RESUMO
Host cell proteins (HCPs) are process-related impurities expressed by the host cells during biotherapeutics' manufacturing, such as monoclonal antibodies (mAbs). Some challenging HCPs evade clearance during the downstream processing and can be co-purified with the molecule of interest, which may impact product stability, efficacy, and safety. Therefore, HCP content is a critical quality attribute to monitor and quantify across the bioprocess. Here we explored a mass spectrometry (MS)-based proteomics tool, the sequential window acquisition of all theoretical fragment-ion spectra (SWATH) strategy, as an orthogonal method to traditional ELISA. The SWATH workflow was applied for high-throughput individual HCP identification and quantification, supporting characterization of a mAb purification platform. The design space of HCP clearance of two polishing resins was evaluated through a design of experiment study. Absolute quantification of high-risk HCPs was achieved (reaching 1.8 and 4.2â¯ppm limits of quantification, for HCP A and B respectively) using HCP-specific synthetic heavy labeled peptide calibration curves. Profiling of other HCPs was also possible using an average calibration curve (using labeled peptides from different HCPs). The SWATH approach is a powerful tool for HCP assessment during bioprocess development enabling simultaneous monitoring and quantification of different individual HCPs and improving process understanding of their clearance.
Assuntos
Anticorpos Monoclonais , Peptídeos , Cricetinae , Animais , Cricetulus , Anticorpos Monoclonais/química , Espectrometria de Massas/métodos , Ensaio de Imunoadsorção Enzimática , Células CHORESUMO
The majority of recombinant adeno-associated viruses (rAAV) approved for clinical use or in clinical trials areproduced by transient transfection using the HEK293 cell line. However, this platform has several manufacturing bottlenecks at commercial scales namely, low product quality (full to empty capsid ratio <20% in most rAAV serotypes), lower productivities obtained after scale-up and the high cost of raw materials, in particular of Good Manufacturing Practice grade plasmid DNA required for transfection. The HeLa-based stable cell line rAAV production system provides a robust and scalable alternative to transient transfection systems. Nevertheless, the time required to generate the producer cell lines combined with the complexity of rAAV production and purification processes still pose several barriers to the use of this platform as a suitable alternative to the HEK293 transient transfection. In this work we streamlined the cell line development and bioprocessing for the HeLaS3-based production of rAAV. By exploring this optimized approach, producer cell lines were generated in 3-4 months, and presented rAAV2 volumetric production (bulk) > 3 × 1011 vg/mL and full to empty capsids ratio (>70%) at 2 L bioreactor scale. Moreover, the established downstream process, based on ion exchange and affinity-based chromatography, efficiently eliminated process related impurities, including the Adenovirus 5 helper virus required for production with a log reduction value of 9. Overall, we developed a time-efficient and robust rAAV bioprocess using a stable producer cell line achieving purified rAAV2 yields > 1 × 1011 vg/mL. This optimized platform may address manufacturing challenges for rAAV based medicines.
Assuntos
Dependovirus , Vetores Genéticos , Humanos , Dependovirus/genética , Células HEK293 , Células HeLa , TransfecçãoRESUMO
Erenumab is a monoclonal antibody (mAb) approved for the preventive treatment of migraine. While preclinical studies on calcitonin gene-related peptide mAbs did not identify any reproductive toxicity, pregnant and breastfeeding women were excluded from the pivotal human studies, and therefore the safety of calcitonin gene-related peptide medications in this population must be studied. So far, postmarketing data of accidental exposures have not brought to light any specific toxicities. Three women treated with erenumab in our series conceived while exposed to the drug. All had previous successful pregnancies, were on erenumab for more than 6 months, and had ≥80% reduction in headache frequency. The one who stopped erenumab only 1 month before conceiving had a spontaneous abortion during the first trimester due to a gestational trophoblastic neoplasia and has since conceived with an uneventful gestation. The other two women stopped treatment during the first trimester, and both pregnancies went to term with no complications. All babies have shown normal development. No plausible explanation relates the mechanism of action of erenumab and the serious complication that occurred in one patient. Continuous follow-up and reporting of all exposures are encouraged to gather safety data on pregnant and nursing women and on the development of the newborns. So far, immediately stopping the drug is advised and may contribute to decreasing the potential risks.
