COVERED: This manuscript aims to summarize all available data concerning the potential efficacy of switching CGRP(r) MoAbs following previous medication discontinuation. Data was sourced by completing a database search for the terms: 'CGRP monoclonal antibody switch OR CGRP monoclonal antibody switching.' EXPERT OPINION: While data considering the potential efficacy of CGRP(r) switching continues to grow, our expert opinion supports the most recent European Headache Federation statement regarding CGRP(r) MoAb prescribing practices, concluding that there remains insufficient data to determine the efficacy of this intervention. As this topic is of significant clinical importance, we recommend a call-to-action to expand on current data considering the therapeutic options for patients that discontinue CGRP(r) MoAb therapy.

Claudins in Cancer: A Current and Future Therapeutic Target.

Claudins are a family of 27 proteins that have an important role in the formation of tight junctions. They also have an important function in ion exchange, cell mobility, and the epithelial-to-mesenchymal transition, the latter being very important in cancer invasion and metastasis. Therapeutic targeting of claudins has been investigated to improve cancer outcomes. Recent evidence shows improved outcomes when combining monoclonal antibodies against claudin 18.2 with chemotherapy for patients with gastroesophageal junction cancer. Currently, chimeric antigen receptor T-cells targeting claudin 18 are under investigation. In this review, we will discuss the major functions of claudins, their distribution in the normal as well as cancerous tissues, and their effect in cancer metastasis, with a special focus on the therapeutic targeting of claudins to improve cancer outcomes.

Preparation and epitope analysis of monoclonal antibodies against African swine fever virus DP96R protein.

BACKGROUND: Many proteins of African swine fever virus (ASFV, such as p72, p54, p30, CD2v, K205R) have been successfully expressed and characterized. However, there are few reports on the DP96R protein of ASFV, which is the virulence protein of ASFV and plays an important role in the process of host infection and invasion of ASFV. RESULTS: Firstly, the prokaryotic expression vector of DP96R gene was constructed, the prokaryotic system was used to induce the expression of DP96R protein, and monoclonal antibody was prepared by immunizing mice. Four monoclonal cells of DP96R protein were obtained by three ELISA screening and two sub-cloning; the titer of ascites antibody was up to 1:500,000, and the monoclonal antibody could specifically recognize DP96R protein. Finally, the subtypes of the four strains of monoclonal antibodies were identified and the minimum epitopes recognized by them were determined. CONCLUSION: Monoclonal antibody against ASFV DP96R protein was successfully prepared and identified, which lays a foundation for further exploration of the structure and function of DP96R protein and ASFV diagnostic technology.

Neonatal and maternal upregulation of antileukoproteinase in horses.

Introduction: The end of gestation, ensuing parturition, and the neonatal period represent highly dynamic phases for immunological changes in both mother and offspring. The regulation of innate immune cells at the maternal-fetal interface during late term pregnancy, after birth, and during microbial colonization of the neonatal gut and other mucosal surfaces, is crucial for controlling inflammation and maintaining homeostasis. Innate immune cells and mucosal epithelial cells express antileukoproteinase (SLPI), which has anti-inflammatory and anti-protease activity that can regulate cellular activation. Methods: Here, we developed and validated new monoclonal antibodies (mAbs) to characterize SLPI for the first time in horses. Peripheral blood and mucosal samples were collected from healthy adults horses and a cohort of mares and their foals directly following parturition to assess this crucial stage. Results: First, we defined the cell types producing SLPI in peripheral blood by flow cytometry, highlighting the neutrophils and a subset of the CD14+ monocytes as SLPI secreting immune cells. A fluorescent bead-based assay was developed with the new SLPI mAbs and used to establish baseline concentrations for secreted SLPI in serum and secretion samples from mucosal surfaces, including saliva, nasal secretion, colostrum, and milk. This demonstrated constitutive secretion of SLPI in a variety of equine tissues, including high colostrum concentrations. Using immunofluorescence, we identified production of SLPI in mucosal tissue. Finally, longitudinal sampling of clinically healthy mares and foals allowed monitoring of serum SLPI concentrations. In neonates and postpartum mares, SLPI peaked on the day of parturition, with mares returning to the adult normal within a week and foals maintaining significantly higher SLPI secretion until three months of age. Conclusion: This demonstrated a physiological systemic change in SLPI in both mares and their foals, particularly at the time around birth, likely contributing to the regulation of innate immune responses during this critical period.

Successful Management of Atypical Hemolytic-Uremic Syndrome in Pregnancy Using Eculizumab: A Case Review.

