Viral clearance capability of monoclonal antibody purification.

Viral clearance steps are routinely included in monoclonal antibody purification processes to safeguard product from potential virus contamination. These steps are often experimentally studied using product-specific feeds and parameters for each project to demonstrate viral clearance capability. However, published evidence suggests that viral clearance capability of many of these steps are not significantly impacted by variations in feed material or process parameter within commonly used ranges. The current investigation confirms robust retrovirus inactivation by low pH treatment and parvovirus removal by second-generation virus filters, independent to individual antibody molecules. Our results also reveal robust retrovirus removal by flowthrough anion exchange chromatography, inside the limits of protein load and host cell protein content. The cumulative viral clearance capability from these steps leads to an excess clearance safety factor of 10,000-fold for endogenous retrovirus-like particles. These results further justify the use of prior knowledge-based modular viral clearance estimation as opposed to repetitive experimentation.

Real-World Clinical Characterisation of Polycythaemia Vera Patients from a Prospective Registry in Portugal: Is Resistance to Hydroxyurea a Reality?

Patients with polycythaemia vera (PV) are at increased risk of thrombosis and haemorrhages. Although hydroxyurea (HU) has been the frontline therapy for patients at high risk of vascular complications, about 25% of patients develop resistance/intolerance to this therapy. The aim of this non-interventional, multicentre cohort study was to understand the clinical characteristics and HU treatment response of Portuguese PV patients. HU resistance/intolerance was defined according to adjusted European LeukemiaNet (ELN) criteria. In total, 134 PV patients with a mean (SD) disease duration of 4.8 (5.0) years were included and followed up for 2 years. At baseline, most patients were ≥60 years old (83.2%), at high risk for thrombotic events (87.2%), and receiving HU therapy (79.1%). A total of 10 thrombotic events and 8 haemorrhagic events were reported, resulting in a 5-year probability of thrombo-haemorrhagic events of 17.2%. Haematocrit (p = 0.007), haemoglobin (p = 0.012) and MPN10 symptom score (12.0 (11.6) vs. 10.3 (9.1); p = 0.041) decreased significantly at the 24-month visit compared to baseline. Overall, 75.9% of patients met at least one of the adjusted ELN criteria for HU resistance, and 14.4% of patients remained on HU throughout the study. The results from this real-world study may help identify the subset of patients at higher risk for disease sequelae who may benefit from earlier second-line treatment.

Exploring features in chromatographic profiles as a tool for monitoring column performance.

In the biopharmaceutical industry, chromatography resins have a finite number of uses before they start to age and degrade, typically due to losses of ligand integrity and/or density. The "health" of a column is predicted and validated by running multiple cycles on representative scale-down models and can be followed by real-time on-going validation during commercial production. Principal Component Analysis (PCA), Partial Least Square (PLS), Similarity Scores and Single One Point-MultiParameter Technique (SOP-MPT) along with machine learning principles were applied to explore the hypothesis that there is predictive capability of latent variables in chromatography absorbance profiles for process performance (step yield) and product quality (aggregates, fragments, host cell proteins (HCP) and DNA, and Protein A ligand). The first stage of this study is described in this paper: a MabSelect SuRe™ chromatography column was cycled with a method to establish the "normal" baseline for process performance and product quality, followed by runs using a harsher NaOH Cleaning in Place (CIP) procedure (with a higher NaOH concentration than that recommended by the vendor) to accelerate resin degradation. The different mathematical analytical tools correlated with resin degradation of the column (reflected in decreasing step yield and binding capacity with increasing running cycle), specifically when using the Wash, Elution and Strip phases of the chromatography method. Monomer, HCP and DNA content were not significantly impacted and therefore a correlation with product quality was inconsequential. Importantly, this work shows proof-of-concept that while more traditional methods of measuring resin integrity such as the height equivalent to a theoretical place (HETP) and Asymmetry (As) measurements could not detect changes in the integrity of the resin, PCA, PLS, Similarity Scores and SOP-MPT (to a lesser extent) applied to the absorbance data were capable of anticipating issues in the chromatography bed by identifying atypical outcomes.