Assuntos
Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina , Peptídeo Relacionado com Gene de Calcitonina , Feminino , Humanos , Recém-Nascido , Gravidez , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados/efeitos adversos , Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina/uso terapêutico , Resultado do TratamentoRESUMO
Recombinant adeno-associated (rAAV) vector-based gene therapy has been the focus of intense research driven by the safety profile and several recent clinical breakthroughs. As of April 2021, there are two rAAV-based gene therapies approved and more than two-hundred active clinical trials (approximately thirty in Phase III). However, the expected increase in demand for rAAV vectors still poses several challenges. Discussed herein are key aspects related to R&D needs and Chemistry, Manufacturing and Control (CMC) efforts required to attend this growing demand. Authors provide their perspective on strategic topics for rAAV-based therapies success: scalability and productivity; improved safety; increased process understanding combined with development of orthogonal bioanalytics that are able to identify, monitor and control Critical Quality Attributes (CQAs) during bioprocessing.
Assuntos
Dependovirus , Vetores Genéticos , Dependovirus/genética , Terapia Genética , Vetores Genéticos/genéticaRESUMO
This article focuses on pleomorphic sarcomas, which are malignant mesenchymal tumors with complex genetic background at the root of their morphologic pleomorphism. They are poorly differentiated tumors that may retain different lines of differentiation, sometimes correlating with clinicopathological or prognostic features. Accurate diagnosis in this group of tumors relies on adequate sampling due to their heterogeneity and assessment with both microscopy and large panels of immunohistochemistry. Molecular analyses have a limited role in their diagnosis as opposed to translocation-related sarcomas but may provide theranostic and important prognostic information in the future.
Assuntos
Sarcoma/patologia , Neoplasias de Tecidos Moles/patologia , Fatores Etários , Biomarcadores Tumorais/genética , Diagnóstico Diferencial , Humanos , Imuno-Histoquímica , Prognóstico , Sarcoma/classificação , Sarcoma/genética , Neoplasias de Tecidos Moles/classificação , Neoplasias de Tecidos Moles/genéticaAssuntos
Contusões/diagnóstico por imagem , Dermatofibrossarcoma/patologia , Dermatofibrossarcoma/cirurgia , Adulto , Assistência ao Convalescente , Antígenos CD34/metabolismo , Contusões/etiologia , Contusões/patologia , Dermoscopia/métodos , Detecção Precoce de Câncer , Feminino , Humanos , Proteínas S100/metabolismo , Resultado do TratamentoRESUMO
We read with interest the article by Sevilla Ribota et al1 that described an unexpected finding of a granular cell tumour (GCT) of the rectum, which was removed by band ligation-assisted mucosectomy. We present a similar case of a GCT of the cecum, which was resected using a different endoscopic procedure.
Assuntos
Neoplasias do Ceco/patologia , Pólipos do Colo/patologia , Tumor de Células Granulares/patologia , Adulto , Neoplasias do Ceco/diagnóstico por imagem , Pólipos do Colo/diagnóstico por imagem , Colonoscopia , Feminino , Tumor de Células Granulares/diagnóstico por imagem , HumanosRESUMO
Pseudomyogenic hemangioendothelioma (PHE) is a rare indolent vascular tumor that typically has a multifocal presentation and involves multiple tissue planes. This report describes a 34-year-old man with multiple infiltrated brown papules and plaques on his left leg that had evolved for 6 months. The skin biopsy revealed a dermal and subcutaneous neoplasm composed of fascicles of spindle cells with atypia and epithelioid cells with prominent nucleoli and abundant eosinophilic cytoplasm. There was no evidence of necrosis, and the mitotic rate was low. There was strong reactivity with cytokeratin AE1/AE3, ERG, and FLI1, multifocal reactivity with smooth muscle actin, and focal reactivity with CD31. There was no expression of keratin MNF116, CAM5.2, CD34, CAMTA1, S100-protein, epithelial membrane antigen, melan-A, HMB-45, factor XIIIa, HHV8, or CD10. The nuclei of neoplastic cells showed intact expression of INI1. The clinical, histological, and immunophenotypical aspects were consistent with a diagnosis of PHE. A lower limb CT scan showed lesions in the skin, muscle, and bone planes. The patient was sent to an oncology center, where he maintains regular clinical and imagiological follow-up.