Hemolytic-uremic syndrome (HUS) is a rare thrombotic microangiopathy characterized by the triad of microangiopathic hemolytic anemia (MAHA), thrombocytopenia, and acute kidney injury. The disease is pathologically marked by fibrinoid necrosis within renal arterioles and glomerular capillaries. HUS can be categorized into typical variants, often linked to Shiga toxin-producing Escherichia coli (STEC) infection, and atypical variants that stem from dysregulation in the alternative complement pathway. Pregnancy is a recognized predisposing condition for HUS due to the potential reduction in complement regulatory proteins and the possibility of heightened maternal immune response. This report illustrates the case of a 36-year-old woman who, at 36 weeks of gestation, faced a breech presentation and was diagnosed with atypical HUS (aHUS) after placental abruption. Following a cesarean section, she developed complications, including a pelvic hematoma and bilateral hydronephrosis. Despite initial suboptimal response to plasmapheresis, the patient exhibited marked clinical improvement with eculizumab treatment, with no evidence of disease relapse.

Airway and Systemic Immunoglobulin Profiling and Immune Response in Adult Asthma.

INTRODUCTION: Immunoglobulins play a vital role in host immune response and in the pathogenesis of conditions like asthma. Therapeutic agents such as monoclonal antibodies target specific elements of the asthmatic inflammatory cascade. Decisions to utilize these medications are often based on systemic inflammatory profiling without direct insight into the airway inflammatory profile. We sought to investigate the relationship between immunoglobulin and cytokine profiles in the airway and systemic immune compartments of adult asthmatics. METHODS: Blood sampling and bronchoscopy with bronchoalveolar lavage (BAL) were performed in 76 well-defined adult asthmatics. Antibody and cytokine profiles were measured in both BAL and serum using ELISA and quantibody arrays. RESULTS: There was no relationship between BAL and serum levels of IgE. This is of significance in an asthma population. For some analytes, correlation analysis was significant (P < 0.05) indicating representativeness of our cohort and experimental setup in those cases. Nevertheless, the predictive power (r2) of the BAL-to-serum comparisons was mostly low except for TNF-α (r2 = 0.73) when assuming a simple (linear) relationship. CONCLUSION: This study highlights the importance of sample site when investigating the roles of immunoglobulins and cytokines in disease pathogenesis and suggests that both localized and systemic immune responses are at play. The prescription of asthma monoclonal therapy is generally based on systemic evaluation of cytokine and immunoglobulin levels. Our research suggests that this approach may not fully reflect the pathophysiology of the disease and may provide insight into why some patients respond to these targeted therapies while others do not.

Identification of B-cell epitope on the N protein of type 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) using monoclonal antibody and construction of epitope-mutated virus.

The escalating epidemic of PRRSV-1 in China has prompted widespread concern regarding the evolution of strains, disparities in pathogenicity to herds, and immunological detection of emerging strains. The nucleocapsid (N) protein, as a highly conserved protein with immunogenic properties in PRRSV, is a subject of intensive study. In this research, the recombinant His-N protein was expressed based on the N gene of PRRSV-1 using a prokaryotic expression system and then administered to BALB/c mice. A cell fusion protocol was implemented between SP2/0 cells and splenocytes, resulting in the successful screening of a monoclonal antibody against the N protein, designated as mAb 2D7, by indirect ELISA. Western Blot analysis and Indirect Immunofluorescence Assay (IFA) confirmed that mAb 2D7 positively responded to PRRSV-1. By constructing and expressing a series of truncated His-fused N proteins, a B-cell epitope of N protein, 59-AAEDDIR-65, was identified. A sequence alignment of two genotypes of PRRSV revealed that this epitope is relatively conserved in PRRSV, yet more so in genotype 1. Cross-reactivity analysis by Western blot analysis demonstrated that the B-cell epitope containing D62Y mutation could not be recognized by mAb 2D7. The inability of mAb 2D7 to recognize the epitope carrying the D62Y mutation was further determined using an infectious clone of PRRSV. This research may shed light on the biological significance of the N protein of PRRSV, paving the way for the advancement of immunological detection and development of future recombinant marker vaccine.

Titer and charge-based heterogeneity multiattribute monitoring of mAbs in cell culture harvest using 2D ProA CEX MS.

Robust monitoring of heterogeneity in biopharmaceutical development is crucial for producing safe and efficacious biotherapeutic products. Multiattribute monitoring (MAM) has emerged as an efficient tool for monitoring of mAb heterogeneities like deamidation, sialylation, glycosylation, and oxidation. Conventional biopharma analysis during mAb development relies on use of one-dimensional methods for monitoring titer and charge-based heterogeneity using non-volatile solvents without direct coupling with mass spectrometry (MS). This approach requires analysis of mAb harvest by ProA for titer estimation followed by separate cation exchange chromatography (CEX) analysis of the purified sample for estimating charge-based heterogeneity. This can take up to 60-90 min due to the required fraction collection and buffer exchange steps. In this work, a native two-dimensional liquid chromatography (2DLC) mass spectrometry method has been developed with Protein A chromatography in the first dimension for titer estimation and cation exchange chromatography (CEX) in the second dimension for charge variant analysis. The method uses volatile salts for both dimensions and enables easy coupling to MS. The proposed 2DLC method exhibits a charge variant profile that is similar to that observed via the traditional methods and takes only 15 min for mass identification of each variant. A total of six charge variants were separated by the CEX analysis after titer estimation, including linearity assessment from 5 µg to 160 µg of injected mAb sample. The proposed method successfully estimated charge variants for the mAb innovator and 4 of its biosimilars, showcasing its applicability for biosimilarity exercises. Hence, the 2D ProA CEX MS method allows direct titer and charge variant estimation of mAbs in a single workflow.