Linfoma pulmonar MALT: reporte de un caso y revisión de la literatura / Pulmonary Malt Lymphoma: A Case Report and Review of the Literature

Pulmonary mucosa-associated lymphoid tissue lymphoma (pMALToma) is an extranodal low-grade B-cell lymphoma which originates from bronchial and/or parenchyma MALT. The authors report a case of an 81-year-old female with incidental finding of multiple bilateral pulmonary consolidations. After being submitted to an extensive workup, the diagnosis of pMALToma was finally established. The authors intend to highlight this rare entity and its challenging workup. It Is characterized by non-specific clinical manifestations and diverse imaging findings frequently leading to the need to perform more invasive techniques for a final diagnosis. (AU)

Evaluating multiproduct chromatography Protein A resin reuse for monoclonal antibodies in biopharmaceutical manufacturing.

In good manufacturing practice (GMP) facilities in the biopharmaceutical industry, chromatography resins are largely underutilized during purification of single drug products during clinical production. Chromatography resins are dedicated to a specific product and disposed of, after only a fraction of their lifetime due to concerns of potential product carryover from one program to another. In this study, we follow a resin lifetime methodology typically used for commercial submissions and apply it to determine the feasibility of purifying different products on a Protein A MabSelect PrismA™ resin. Three distinct monoclonal antibodies were used as model molecules. Column performance was monitored through chromatogram profiles, yield, clearance capability of selected media components, pressure and product quality. A protein carryover study was designed to demonstrate that the column cleaning procedures reduced protein carryover to safe cleanliness levels regardless of multiple product contact cycles and the order in which the mAbs are captured. Data show that up to 90 total cycles (30 cycles per antibody), there was negligible protein carryover and impact on process performance. Product quality was consistent, with the only meaningful trends found for the leached Protein A ligand, without affecting the conclusion of the study. While the study was restricted to three antibodies, the proof of concept for resin reuse was demonstrated.

Modification of Sugar Profile and Ripening in Atemoya (Annona × atemoya Mabb.) Fruits through Copper Hydroxide Application.

Atemoya (Annona × atemoya Mabb.), a climacteric fruit of the Annonaceae family, is becoming increasingly popular due to its organoleptic and nutritional properties. Anthracnose, a fungus of the Colletotrichum genus, is one of the most serious diseases in orchards, causing significant damage if not controlled, so producers use phytosanitary products. The current study sought to investigate the quality of atemoya fruits after harvest in an orchard with anthracnose controlled by Cu(OH)2 application: T1-no Cu(OH)2, T2-7.8 mL Cu(OH)2 L1 divided into two applications, T3-15.6 mL Cu(OH)2 L1 divided into four, T4-8.0 mL Cu(OH)2 L1 divided into eight, and T5-13.0 mL Cu(OH)2 L1 divided into thirteen applications. The sugar profile of fruits was examined, as well as MDA, H2O2, and quality parameters such as pH, mass, soluble solids, titratable acidity, and maturation index. MDA, such as H2O2, can function as a signal molecule. Eight applications of 1.0 mL L-1 Cu(OH)2 resulted in increased concentrations of H2O2 and MDA, signal molecules involved in sugar modification profiles such as glucose, fructose, and trehalose. It also had a high titratable acidity, a lower maturation index, better fruit quality, and a longer shelf life.

Using Sensitivity Analysis to Simplify the Virus Safety Factor Calculation in the Manufacture of Biopharmaceuticals.

Virus safety of biopharmaceuticals produced in cells of animal origin is governed by regulatory guidelines. It is ensured through raw material controls, cell substrate testing, and evaluation of the purification process for virus clearance capability. An additional control for cell lines that contain endogenous viruses is the virus safety factor (VSF) calculation, to demonstrate that the virus clearance exceeds the amount of potential endogenous virus in a dose of product. Product-specific input data (product titer, process yield, intended dose, purification process virus clearance capability, and the measured titer of endogenous virus produced by the cells) are typically used for the calculation. A wide range of relevant data was obtained from the production of monoclonal antibodies in Chinese Hamster Ovary (CHO) cells, and a sensitivity analysis was performed by using Monte Carlo simulations to determine which input data had the most significant impact on the range and distribution of the VSF. The sensitivity analysis suggested that the VSF calculation can be streamlined to include virus clearance capability, the endogenous virus titer, and dose while excluding product titer and process yield. Furthermore, the simulated VSF exceeded 4 log10 in 96% of the simulations, providing a high level of assurance of virus safety for antibodies produced in CHO cells and purified within specified operational parameters.

Roles of Calcium Signaling in Gene Expression and Photosynthetic Acclimatization of Solanum lycopersicum Micro-Tom (MT) after Mechanical Damage.