Assuntos
Hemangioendotelioma/diagnóstico , Neoplasias Cutâneas/diagnóstico , Neoplasias de Tecidos Moles/diagnóstico , Adulto , Humanos , Perna (Membro) , MasculinoRESUMO
A 10-year-old male neutered Persian cat was presented with an abdominal mass and history of weakness. Blood smear examination found marked elliptocytosis, and serum biochemical analysis revealed hypokalemia, hypochloremia, increased creatine kinase activity, and a high aldosterone concentration. Cytologic examination of the mass revealed neoplastic endocrine cells with moderate criteria of malignancy, favoring adrenocortical neoplasia. The adrenal mass was surgically excised and histologically characterized by lobules of mildly pleomorphic, polygonal neoplastic cells with moderate to abundant, occasionally granular, eosinophilic cytoplasm. Lobules were separated by fine fibrovascular trabeculae, and numerous cystic cavities containing amorphous eosinophilic material that stained positive with Alcian blue and periodic acid-Schiff were seen. Neoplastic cells were multifocally positive for cytochrome P450 aldosterone synthase. Based on clinicopathologic and immunohistochemical findings the present case was diagnosed as an aldosterone-producing adrenocortical carcinoma with myxoid differentiation. While this entity has not been reported in cats, myxoid differentiation of adrenocortical carcinomas has been found in other species and can pose a major diagnostic challenge on microscopic examination.
Assuntos
Neoplasias do Córtex Suprarrenal/veterinária , Carcinoma Adrenocortical/veterinária , Aldosterona/metabolismo , Doenças do Gato/patologia , Córtex Suprarrenal/metabolismo , Córtex Suprarrenal/patologia , Neoplasias do Córtex Suprarrenal/diagnóstico , Neoplasias do Córtex Suprarrenal/patologia , Carcinoma Adrenocortical/diagnóstico , Carcinoma Adrenocortical/patologia , Animais , Biópsia/veterinária , Doenças do Gato/diagnóstico , Gatos , MasculinoRESUMO
Multiple cutaneous and uterine leiomyomatosis (MCUL), also known as Reed's syndrome, is a rare genodermatosis, with an autosomal dominant pattern of inheritance. It results from a germline heterozygous mutation of fumarate hydratase gene, that is classified as a tumor suppressor gene. Hereditary leiomyomatosis and renal cell cancer is characterized by the association of MCUL with renal cell carcinoma. We report a case of a 57-year-old woman, with multiple cutaneous leiomyomas as the presenting sign of Reed's syndrome.
Assuntos
Neoplasias Meníngeas/cirurgia , Meningioma/cirurgia , Neoplasias dos Seios Paranasais/cirurgia , Idoso de 80 Anos ou mais , Seio Etmoidal , Seio Frontal , Humanos , Imageamento por Ressonância Magnética , Masculino , Neoplasias Meníngeas/diagnóstico , Meningioma/diagnóstico , Invasividade Neoplásica , Procedimentos Neurocirúrgicos/métodos , Neoplasias dos Seios Paranasais/diagnóstico , Seios Paranasais/cirurgiaRESUMO
Mucins are high-molecular weight glycoproteins (0.25-20 MDa) containing one or more domains that are heavily O-glycosylated. Their implications as targets for cancer treatment have increased the interest in these glycoproteins, mainly in the fields of vaccines and antibodies. However, mucins present high heterogeneity, posing challenges that affect purification processes and quality control analysis. In that sense, it is necessary to develop and improve downstream processes and analytical methods to characterize these products. Here a tool based on biolayer interferometry analysis to improve mucin's detection and quantification in a fast, simple and label free-way is presented. Taking advantage of lectin recognition of mucins' carbohydrate structures, several lectins were evaluated and immobilized on streptavidin biosensors. Different assay conditions were optimized and the most suitable lectin, Aleuria aurantia lectin (AAL), was selected. Bovine Submaxillary Gland and human MUC5B mucins were used as proof of concept and were successfully detected and quantified at different stages of purification. High sensitivity levels were achieved with LOD and LOQ of 3.8 µg mL-1 and 11.7 µg mL-1 for BSM, and 0.2 µg mL-1 and 0.6 µg mL-1 for MUC5B. AAL binding specificity was also confirmed with fucose competition assays. Our method represents an advance on mucins detection and quantification since the existing methods present several disadvantages for process development. Hereafter, it can be applied to the optimization of new or already established downstream processes for mucins' purification.
