Clinical and neuroradiographic features of fentanyl inhalation-induced leukoencephalopathy.

A man in his late 40s with no known past medical history was unresponsive for an unknown period of time. Crushed pills and white residue were found on a nearby table. On presentation he was obtunded and unresponsive to verbal commands but withdrawing to painful stimuli. The initial urine drug screen was negative, but a urine fentanyl screen was subsequently positive with a level of 137.3 ng/mL. MRI of the brain showed reduced diffusivity and fluid attenuated inversion recovery (FLAIR) hyperintensity symmetrically in the bilateral supratentorial white matter, cerebellum and globus pallidus. Alternative diagnoses such as infection were considered, but ultimately the history and workup led to a diagnosis of fentanyl-induced leukoencephalopathy. Three days after admission the patient became able to track, respond to voice and follow basic one-step commands. The patient does not recall the mechanism of inhalation. While there are case reports of heroin-induced leukoencephalopathy following inhaled heroin use and many routes of fentanyl, this is the first reported case of a similar phenomenon due to fentanyl inhalation.

Turning the world upside down: reversal-of-vision metamorphopsia in a patient with syncope.

An elderly man with recurrent syncope was admitted with a globe rupture following a syncopal attack. After an initial unremarkable evaluation, the patient reported inversion of the room's wall clock during a bedside evaluation. This symptom is called reversal-of-vision metamorphopsia (RVM) and is a rare visual disturbance that typically results from organic processes localised to the retina and/or posterior cortex of the brain or in some cases is psychogenic in nature. In this case, both the syncope and RVM were caused by impaired circulation in the posterior cortex, and management included an antiplatelet agent, statin and permissive blood pressure targets, which resulted in the correction of RVM.

Cross-sectional study evaluating the impact of SARS-CoV-2 variants on Long COVID outcomes in UK hospital survivors.

OBJECTIVES: COVID-19 studies report on hospital admission outcomes across SARS-CoV-2 waves of infection but knowledge of the impact of SARS-CoV-2 variants on the development of Long COVID in hospital survivors is limited. We sought to investigate Long COVID outcomes, aiming to compare outcomes in adult hospitalised survivors with known variants of concern during our first and second UK COVID-19 waves, prior to widespread vaccination. DESIGN: Prospective observational cross-sectional study. SETTING: Secondary care tertiary hospital in the UK. PARTICIPANTS: This study investigated Long COVID in 673 adults with laboratory-positive SARS-CoV-2 infection or clinically suspected COVID-19, 6 weeks after hospital discharge. We compared adults with wave 1 (wildtype variant, admitted from February to April 2020) and wave 2 patients (confirmed Alpha variant on viral sequencing (B.1.1.7), admitted from December 2020 to February 2021). OUTCOME MEASURES: Associations of Long COVID presence (one or more of 14 symptoms) and total number of Long COVID symptoms with SARS-CoV-2 variant were analysed using multiple logistic and Poisson regression, respectively. RESULTS: 322/400 (wave 1) and 248/273 (wave 2) patients completed follow-up. Predictors of increased total number of Long COVID symptoms included: pre-existing lung disease (adjusted count ratio (aCR)=1.26, 95% CI 1.07, 1.48) and more COVID-19 admission symptoms (aCR=1.07, 95% CI 1.02, 1.12). Weaker associations included increased length of inpatient stay (aCR=1.02, 95% CI 1.00, 1.03) and later review after discharge (aCR=1.00, 95% CI 1.00, 1.01). SARS-CoV-2 variant was not associated with Long COVID presence (OR=0.99, 95% CI 0.24, 4.20) or total number of symptoms (aCR=1.09, 95% CI 0.82, 1.44). CONCLUSIONS: Patients with chronic lung disease or greater COVID-19 admission symptoms have higher Long COVID risk. SARS-CoV-2 variant was not predictive of Long COVID though in wave 2 we identified fewer admission symptoms, improved clinical trajectory and outcomes. Addressing modifiable factors such as length of stay and timepoint of clinical review following discharge may enable clinicians to move from Long COVID risk stratification towards improving its outcome.

Real world use of dolutegravir two drug regimens.