A momentary increase in cytoplasmic Ca2+ generates an oscillation responsible for the activation of proteins, such as calmodulin and kinases, which interact with reactive oxygen species (ROS) for the transmission of a stress signal. This study investigated the influence of variations in calcium concentrations on plant defense signaling and photosynthetic acclimatization after mechanical damage. Solanum lycopersicum Micro-Tom was grown with 0, 2 and 4 mM Ca2+, with and without mechanical damage. The expression of stress genes was evaluated, along with levels of antioxidant enzymes, hydrogen peroxide, lipid peroxidation, histochemistry, photosynthesis and dry mass of organs. The ROS production generated by mechanical damage was further enhanced by calcium-free conditions due to the inactivation of the oxygen evolution complex, contributing to an increase in reactive species. The results indicated that ROS affected mechanical damage signaling because calcium-free plants exhibited high levels of H2O2 and enhanced expression of kinase and RBOH1 genes, necessary conditions for an efficient response to stress. We conclude that the plants without calcium supply recognized mechanical damage but did not survive. The highest expression of the RBOH1 gene and the accumulation of H2O2 in these plants signaled cell death. Plants grown in the presence of calcium showed higher expression of SlCaM2 and control of H2O2 concentration, thus overcoming the stress caused by mechanical damage, with photosynthetic acclimatization and without damage to dry mass production.

Factors affecting robustness of anion exchange chromatography: Selective retention of minute virus of mice using membrane media.

Mobile and stationary phase factors were investigated in order to identify conditions for effective capture of minute virus of mice (MVM), a potential adventitious contaminant in biomanufacturing, using anion exchange membrane chromatography (AEX). The initial study was conducted for Membrane A for a range of feed conditions using bovine serum albumin (BSA) as a model protein mimicking acidic host-cell proteins (HCPs) competitive for virus binding. The effects of pH (6-8), salt concentration (0-150 mM NaCl) and level of BSA (0-10 g/L) were systematically investigated. It was found that higher BSA concentration has the most negative impact on MVM binding followed by the increased conductivity of the feed solution. The effect of pH on MVM binding is also detected but has a less impact compared to other two factors in the range of feed conditions investigated. In addition to Membrane A, three other AEX membranes (Membrane B, C and D) were investigated for MVM binding at a selected feed condition. Based on properties of the membranes investigated, it was found that ligand charge density has the most significant impact on MVM binding performance of AEX membranes from stationary phase perspective.

Antifungal activity of liriodenine on clinical strains of Cryptococcus neoformans and Cryptococcus gattii species complexes.

Background: Cryptoccocal meningitis continues to present high incidence among AIDS patients. The treatment of choice is the synergistic combination of flucytosine (5-FC) with amphotericin B deoxycholate (AmBd) or its lipid formulations. However, 5-FC is unavailable in many countries and AmB demands hospitalization. The combination of AmB with the fungistatic fluconazole (FLC) or the use of high FLC daily doses alone became the choice. Nonetheless, sterilization of cerebrospinal fluid is delayed with FLC monotherapy, mainly with high fungal burden. These findings suggest the search for new antifungal compounds, such as liriodenine. Methods: Liriodenine antifungal activity was evaluated by three procedures: determining the minimum inhibitory concentration (MIC) on 30 strains of the Cryptococcus neoformans (C. neoformans) complex and 30 of the Cryptococcus gattii (C. gattii) complex, using EUCAST methodology and amphotericin B deoxycholate as control; performing the time-kill methodology in two strains of the C. neoformans complex and one of the C. gattii complex; and injury to cryptococcal cells, evaluated by transmission electron microscopy (TEM). Liriodenine absorption and safety at 0.75 and 1.50 mg.kg-1 doses were evaluated in BALB/c mice. Results: Liriodenine MICs ranged from 3.9 to 62.5 µg.mL-1 for both species complexes, with no differences between them. Time-kill methodology confirmed its concentration-dependent fungicidal effect, killing all the strains below the limit of detection (33 CFU.mL-1) at the highest liriodenine concentration (32-fold MIC), with predominant activity during the first 48 hours. Liriodenine induced severe Cryptococcus alterations - cytoplasm with intense rarefaction and/or degradation, injury of organelles, and presence of vacuoles. Liriodenine was better absorbed at lower doses, with no histopathological alterations on the digestive tract. Conclusion: The fungicidal activity confirmed by time-kill methodology, the intense Cryptococcus injury observed by TEM, the absorption after gavage administration, and the safety at the tested doses indicate that the liriodenine molecule is a promising drug lead for development of anticryptococcal agents.