Assuntos
Modelos Moleculares , Mucinas/metabolismo , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , GlicosilaçãoRESUMO
14-3-3σ is a protein expressed in many epithelial tissues associated with essential cell functions, including cell-cycle control, apoptosis, and cytoskeletal integrity. There is a paucity of knowledge of the tumorigenesis of canine renal cell carcinomas (RCCs), and the histological origin of this tumor has not been established. This study analyzed the expression of 14-3-3σ, Ki-67, cytokeratins, and vimentin in 40 canine RCCs. Aberrant expression of 14-3-3σ was demonstrated in 15 (38%) cases and was associated with a significantly shorter survival time ( P < .002). In contrast to canine RCC, normal kidney did not express 14-3-3σ. The Ki-67 proliferation index did not show utility as a prognostic factor. The distal convoluted tubular epithelium in normal kidneys coexpressed cytokeratins and vimentin, and thus maintenance of this coexpression pattern in canine RCC suggests that most tumors arise from the distal segment of the nephron. These results suggest that 14-3-3σ is a potential negative prognostic factor and a possible therapeutic target.
Assuntos
Proteínas 14-3-3/metabolismo , Carcinoma de Células Renais/veterinária , Doenças do Cão/metabolismo , Neoplasias Renais/veterinária , Animais , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Doenças do Cão/patologia , Cães , Feminino , Queratinas/metabolismo , Antígeno Ki-67/metabolismo , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Masculino , Estudos Retrospectivos , Vimentina/metabolismoRESUMO
Wells syndrome (WS) or eosinophilic cellulitis is a rare, idiopathic, inflammatory dermatosis. The typical clinical presentation is urticarial plaque without preferential location that usually heals without scarring. We present a 62-year-old man with history of lung cancer that had undergone a right superior lobectomy 12 months previously. The patient had a relapsing dermatosis beginning about 6 months before the diagnosis of the lung cancer, characterised by pruritic, erythematous plaques located on the trunk and arms. These lesions spontaneously resolved within a few weeks without scarring. A skin biopsy revealed findings compatible with WS. Several diseases have been associated with WS. These include haematological diseases, fungal, parasitic and viral infections, drug reactions and rarely non-haematological malignancies. We present a case of this rare syndrome in a patient with history of lung cancer that we believe acted as a triggering event. To our knowledge, this is the second case reporting this association.
Assuntos
Celulite (Flegmão)/patologia , Clobetasol/uso terapêutico , Fármacos Dermatológicos/uso terapêutico , Eosinofilia/patologia , Loratadina/uso terapêutico , Neoplasias Pulmonares/patologia , Dermatopatias/patologia , Tacrolimo/uso terapêutico , Administração Tópica , Biópsia , Celulite (Flegmão)/tratamento farmacológico , Celulite (Flegmão)/etiologia , Eosinofilia/tratamento farmacológico , Eosinofilia/etiologia , Humanos , Neoplasias Pulmonares/complicações , Neoplasias Pulmonares/cirurgia , Masculino , Pessoa de Meia-Idade , Recidiva , Resultado do TratamentoRESUMO
BACKGROUND: Although the association between intraductal papillary mucinous neoplasm of the pancreas (IPMN) and pancreatic neuroendocrine tumor (PNET) has been increasingly reported, whether this association is real or coincidence remains unclear. We report a case of synchronous IPMN and a PNET which were diagnosed preoperatively and discuss the tumorigenesis, clinicopathological features and management of these rare tumors based on the published literature. CASE REPORT: A 56-year-old male was incidentally diagnosed with a 14 mm branch duct IPMN and a 3.6 mm non-functional PNET during an evaluation due to persistent upper abdominal pain via endoscopic ultrasound. Close follow-up of the patient was decided as the IPMN had no worrisome features. A review of twenty-two previously reported cases of synchronous IPMN and PNET indicated that: a) only seven cases were diagnosed preoperatively; b) abdominal pain was the main presenting symptom; c) IPMN was the dominant tumor and presented with low grade dysplasia; d) the PNET was small and non-functional and had an indolent behavior; and e) only one case underwent radiologic follow-up. DISCUSSION: IPMN are associated with other pancreatic and extrapancreatic malignancies. Thus, the entire pancreatic parenchyma should be examined closely during the evaluation of an IPMN in order to exclude other pancreatic lesions, for example, a PNET.