BACKGROUND: Since 2015, we prescribed dolutegravir (DTG)-based two drug regimens (DTG-2DR) for 620 people [total cohort 3133 (19.8%)]. METHOD: Clinic database search 1 January 15 to 31 October 21. Demographic, tolerability and HIV related data analysed. RESULTS: In total, 620 people identified; 561 had complete data. 446 male (79.5%); median age 54 years (interquartile range 46, 59). 343 (61.1%) MSM. Nine people who initiated naïvely achieved viral suppression (100%). 546/552 (99.0%) switched or continued and were suppressed at data censor. 460/552 (83.3%) received DTG-lamivudine (DTG/3TC), 74/552 (13.4%) received DTG-rilpivirine (DTG/RPV) and 18/552 (3.3%) received DTG-emtricitabine (DTG/FTC). 70 (12.5%) switched off DTG-2DR (55 DTG/3TC, 13 DTG/RPV, two DTG/FTC) due to side-effects. 41 episodes of blip (1 off >50 copies/ml) occurred in 30 people (5.3%). 11/41 on DTG-RPV [ n  = 7 multi-tablet regimen (MTR), n  = 4 single tablet regimen (STR)]. 27/41 DTG-3TC, 3/41 DTG/FTC ( n  = 26 MTR, n  = 4 STR). Six people (1.1%) failed (confirmed viral load >200 copies/ml or persistent low level viraemia) ( n  = 4 DTG-3TC STR, n  = 1 DTG-3TC MTR, n  = 1 DTG-RPV MTR). Four failures due to low level viraemia, one due to non-adherence and one due to high viral load. Resistance tests performed for 5/6 - mutations detected only in latter person with high viral load failure (on DTG-3TC MTR) who developed triple class resistance. CONCLUSION: Majority of experience is in DTG/3TC stable switch. Minority of patients developed side-effects. Low number of virological failures, one developed integrase inhibitor resistance. Viral failure associated with MTR, commensurate with trial data showing no failure with resistance if DTG/3TC STR used. Overall DTG-2DR demonstrates high efficacy in real-world setting.

Implementation of a Nurse-Driven Medication Ordering Protocol to Improve Clinician and Nursing Experiences.

ABSTRACT: Paging and text messaging to request new orders remain common means of communication between clinicians and nurses in the hospital setting. However, sending and triaging multiple pages can lead to interruptions in other clinical duties. A medication order delegation protocol allowing for nurse-driven ordering of low-risk medications was developed with an objective of decreasing potentially avoidable pages. The aim of this study was to evaluate the impact of implementing this protocol on nurse and clinician perceptions of clerical burden and satisfaction. A survey assessing satisfaction with the process of obtaining medications in this protocol and the perception of clerical burden associated with ordering them before and after delegation protocol implementation was completed by over 160 clinicians and nurses. Survey respondents reported increased satisfaction and decreased clerical burden associated with the implementation of the delegation protocol. These results suggest the potential for delegation protocols to limit clerical burden associated with paging.

Leveraging light chain binding avidity for control of mispaired byproducts during production of asymmetric bispecific antibodies.

Many biotherapeutic formats leverage antibody light chain affinity chromatography to enable robust manufacturing processes and to streamline process development. These include multi-specific antibody and antibody fragment platforms which are often designed for specific capture purification methods that can provide efficient removal of commonly expressed product-related impurities. Recently, several accounts of product-related impurity separation by leveraging binding avidity during affinity chromatography have been described in the literature. However, a more comprehensive evaluation of avidity-based separations, particularly for light chain affinity media with specificity for constant regions of antibody light chains, is valuable for development of emerging multi-specific and fragment antibody formats. Results in this work demonstrate the capability of camelid antibody-based light chain affinity media to separate asymmetric bispecific antibody heterodimers from impurities possessing more than one light chain of the same class that the media binds to, including mispaired variants, aggregates, and fragment impurities. Largest resolution for respective mispaired species were provided by CaptureSelect KappaXP and LambdaXP chromatography media. The addition of elution modifiers provided increased impurity separation, with CaptureSelect KappaXP requiring up to 500 mM concentrations of elution modifiers to produce substantial improvements to resolution, and LambdaXP showing much higher sensitivity. Isocratic elution methods developed for lambda light chain affinity chromatography media provided near complete removal of mispaired variants, and substantial removal of aggregates and fragment impurities. Addition of just 20 mM of elution modifiers such as NaCl are shown to drive increased binding strength and separation of heterodimer species from impurities on CaptureSelect LambdaXP. These results provide scalable and transferable methods for product-related impurity control for various biotherapeutic modalities by lambda light chain affinity chromatography.