Anti-Netrin-1 decorated nanoparticles combined with chemotherapy for the treatment of triple-negative breast cancer.

Nanoparticle's success as drug delivery systems for cancer treatment has been achieved through passive targeting mechanisms. However, tumor heterogeneity and rapid drug clearance limit the treatment efficacy. Improved outcomes and selective drug release can be achieved by grafting ligands at the surface of nanocarriers that bind molecules overexpressed in the tumor microenvironment (TME). In this work, we developed a docetaxel-loaded nanoemulsions (NEs) binding an anti-netrin-1 monoclonal antibody (NP137) to selectively target the netrin-1 protein overexpressed in many different tumors. The goal is to refine a combined approach utilizing NP137 and docetaxel as an improved tumor-targeting chemotherapeutic agent for addressing triple-negative breast cancer (TNBC). Several factors have been considered for the optimization of the active targeted drug delivery system via the click-chemistry conjugation, as the impact of PEGylated surfactant that stabilize the NEs shell on conjugation efficiency, cytocompatibility with EMT6 cell line and colloidal stability over time of NEs. Results showed that a 660 Da PEG chain length contributed to NEs colloidal stability and had no impact on cell viability or on the antibody binding ability for its ligand after surface conjugation. Moreover, docetaxel was encapsulated into the oily core of NEs, with an encapsulation efficiency of 70 %. To validate our treatment strategy in vivo, the 4T1 murine breast cancer model was used. As a result, the comparison of active-targeted and non-targeted NEs revealed that only active-targeted NE could decrease the tumor growth rate.

Clinicopathological Characteristics of Light and Heavy Chain Deposition Disease: A Case Series.

RATIONALE & OBJECTIVE: Light and heavy chain deposition disease (LHCDD) is a rare form of monoclonal immunoglobulin deposition disease (MIDD), and limited clinical data are available characterizing this condition. We described the clinicopathological characteristics and outcomes of LHCDD. STUDY DESIGN: Case series. SETTING & PARTICIPANTS: 13 patients with biopsy-proven LHCDD, diagnosed between January 2008 to December 2022, at two Chinese medical centers. FINDINGS: Among the 13 patients described, 6 were men and 7 were women, with a mean age of 52.6 ± 8.0 years. Patients presented with hypertension (76.9%), anemia (84.6%), elevated serum creatinine (84.6%, median serum creatinine 1.7 mg/dL), proteinuria (100%, average urine protein 3.0g/24h), nephrotic syndrome (30.8%) and microscopic hematuria (76.9%). Serum immunofixation electrophoresis showed monoclonal immunoglobulin for 11 (84.6%) patients. Serum free light chain (FLC) ratios were abnormal in 11 (84.6%) patients, and heavy/light chain (HLC) ratios were abnormal in 9 of 10 (90%) patients with available data. Five patients were diagnosed with multiple myeloma. A histological diagnosis of nodular mesangial sclerosis was made in 10 (76.9%) patients. Immunofluorescence demonstrated deposits of IgG subclass (γ-κ/γ-λ:4/3) in 7 patients, and IgA (α-κ/α-λ:2/3) in 5 patients. Six patients underwent IgG subclass staining (γ1/γ2/γ3:3/2/1). The deposits of IgD-κ were confirmed by mass spectrometry in 1 patient. Among 12 patients for whom data were available over a median of 26.5 months, 11 received chemotherapy, and 1 received conservative treatment. One patient died. Three (25%) patients progressed to kidney failure. Among the 9 patients evaluable for hematological and kidney disease progression, five (56%) had a hematologic response and one (11%) achieved improvement in kidney disease. LIMITATIONS: Retrospective descriptive study, limited number of patients, UPEP or UIFE missing for most patients. CONCLUSIONS: In this case series of LHCDD, light and heavy chain deposition in kidney tissues were most frequent with monoclonal IgG1-κ. Among patients with evaluable data, more than half had hematologic response but a kidney response was uncommon.

Higher order structure characterization of two interdomain disulfide bond variants of a bispecific monoclonal antibody.

Characterization and understanding of protein higher order structure (HOS) is essential at all stages of biologics development. Here, two folding variants of a bispecific monoclonal antibody, the correctly folded form and an alternative configuration with reduced potency, were characterized by several HOS characterization techniques. Specifically, differential scanning calorimetry (DSC), circular dichroism (CD), Fourier-transform infrared spectroscopy (FTIR), Raman and Raman optical activity (ROA) spectroscopy were used together to elucidate the impacts of disulfide bond scrambling in the fused scFv domains on the structure and thermal stability of the antibody. This study illustrates the importance of selecting appropriate biophysical characterization techniques based on the nature and magnitude of the HOS change.