Understanding the effects of system differences for parameter estimation and scale-up of high throughput chromatographic data.

In this paper, we evaluate how employing fraction collection and multistep gradients with RoboColumns® (Repligen, formally Atoll) affects both comparison to benchtop experimental data and column simulation parameter estimation. These operational differences arise from the RoboColumn® system (operated on an automated liquid handling device) requiring offline analysis for determination of elution profiles rather than the continuous in-line UV curves obtained with larger scale systems. In addition, multistep gradients are used to model the smooth linear gradients of larger scale systems because sequential injections are used to provide liquid flow. Comparisons of two sets of column simulations was first carried out to demonstrate that fraction collection reduced the first moments of the elution peaks by 1/2 of the fraction volumes. Additional column simulations determined that the effect of a multistep gradient approximation on retention volume was dependent upon the gradient step length. An empirical transformation was then developed to correct the first moments obtained from gradient experimental data using the RoboColumn® system. These corrected values provided a more direct comparison of the experimental data at different scales and resulted in a significant improvement in agreement with results obtained using a 20 mL benchtop column. Linear steric mass-action (SMA) parameters were then estimated using the corrected values and employed to successfully predict the performance of the benchtop system data. Finally, these parameters were demonstrated to be well suited for modeling the RoboColumn® gradient data when properly accounting for multistep gradients and fraction collection. This work continues previous investigations into understanding system differences associated with robotic liquid handling devices and proposes a methodology for properly accounting for operational differences to predict operation at larger scales using conventional chromatography systems.

Chronic Lymphocytic Leukaemia: Rare Involvement of the Central Nervous System.

Involvement of the central nervous system, although uncommon, is one of the most frequent extramedullary manifestations of chronic lymphocytic leukaemia (CLL). Various conditions can lead to neurological symptoms in CLL patients and distinguishing between clinically significant CLL involvement of the CNS and other aetiologies can be challenging. The authors report the case of a 90-year-old woman with a previous diagnosis of low-risk CLL who presented to the emergency room with altered mental status. After the most frequent causes were ruled out and considering the underlying disease, CNS infiltration by clonal B-cells was hypothesised and later confirmed. Treatment was initiated, but the patient died soon afterwards. LEARNING POINTS: Central nervous system involvement in chronic lymphocytic leukaemia (CLL) is rare and characterized by a heterogeneous clinical presentation.CNS localisation of CLL should be considered in patients with any neurological symptom, irrespective of the stage and activity of CLL.Brain imaging and cerebrospinal fluid examination should be performed.

Subserous Type of Eosinophilic Colitis: A Rare Disease.

Eosinophilic colitis is a rare heterogeneous inflammatory disorder. The pathogenesis is not well understood even though it seems to be multifactorial, with hypersensitivity as a major contributor. The clinical presentation depends on the eosinophilic infiltration of different sections within the gastrointestinal tract. Diagnosis is based on the presence of peripheral eosinophilia and histopathological evidence of colon wall eosinophilic infiltration. The authors present the case of a woman with a predominantly subserous pattern of eosinophilic colitis potentially triggered by intake of an Ulmus rubra-rich product. LEARNING POINTS: Subserous eosinophilic colitis (EC) is a rare inflammatory gastrointestinal disease that should be suspected when peripheral eosinophilia is accompanied by eosinophil-rich ascites and confirmed by the presence of eosinophilic infiltration of the colon wall.Although the aetiology of EC is not fully understood, a combination of genetic predisposition, dysbiosis and environmental factors (e.g., ingested allergens) likely contribute to it.The subserous type has the best prognosis and responds well to corticosteroids, with fewer relapses than other types.

Chemophenetics as a Tool for Distinguishing Morphotypes of Annona emarginata (Schltdl.) H. Rainer.

Annona emarginata (Annonaceae) has two morphological variations (A. emarginata 'mirim' and A. emarginata 'terra-fria'). The species has agricultural value and produces specialized metabolites of pharmacological interest. The objective of this work was to analyze whether chemical and morphological differences contribute to differentiate A. emarginata 'terra-fria' from A. emarginata 'mirim', as chemophenetic variations of A. emarginata. The analysis of chemical compounds was based on the quantification, profile of root alkaloids and on the leaves volatile profile, together with morphometric analyses of the leaf blade. The samples were collected in three phenological stages (flowering, fruiting, and vegetative) at two places in São Paulo, Brazil. Differences in the composition of the alkaloid profile and leaf volatiles (in both places and in the three phenological stages) allowed us to separate the two morphotypes by multivariate statistical analysis. These differences agreed with the leaf blade morphology and flower color. This first chemophenetic report of A. emarginata demonstrates that, in addition to morphological variations, the specialized metabolism of roots and leaves can be phytochemical characters, which suggest the existence of at least morphochemotypes of A. emarginata.