Formation of transient highly-charged mAb clusters strengthens interactions with host cell proteins and results in poor clearance of host cell proteins by protein A chromatography.

Protein A chromatography with a high salt wash usually leads to robust clearance of host cell proteins (HCPs) in most recombinant monoclonal antibodies (mAbs), but a small subset of recalcitrant mAbs show significant HCP copurification. In this study, we carried out systematic studies using 4 different mAbs to explore the HCP copurification mechanism. HCP identification results revealed that the 3 high-HCP mAbs had many common HCPs which do not copurify with the low-HCP mAb, suggesting a similar mechanism is at play. Through wash evaluation, surface patch analysis, chain-swapping, domain evaluation, and structure-guided mutations, several charged residues in each mAb were found which correlated with HCP copurification. Surprisingly, these residues are also critical for self-association propensity. We observed an inverse correlation between diffusion interaction parameter and HCP copurification. Each of the high-HCP mAbs could form dynamic clusters consisting of 3∼6 mAb molecules. Therefore, a mAb cluster can exhibit higher net positive charges on the order of 3 to 6, compared with the individual mAb. In Protein A chromatography, high-HCP mAbs had elution tailing which contained high level of HCPs. Addition of Arginine-HCl or point mutations preventing cluster formation effectively reduced HCP copurification and elution tailing. Based on these results, we propose a novel HCP-copurification mechanism that formation of mAb clusters strengthens charge-charge interactions with HCPs and thus compromises HCP removal by Protein A chromatography. Besides arginine, histidine under acidic pH conditions prevented cluster formulation and resulted in effective HCP removal. Finally, structure-guided protein engineering and solution screening by using cluster size as indicator are useful tools for managing mAbs with high-HCP issues.

Pharmacological Potential of Lathyrane-Type Diterpenoids from Phytochemical Sources.

Lathyrane diterpenoids are one of the primary types of secondary metabolites present in the genus Euphorbia and one of the largest groups of diterpenes. They are characterized by having a highly oxygenated tricyclic system of 5, 11 and 3 members. These natural products and some synthetic derivatives have shown numerous interesting biological activities with clinical potential against various diseases, such as cytotoxic activity against cancer cell lines, multi-drug resistance reversal, antiviral properties, anti-inflammatory activity and their capability to induce proliferation or differentiation into neurons of neural progenitor cells. The structure of the lathyrane skeleton could be considered privileged because its framework is able to direct functional groups in a well-defined space. The favorable arrangement of these makes interaction possible with more than one target. This review aims to highlight the evidence of lathyranes as privileged structures in medicinal chemistry. Chemical structures of bioactive compounds, the evaluation of biological properties of natural and semisynthetic derivatives, and the exploration of the mechanisms of action as well as target identification and some aspects of their targeted delivery are discussed.

Identification of compendial nonionic detergents for the replacement of Triton X-100 in bioprocessing.

We have systematically investigated six compendial nonionic detergents as potential replacements for Triton ×-100 in bioprocessing applications. Use of compendial raw materials in cGMP bioprocessing is advantageous for a variety of reasons including material specifications developed to meet stringent pharmaceutical product quality requirements, regulatory familiarity and comfort, and availability from vendors experienced supplying the biopharmaceutical industry. We first examine material properties of the detergents themselves including melting point and viscosity. Process performance and product contact in real-world bioprocess applications are then investigated. Lastly, we test the detergents in virus inactivation (VI) experiments with recombinant proteins and adeno-associated virus. Two of the detergents tested, PEG 9 Lauryl Ether and PEG 6 Caprylic/Capric Glycerides, showed favorable properties that make them attractive for use as potential Triton X-100 replacements. Process performance testing indicated negligible impact of the detergents on product yield, purity, and activity compared to a control with no detergent. Importantly, both PEG 9 Lauryl Ether and PEG 6 Caprylic/Capric Glycerides demonstrated very fast VI kinetics with complete inactivation of XMuLV observed in less than 1 min at a target 1% detergent concentration. Potential advantages and disadvantages of both candidate detergents for use in cGMP bioprocessing are summarized and discussed.

Ultrasonic Transducers Made From Freeze-Cast Porous Piezoceramics.

