Production of an anti-TNFα antibody in murine myeloma cells by perfusion culture.

Infliximab is a mouse/human chimeric IgG1 monoclonal antibody which recognizes the proinflammatory cytokine, tumor necrosis factor α (TNFα), and inhibits receptor interactions, thereby decreasing inflammation and autoimmune response in patients. This monoclonal antibody has been successfully used to treat rheumatoid arthritis, ankylosing spondylitis, and psoriatic arthritis. However, the high treatment cost limits patient access to this biotherapy. One alternative to this problem is the use of biosimilars. In this work, we describe the stable expression and physicochemical characterization of an anti-TNFα antibody. While infliximab is produced in recombinant murine SP2/0 cells, our anti-TNFα IgG antibody was expressed in recombinant murine NS0 myeloma cells. The best anti-TNFα antibody-expressing clone was selected from three clone candidates based on the stability of IgG expression levels, specific productivity as well as TNFα-binding activity compared to commercial infliximab. Our results indicate that the selected cell clone, culture medium, and fermentation mode allowed for the production of an anti-TNFα antibody with similar characteristics to the reference commercially available product. An optimization of the selected culture medium by metabolomics may increase the volumetric productivity of the process to satisfy the demand for this product. Further experiments should be performed to evaluate the biological properties of this anti-TNFα antibody. KEY POINTS: • An anti-TNFα antibody was produced in NS0 cells using perfusion culture. • A proprietary chemically defined culture medium was used to replace commercially available protein-free medium. • The purified anti-TNFα antibody was comparable to the reference marketed product.

Screening and selection strategy for the establishment of biosimilar to trastuzumab-expressing CHO-K1 cell lines.

The high prices of biopharmaceuticals or biologics used in the treatment of many diseases limit the access of patients to these novel therapies. One example is the monoclonal antibody trastuzumab, successfully used for breast cancer treatment. An economic alternative is the generation of biosimilars to these expensive biopharmaceuticals. Since antibody therapies may require large doses over a long period of time, robust platforms and strategies for cell line development are essential for the generation of recombinant cell lines with higher levels of expression. Here, we obtained trastuzumab-expressing CHO-K1 cells through a screening and selection strategy that combined the use of host cells pre-adapted to protein-free media and suspension culture and lentiviral vectors. The results demonstrated that the early screening strategy obtained recombinant CHO-K1 cell populations with higher enrichment of IgG-expressing cells. Moreover, the measurement of intracellular heavy chain polypeptide by flow cytometry was a useful metric to characterize the homogeneity of cell population, and our results suggest this could be used to predict the expression levels of monoclonal antibodies in early stages of cell line development. Additionally, we propose an approach using 25 cm2 T-flasks in suspension and shaking culture conditions as a screening tool to identify high producing cell lines. Finally, trastuzumab-expressing CHO-K1 clones were generated and characterized by batch culture, and preliminary results related to HER2-recognition capacity were successful. Further optimization of elements such as gene optimization, vector selection, type of amplification/selection system, cell culture media composition, in combination with this strategy will allow obtaining high producing clones.

Taquipnea persistente en un lactante por hiperplasia de células neuroendocrinas pulmonares / Persistent tachypnea of infancy and pulmonary neuroendocrine cells hyperplasia

Introducción. Sobre la base de un caso clínico se presenta la descripción de un cuadro intersticial pulmonar por hiperplasia de células neuroendocrinas en un lactante. Método. Seguimiento clínico desde el sexto mes hasta los 17 meses con extensos estudios para descartar otras patologías semejantes. El diagnóstico definitivo fue determinado por taquipnea persistente y rales crepitantes con imágenes en vidrio esmerilado características, en lóbulo medio y língula. La biopsia pulmonar fue “normal” a la observación con microscopio óptico a pesar de las imágenes evidentes en TAC. La tinción con bombesina demostró acumulaciones anormales de células neuroendocrinas de 10,9% en bronquiolos e hiperinsuflación (Children’s Hospital of Colorado). Resultados. Se confirmó en este lactante con síntomas de cuadro intersticial la patología pulmonar sospechada: hiperplasia de células neuroendocrinas. Ésta debe ser sospechada en un lactante con: taquipnea, rales crepitantes persistentes, posible desnutrición, radiografía hiperinsuflada, tomografía que muestra imágenes en mosaico tipo vidrio esmerilado especialmente en lóbulo medio y língula. Biopsia pulmonar casi normal y aumento porcentual de células neuroendocrinas en bronquios periféricos.