Severe Aplastic Anemia as First Manifestation of Classical Hodgkin Lymphoma.

Autoimmune cytopenia, a known paraneoplastic complication of lymphoid neoplasms, may occur before, concurrently, at relapse, or even years after completion of lymphoma treatment. In the case of Hodgkin lymphoma (HL), it is thought that immune dysregulation, typical of this neoplasm, may be involved in the genesis of these manifestations. We report a 57-year-old male presenting with stage IIIA, International Prognostic Score (IPS) 4, nodular sclerosis HL, and severe AA (SAA) confirmed on the histologic exam of the bone marrow that showed severe marrow hypoplasia due to a decrease in the elements of the three cell linages with left shift of the myeloid maturation. Immunosuppression with steroids and cyclosporine A was started. Eltrombopag and G-CSF were also added. In spite of prompt initiation of immunosuppressive therapy, the patient presented an unfavorable outcome with progressive pancytopenia and severe acute cerebral hemorrhagic event. The patient died 59 days after admission. Although autoimmune disorders are described in HL, its concomitant diagnosis is extremely rare. Our case shows a rare instance of SAA as the first manifestation of HL.

Antifungal activity of liriodenine on agents of systemic mycoses, with emphasis on the genus Paracoccidioides.

BACKGROUND: Endemic systemic mycoses remain a health challenge, since these opportunistic diseases are increasingly infecting immunosuppressed patients. The simultaneous use of antifungal compounds and other drugs to treat infectious or non-infectious diseases has led to several interactions and undesirable effects. Thus, new antifungal compounds should be investigated. The present study aimed to evaluate the activity of liriodenine extracted from Annona macroprophyllata on agents of systemic mycoses, with emphasis on the genus Paracoccidioides. METHODS: The minimum inhibitory concentration (MIC) and minimum fungicide concentration (MFC) were determined by the microdilution method. The cellular alterations caused by liriodenine on a standard P. brasiliensis (Pb18) strain were evaluated by transmission and scanning electron microscopy. RESULTS: Liriodenine was effective only in 3 of the 8 strains of the genus Paracoccidioides and in the Histoplasma capsulatum strain, in a very low concentration (MIC of 1.95 µg.mL-1); on yeasts of Candida spp. (MIC of 125 to 250 µg.mL-1), including C. krusei (250 µg.mL-1), which has intrinsic resistance to fluconazole; and in Cryptococcus neoformans and Cryptococcus gattii (MIC of 62.5 µg.mL-1). However, liriodenine was not effective against Aspergillus fumigatus at the studied concentrations. Liriodenine exhibited fungicidal activity against all standard strains and clinical isolates that showed to be susceptible by in vitro tests. Electron microscopy revealed cytoplasmic alterations and damage to the cell wall of P. brasiliensis (Pb18). CONCLUSION: Our results indicate that liriodenine is a promising fungicidal compound that should undergo further investigation with some chemical modifications.

Pressure-Flow experiments, packing, and modeling for scale-up of a mixed mode chromatography column for biopharmaceutical manufacturing.

To obtain consistent chromatographic behavior, it is important to develop resin packing methods in accordance with the characteristics of each resin. Resins, particularly those with a significant level of compressibility, require proper knowledge of the packing methodology to ensure scalable performance. The study demonstrates the applicability of pressure-flow modeling based on the Blake-Kozeny equation for cellulose based resins, using the MEP HyperCel (Pall) resin as a case study. This approach enabled the understanding of the appropriate bed compressibility and the determination of the minimum column diameter that can predict bed integrity during commercial manufacturing scale operation. Studies suggested that scale-dependent wall effects become negligible for column diameters exceeding 20 cm. Pressure-flow modeling produced a minimum compression recommendation of 0.206 for the MEP HyperCel resin. Columns with diameters up to 80 cm packed with this bed compression yielded incompressible beds with pressure-flow curves consistent with model predictions. Model parameter (particle diameter, viscosity, porosity) values were then varied to demonstrate how changing operating conditions influence model predictions. This analysis supported the successful troubleshooting of unexpected high pressures at the commercial manufacturing scale using MEP HyperCel resin, further supporting the applicability of this approach.