Porous and composite piezoelectric ceramics are of interest for underwater ultrasonic transducers due to their improved voltage sensitivity and acoustic matching with water, compared with their dense counterparts. Commonly, these materials are fabricated by dice-and-fill of sintered blocks of polycrystalline piezoceramic, which results in a high volume of waste. The freeze-casting technique offers a low waste and scalable alternative to the dice-and-fill method to produce porous piezoceramics with highly orientated, anisometric pores. In this article, we have fabricated underwater ultrasonic transducers from freeze-cast lead zirconate titanate (PZT) with a range of porosities. The porous PZT samples were characterized in terms of their piezoelectric and dielectric properties before being encapsulated for acoustic performance testing in water. Off resonance, the on- axis receive sensitivity of the manufactured devices was approximately [Formula: see text]; the transmit voltage response (TVR) was in the range of approximately [Formula: see text] at 60 kHz to [Formula: see text] at 180 kHz. The most porous transducer devices (0.51, 0.43, and 0.33 pore fraction) exhibited primarily a thickness mode resonance, whereas the least porous transducers (0.29 pore fraction and dense benchmark) exhibited an undesired radial mode, which was observed as an additional resonant peak in the electrical impedance measurements and lateral off-axis lobes in the acoustic beampatterns. Our results show that the acoustic sensitivities and TVRs of the porous freeze-cast transducers are comparable to those of a dense pressed transducer. However, the freeze-cast transducers with porosity exceeding 0.30 pore fraction were shown to achieve an effective structure with aligned porosity that suppressed undesired radial mode resonances.

Identification of a CE-SDS shoulder peak as disulfide-linked fragments from common CH2 cleavages in IgGs and IgG-like bispecific antibodies.

Fragmentation is a well-characterized degradation pathway of therapeutic antibodies and is usually monitored by capillary electrophoresis-sodium dodecyl sulfate (CE-SDS). Although fragments due to cleavage in CH2 domains linked by intrachain disulfide bonds are common and can be detected by reduced reversed-phase - liquid chromatography mass spectrometry (RP-LCMS) and reduced CE-SDS methods, their separation in nonreduced CE-SDS (nrCE-SDS) has not been reported but speculated as comigrating with intact IgG. A shoulder peak in nrCE-SDS was observed in the stability samples of an IgG-like bispecific antibody and was determined to be mainly caused by fragments from clipping at the C-terminus of leucine (L)306 or L309 (EU numbering) in the CH2 domain of both heavy chains (HCs) and, to a lesser degree, at the C-terminus of L182 in the CH1 domain of the knob HC. Subunit LCMS analysis verified that the crystallizable fragment contained variants with one or multiple mass additions of ~18 Da due to clipping. Further investigation revealed that CH2 clippings at L306 and L309 were largely due to proteolytic activity, and cleavages were present at various levels in all in-house IgG1 and IgG4 molecules studied. Our study shows that CH2 domain cleavages, with complementary fragments still linked by intrachain disulfide, can be electrophoretically resolved as a front shoulder of the main peak in nrCE-SDS. Given the high occurrence of CH2 cleavages in antibodies, these findings will have broad applicability and could help manufacturers of therapeutic antibodies in process improvement, product characterization, investigations, formulation stability, and stability comparability studies.

Parameter Reduction and Optimisation for Point Cloud and Occupancy Mapping Algorithms.

Occupancy mapping is widely used to generate volumetric 3D environment models from point clouds, informing a robotic platform which parts of the environment are free and which are not. The selection of the parameters that govern the point cloud generation algorithms and mapping algorithms affects the process and the quality of the final map. Although previous studies have been reported in the literature on optimising major parameter configurations, research in the process to identify optimal parameter sets to achieve best occupancy mapping performance remains limited. The current work aims to fill this gap with a two-step principled methodology that first identifies the most significant parameters by conducting Neighbourhood Component Analysis on all parameters and then optimise those using grid search with the area under the Receiver Operating Characteristic curve. This study is conducted on 20 data sets with specially designed targets, providing precise ground truths for evaluation purposes. The methodology is tested on OctoMap with point clouds created by applying StereoSGBM on the images from a stereo camera. A clear indication can be seen that mapping parameters are more important than point cloud generation parameters. Moreover, up to 15% improvement in mapping performance can be achieved over default parameters.

Role of configurational flexibility on the adsorption kinetics of bivalent bispecific antibodies on porous cation exchange resins.