Introduction. A clinical case of a 6 month old infant with symptoms of interstitial lung disease is presented. Methodology. Follow up until 17 months of age is described. Extensive studies were included to rule out other similar pathologies of infancy. Final diagnosis was determined by characteristic clinical symptomatology of persistent tachypnea and crepitant rales, hyperinflated chest x rays, CT scan presenting ground glass opacities in the middle lobe and lingula. Lung biopsy was nearly normal and bombesin staining showed increased percentage of neuroendocrine bronchial cells (10.9%) (Children’s Hospital of Colorado). Results. NEHI was confirmed in this 6 months old infant. This diagnosis should be suspected in a tachypneic infant, with persistent fine rales, possible undernutrition, chest x-rays hyperinflation and CT scan with ground glass opacities more characteristically in middle lobe and lingula. Lung biopsy is near normal but staining with bombesin shows increased number of neuroendocrine cells and neuroendocrine bodies.

Enfermedad pulmonar intersticial en la niñez. Ocho años de experiencia / Childhood interstitial lung disease. Eight years experience

Se presenta la casuística de 8 años (2005-2013) de 27 enfermos con cuadros intersticiales en seguimiento efectivo. No fueron incluidas las displasias bronco-pulmonares, el daño pulmonar crónico por virus, la fibrosis quística y la aspiración recurrente. Método. Revisión de Historias Clínicas; tomografías computadas y biopsias pulmonares en actividad multidisciplinaria coordinada. Se modificaron las técnicas quirúrgicas de biopsia, radiología (tomografía computada) y se consultó a centros extranjeros con mayor experiencia en 14 casos. Resultados. Se presentan resultados de tomografías y de la clínica. La patología diagnosticada más frecuente fue la hemorragia pulmonar con 4 hemosiderosis pulmonares idiopáticas y 2 de otro origen (sangrado por celiaquía y hemorragia pulmonar del lactante). La segunda frecuencia, 4 casos, correspondió a posibles defectos del surfactante con genética incluida. Está representado casi todo el espectro de la patología intersticial. Los cuadros intersticiales pulmonares en la infancia seguramente son más frecuentes que lo supuesto hasta la fecha. Deben ser diagnosticados y tratados por equipos multidisciplinarios con énfasis especial en la técnica de biopsia, la tomografía computada y el futuro desarrollo de estudios genéticos avanzados...

Twenty-seven cases of interstitial lung disease in children with complete follow-up are presented. Bronchopulmonary dysplasia, viral chronic lung damage, cystic fibrosis and recurrent aspiration were not included. Methods. Detailed revision of 27 clinical records during the period 2005-2013. Results. computerized tomography, clinical symptoms, lung biopsies and diagnosis are shown. Coordinated multidisciplinary organization is underlined as necessary. Foreign Centers with larger experience were consulted in 14 cases; biopsy technique as well as computed tomography were accordingly modified. Most frequent finding was pulmonary hemorrhage of different origin: 4 idiopathic pulmonary hemosiderosis cases; one bleeding due to celiac disease and one acute pulmonary hemorrhage of infancy. Four cases of possible surfactant deficiencies were suspected (genetic studies included). Almost the whole spectrum of interstitial lung disease was found and surely it shall be found to be more frequent as long as a coordinated multidisciplinary effort is undertaken...

Heat shock proteins HSP27 and HSP70 in unilateral obstructed kidneys.

Unilateral ureteropelvic junction obstruction (UPJO), a condition involving injury to the kidney, leads to vasoconstriction, macrophage infiltration, oxidative stress, and tubulointerstitial fibrosis. In the present study we analyzed in 22 children, the impact of UPJO on expression of HSP27 and HSP70 and related the changes to renal function and duration of obstruction. Kidney function was evaluated, and renal biopsies were obtained for immunohistochemical and western blot analyses at the time of surgery. Kidney tissue from 5 children of similar age, removed because of various malignancies, was used as control. Increased HSP27, with an expression pattern related to the duration of the obstruction, was noted in proximal tubules (PTs), cortical collecting ducts (CCDs), and medullary collecting ducts. Strong HSP27 staining in cytoplasm and nuclei of these segments appeared in children with kidney obstruction for 2.1+/-0.41 years. HSP70 showed a close spatial expression pattern with that of HSP27. The increased staining of HSP70 in PTs and CCDs was related to the decreased glomerular filtration rate. The constitutive/cognate form of HSP70 (HSC72) was absent. These results support the concept that HSP27 and HSP70 are involved in the adaptive response of the human kidney to congenital UPJO.