Complexities of Diagnosing and Treating a Mesenteric Cyst in a Chronic Myeloid Leukaemia Patient.

Mesenteric cysts are a rare nosologic entity, the diagnosis of which is complex due to their nonspecific presentation. They may emerge from any part of the mesentery and grow to any size, thus conditioning a wide range of clinical manifestations that renders them easily mistaken for different gastrointestinal pathologies. Diagnosis encompasses a mixture of clinical suspicion, imaging techniques and sometimes surgery, and curative treatment is based on complete surgical resection of the cyst. We hereby present a case of a mesenteric cyst that developed on the anterior abdominal wall of a 59-year-old man awaiting allogeneic bone marrow transplantation after being diagnosed with chronic myeloid leukaemia. He was admitted to the emergency room with complaints of an increased abdominal perimeter and increased weight, not associated with alterations to his dietary or physical exercise habits. Suspecting ascites in the context of leukaemic progression, the patient was admitted to the medical ward; however, subsequent study identified a mesenteric cyst as the most probable diagnosis and the patient was proposed to undergo surgery. He underwent laparotomic cyst excision without complications and the histological evaluation of the surgical specimen confirmed the diagnosis. LEARNING POINTS: Although rare entities, mesenteric cysts should be suspected whenever nonspecific gastrointestinal symptoms are present and liquid collections are identified.Treatment consists of complete surgical excision, which, although not free of complications, is usually safe and the recurrence rate is very low.In light of the lymphatic theory with respect to the aetiology of mesenteric cysts, this nosologic entity should be highly suspected in oncologic patients where lymphangiogenesis is potentiated.

Highland games: A benchmarking exercise in predicting biophysical and drug properties of monoclonal antibodies from amino acid sequences.

Biopharmaceutical product and process development do not yet take advantage of predictive computational modeling to nearly the degree seen in industries based on smaller molecules. To assess and advance progress in this area, spirited coopetition (mutually beneficial collaboration between competitors) was successfully used to motivate industrial scientists to develop, share, and compare data and methods which would normally have remained confidential. The first "Highland Games" competition was held in conjunction with the October 2018 Recovery of Biological Products Conference in Ashville, NC, with the goal of benchmarking and assessment of the ability to predict development-related properties of six antibodies from their amino acid sequences alone. Predictions included purification-influencing properties such as isoelectric point and protein A elution pH, and biophysical properties such as stability and viscosity at very high concentrations. Essential contributions were made by a large variety of individuals, including companies which consented to provide antibody amino acid sequences and test materials, volunteers who undertook the preparation and experimental characterization of these materials, and prediction teams who attempted to predict antibody properties from sequence alone. Best practices were identified and shared, and areas in which the community excels at making predictions were identified, as well as areas presenting opportunities for considerable improvement. Predictions of isoelectric point and protein A elution pH were especially good with all-prediction average errors of 0.2 and 1.6 pH unit, respectively, while predictions of some other properties were notably less good. This manuscript presents the events, methods, and results of the competition, and can serve as a tutorial and as a reference for in-house benchmarking by others. Organizations vary in their policies concerning disclosure of methods, but most managements were very cooperative with the Highland Games exercise, and considerable insight into common and best practices is available from the contributed methods. The accumulated data set will serve as a benchmarking tool for further development of in silico prediction tools.

Encephalopathy after Bendamustine Treatment: A Rare Side Effect?

Bendamustine is a chemotherapeutic drug associated with frequent haematological and gastrointestinal adverse effects and, more rarely, neurological toxicity. We present the case of a 79-year-old man with follicular lymphoma, grade 2, Ann-Arbor stage IV-A, FLIPI 4, high risk, with bulky disease and vital organ compression, treated with R-CHOP and then rituximab-bendamustine, who developed encephalopathy approximately 2 months after the last cycle. After ruling out other possible aetiologies, we assumed the patient's encephalopathy was due to a delayed bendamustine side effect, possibly aggravated by the concomitant administration of rituximab. The authors hope to alert clinicians to this rare adverse effect, its difficult identification and the need to rule out other aetiologies. LEARNING POINTS: Bendamustine neurotoxicity is a rare side effect.Bendamustine neurotoxicity should be considered a diagnosis of exclusion in a patient with progressive encephalopathy treated with the drug.Drug cessation is the mainstay of treatment in cases of bendamustine-induced encephalopathy.