The adsorption kinetics of a monoclonal antibody (mAb) used as a reference and of bivalent bispecific antibodies (BiSAb) on a macroporous cation exchanger is studied experimentally by examining the transient patterns of bound protein within the particles using confocal microscopy for a range of protein concentrations, buffer concentrations and pH, and temperatures. The mAb adsorption kinetics is controlled by pore diffusion and conforms to the classical shrinking core model. While the mAb adsorption rate increases with temperature, the ratio of effective and free solution diffusivity, De /D0, remains constant and has a value of 0.20. The BiSAb's structure is comprised of scFv domains that are genetically fused to a framework IgG through flexible peptide linkers which results in conformational flexibility leading to multiple binding forms with varying affinity for the adsorbent surface. As a result, adsorption of the BiSAbs shows complex patterns of total bound protein within the particles. These BiSAb adsorption patterns are influenced by buffer ionic strength, pH, and temperature in unique ways. Sharper intraparticle profiles are observed for conditions where the binding strength is greater (lower buffer concentration and/or pH) or when the protein is chemically crosslinked to restrict configurational flexibility. Temperature affects the BiSAb pore diffusivity as well as the interconversion kinetics. While the effects of temperature on BiSAb transport are also described by a constant De /D0 = 0.15, the temperature also affects the rate of interconversion between binding forms leading to faster equilibration at higher temperatures. A phenomenological model indicates that the interplay of pore diffusion and adsorption with the kinetically limited interconversion between binding forms is responsible for the experimental trends.

A one-week internal medicine procedure rotation designed to increase procedural opportunities and competency.

Background: Mounting literature describes increased procedure volume and improvement in procedural skills following implementation of procedural curricula and standardized rotations, generally requiring at least two weeks and incorporating dedicated lecture and didactic efforts. It is unknown whether shorter rotations that feature self-directed curricula can achieve similar outcomes.Methods: House staff participated in a one-week procedure rotation that coincided with preexisting non-clinical blocks ('jeopardy'). It provided an online curriculum as well as opportunities to perform procedures under interprofessional supervision. Inpatient procedure volumes were tallied before and after implementation of the rotation. During the first year of the rotation (academic year 2013-2014), house staff completed a knowledge-based quiz and a Likert-based survey (range 1-5) addressing confidence in performing procedures and satisfaction in procedural training. Results: Ninety-five of 99 house staff participated in the intervention (96% response rate). The total number of procedures performed by the Division of Hospital Medicine increased from an average of 74 per year over the four years prior to the introduction of the rotation to 291 per year during the third year of the rotation. The knowledge-based quiz score improved from a pre-intervention mean value of 50% to a post-intervention mean value of 61% (P = 0.020). Confidence in performing procedures improved from a pre-intervention mean value of 2.37 to a post-intervention mean value of 2.59 (P < 0.001). Satisfaction with procedural training improved from a pre-intervention mean value of 2.48 to a post-intervention mean value of 2.69 (P < 0.001).Conclusions: A one-week procedure rotation with a self-directed curriculum was introduced into the curriculum of an internal medicine residency program and was associated with increased procedure volume and sustained improvement in house staff knowledge, confidence, and satisfaction with procedural training.

Challenges for design of aggregation-resistant variants of granulocyte colony-stimulating factor.

Non-native protein aggregation is a long-standing issue in pharmaceutical biotechnology. A rational design approach was used in order to identify variants of recombinant human granulocyte colony-stimulating factor (rhG-CSF) with lower aggregation propensity at solution conditions that are typical of commercial formulation. The approach used aggregation-prone-region (APR) predictors to select single amino acid substitutions that were predicted to decrease intrinsic aggregation propensity (IAP). The results of static light scattering temperature-ramps and chemical unfolding experiments demonstrated that none of the selected variants exhibited improved aggregation resistance, and the apparent conformational stability of each variant was lower than that of WT. Aggregation studies under partly denaturing conditions suggested that the IAP of at least one variant remained unaltered. Overall, this study highlights a general challenge in designing aggregation resistance for proteins, due to the need to accurately predict both APRs and conformational stability.

Chromatographic and adsorptive behavior of a bivalent bispecific antibody and associated fragments.