Assuntos
Adenocarcinoma Mucinoso/complicações , Neoplasias Císticas, Mucinosas e Serosas/complicações , Tumores Neuroendócrinos/complicações , Neoplasias Pancreáticas/complicações , Adenocarcinoma Mucinoso/diagnóstico por imagem , Carcinoma Ductal Pancreático/cirurgia , Endossonografia , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Císticas, Mucinosas e Serosas/diagnóstico por imagem , Tumores Neuroendócrinos/diagnóstico por imagem , Neoplasias Pancreáticas/diagnóstico por imagem , Tomografia Computadorizada por Raios XAssuntos
Humanos , Feminino , Adulto , Complicações Pós-Operatórias/diagnóstico por imagem , Obstrução do Fluxo Ventricular Externo/diagnóstico por imagem , Falso Aneurisma/diagnóstico por imagem , Implante de Prótese de Valva Cardíaca/efeitos adversos , Aneurisma Cardíaco/diagnóstico por imagem , Complicações Pós-Operatórias/cirurgia , Valva Pulmonar/transplante , Obstrução do Fluxo Ventricular Externo/cirurgia , Obstrução do Fluxo Ventricular Externo/etiologia , Falso Aneurisma/cirurgia , Aneurisma Cardíaco/cirurgiaRESUMO
To deliver the required cell numbers and doses to therapy, scaling-up production and purification processes (at least to the liter-scale) while maintaining cells' characteristics is compulsory. Therefore, the aim of this work was to prove scalability of an integrated streamlined bioprocess compatible with current good manufacturing practices (cGMP) comprised by cell expansion, harvesting and volume reduction unit operations using human mesenchymal stem cells (hMSC) isolated from bone marrow (BM-MSC) and adipose tissue (AT-MSC). BM-MSC and AT-MSC expansion and harvesting steps were scaled-up from spinner flasks to 2L scale stirred tank single-use bioreactor using synthetic microcarriers and xeno-free medium, ensuring high cellular volumetric productivities (50×106cellL-1day-1), expansion factors (14-16 fold) and cell recovery yields (80%). For the concentration step, flat sheet cassettes (FSC) and hollow fiber cartridges (HF) were compared showing a fairly linear scale-up, with a need to slightly decrease the permeate flux (30-50 LMH, respectively) to maximize cell recovery yield. Nonetheless, FSC allowed to recover 18% more cells after a volume reduction factor of 50. Overall, at the end of the entire bioprocess more than 65% of viable (>95%) hMSC could be recovered without compromising cell's critical quality attributes (CQA) of viability, identity and differentiation potential. Alongside the standard quality assays, a proteomics workflow based on mass spectrometry tools was established to characterize the impact of processing on hMSC's CQA; These analytical tools constitute a powerful tool to be used in process design and development.
Assuntos
Reatores Biológicos , Técnicas de Cultura de Células/métodos , Células-Tronco Mesenquimais/citologia , Proteoma , Regulação para Baixo , Humanos , Espectrometria de Massas , Células-Tronco Mesenquimais/metabolismo , Proteoma/análise , Proteoma/metabolismo , Proteômica , Regulação para CimaRESUMO
Narrow-bandwidth light treatments may be used to manipulate plant growth, development and metabolism. In this report LED-based light treatments were used to affect yield and metabolic content of sweet basil (Ocimum basilicum L. cv "Ceasar") grown in controlled environments. This culinary herb produces an aroma highly appreciated by consumers, primarily composed of terpenes/terpenoids, phenylpropanoids, and fatty-acid- derived volatile molecules. Basil plants were grown under narrow-bandwidth light conditions, and leaf area, height, mass, antioxidant capacity and volatile emissions were measured at various time points. The results indicate reproducible significant differences in specific volatiles, and in biochemical classes of volatiles, compared to greenhouse grown plants. For example, basil plants grown under blue/red/yellow or blue/red/green wavelengths emit higher levels of a subset of monoterpenoid volatiles, while a blue/red/far-red treatment leads to higher levels of most sesquiterpenoid volatile molecules. Specific light treatments increase volatile content, mass, and antioxidant capacity. The results show that narrow-bandwidth illumination can induce discrete suites of volatile classes that affect sensory quality in commercial herbs, and may be a useful tool in improving commercial production.