The elution and adsorptive behavior of a bivalent bispecific antibody (BiSAb), comprising an IgG1 framework with a scFv domain genetically fused to each heavy chain C-terminus via flexible linkers, and of two associated fragments were studied on two cation exchange chromatography media - ProPac WCX-10, which is pellicular and suitable for analytical use, and Nuvia HR-S, which is macroporous and suitable for preparative and process scale uses. Both fragments were identified by MS as missing one of the two scFv domains and its flexible linker, but one of them also contains an additional C-terminal lysine. The separation of these fragments on both resins occurs as a result of differences in non-specific ligand-protein interactions that are modulated by the salt concentration. For the ProPac WCX-10 column, complex, multipeak elution behaviors are observed, since, as a result of the linker flexibility, both the intact molecule and the fragments appear to exist in multiple binding configurations with each scFv domains either collapsed onto the IgG framework or extended away from it. With a residence time of 2.5 min and at 21 °C, two peak elution is observed for the fragments which contain a single linked scFv and three peak elution for the intact molecule which contains two linked scFvs. This behavior is affected by residence time, temperature, and hold time. Increasing the residence time to 25 min or increasing temperature to 40°C results in elution of a single, merged peak for each of the protein species. For Nuvia HR-S, the broader peaks, obtained as a result of mass transfer limitations, tend to obscure the multipeak elution behavior. Nevertheless, even for this resin, the effects of configurational flexibility are still manifested at the single-particle scale and affect the evolution of the patterns of protein binding within individual resin particles as evident from confocal microscopy observations.

Characterization and Monitoring of a Novel Light-heavy-light Chain Mispair in a Therapeutic Bispecific Antibody.

Site-specific cysteine engineering, along with other genetic mutations, is broadly implemented in bispecific antibodies (bsAb). Thus far, homodimer, half hole antibody, one-light chain mispaired and light chain swapped variants have been reported as chain-pairing variants for the asymmetric IgG-like bispecific antibodies. Here we report a novel mispair in which the CH3 engineered cysteine on the hole heavy chain (HC) of a knob-into-hole (KiH) bsAb is linked to the engineered cysteine in CL through a disulfide bond, forming a LHL species in a bsAb construct. Due to its impact on bioactivity, it is critical to implement an analytical strategy to monitor this CQA and mitigate risk for the future products. A set of orthogonal physicochemical assays that include hydrophobic interaction chromatography (HIC), capillary electrophoresis sodium dodecyl sulfate (CE-SDS), reverse phase liquid chromatography ultra-performance chromatography mass spectrometry (RP-UPLC MS) and disulfide bond mapping have been utilized to monitor and characterize this chain-pairing impurity for manufacturing process control and product release. Our data shows the LHL mispair in condition medium (CM) is approximately 1.3 - 1.9%. LambdaFabSelect affinity chromatography removes two major chain-pairing variants in CM - i.e. the hole-hole homodimer and hole half-antibody, while retaining the LHL species. Process improvement in Capto Q (anion exchange) and HS50 (cation exchange) chromatography steps removes LHL to as low as 0.2% in the final product. We have demonstrated an orthogonal analytical methodology that is capable of characterizing and monitoring bsAb mispairing, suitable for use in manufacturing process control and product release, and can be potentially implemented for similar bsAb constructs with engineered disulfide bonds.

Development of a mechanism of action-reflective, dual target cell-based reporter bioassay for a bispecific monoclonal antibody targeting human CTLA-4 and PD-1.

T-cell-mediated immunotherapy has generated much excitement after the success of therapeutic biologics targeting immune checkpoint molecules. Bispecific antibodies (BsAbs) that recognize two antigen targets are a fast-growing class of biologics offering promising clinical benefits for cancer immunotherapy. Due to the complexity of the molecule structure and the potential mechanism of action (MOA) that involves more than one signaling pathway, it is critical to develop appropriate bioassays for measuring potency and characterizing the biological properties of BsAbs. Here, we present a dual target, cell-based reporter bioassay for a BsAb that binds human CTLA-4 and PD-1 and targets two subsequent signaling pathways that negatively regulate T-cell activation. This reporter bioassay is capable of measuring the potency of both antigen target arms in one assay, which would not be achievable using two single target bioassays. This dual target reporter bioassay demonstrates good performance characteristics suitable for lot release, stability testing, critical quality attribute assessment, and biological properties characterization of the CTLA-4/PD-1 BsAb. Furthermore, this assay can capture the synergistic effect of anti-CTLA-4 and anti-PD-1 activity of the BsAb. Compared to single target assays, this dual target bioassay could better reflect the potential MOA of BsAbs and could be used for evaluation of other bispecific biologics, as well as antibody combination therapies.