ABSTRACT
Immunoglobulin M (IgM) is a pentameter of approximately 950 kDa and Each subunit is composed of two heavy chains of about 65 kDa, two light chains of 25 kDa and one J or junction chain, with a molecular weight of about 15 kDa. IgM is a promising therapeutic candidate. In the world the first human IgM-enriched immunoglobulin is Pentaglobin ® , it is superior to normal IgG preparations in eliminating infectious pathogens and neutralizing their endoexotoxins. In this work we purified IgM from human plasma with a Sepharose 4FF size exclusion column and modified the cut defined in previous experiments of fraction 2 (IgM enriched fraction), aiming at higher purity and elimination of contaminating proteins, we applied the fraction in the anionic exchange column ANX Sepharose FF and in the affinity column IMAC Co2+. Objectives: To verify the IgM purification strategy using the filtration gel columns, followed by ion exchange and metal affinity chromatography after modifying the location of the cut of the filtration gel column fraction and analyze IgM yield after this modification. Materials and Methods: Plasma was applied directly to the Sepharose 4FF filtration gel column. The IgM rich fraction was subsequently applied to the ANX Sepharose FF anion exchange column, the fraction with the highest IgM recovery was applied to an IMAC Co2+ metal affinity column at pH 5.0 and pH 6.0 O collected from all fractions were analyzed by the Bradford test, polyacrylamide gel and turbidimetry. Results and discussion: The new cut of fraction 2 of Sepharose 4 FF was not higher than the IgM yield, but twice as much total immunoglobulins were recovered, in the ANX column Sepharose 4 FF was recovered 77% of IgM in the fraction 350mM-1, but in this fraction it contains only 60% of immunoglobulins (IgM and IgA), requiring a new purification step, and IgG and Albumin do not bind strongly to the column, eluted in the flowthrough, allowing a good separation of these proteins. The 350mM fraction was applied to the IMAC Co2+ affinity column to increase igM purity at pH 5.0 and 6.0, the IgM recovery in the flowthroug is similar, but in the polyacrylamide gel of pH 6.0 it is possible to analyze that the high mass proteins bind more strongly to the column than at pH 5.0, and in quantitative tests the purification factor of pH 6.0 is higher than pH 5.0.Conclusion: The new cut of fraction 2 of the Sepharose 4FF column was not efficient in reducing contaminant proteins and IgM yield, the ANX Sepharose FF column is efficient in separating IgG and albumin from IgM even with higher protein load, the metal affinity column IMAC Co2+ was not effective in separating total IgM from contaminant proteins even at different pH's employed, and new strategies and studies for increasing immunoglobulin purity need to be developed.
A imunoglobulina M (IgM) é um pentâmero de aproximadamente 950 kDa e cada subunidade é composta por duas cadeias pesadas de cerca de 65 kDa, duas cadeias leves de 25 kDa e uma cadeia J ou de junção, com um peso molecular de cerca de 15 kDa. IgM é como candidato terapêutico promissor. No mundo a primeira imunoglobulina humana enriquecida com IgM é Pentaglobin ® , ele é superior às preparações normais de IgG na eliminação de patógenos infecciosos e neutralização de suas endo-exotoxinas. Neste trabalho purificamos IgM do plasma humano com uma coluna de exclusão por tamanho Sepharose 4FF e modificamos o corte definido em experimentos anteriores da fração 2 (fração enriquecida com IgM), visando de maior pureza e eliminação de proteínas contaminantes, aplicamos a fração na coluna de troca aniônica ANX Sepharose FF e na coluna de afinidade IMAC Co2+. Objetivos: Verificar a estratégia de purificação de IgM empregando as colunas de gel filtração, seguida de troca iônica e cromatografia de afinidade a metal após a modificação da localização do corte da fração da coluna de gel filtração e analisar rendimento de IgM após esta modificação. Materiais e Métodos: O plasma foi aplicado diretamente na coluna de gel filtração Sepharose 4FF. A fração rica em IgM e foi posteriormente aplicado na coluna de troca aniônica ANX Sepharose FF, a fração com maior recuperação de IgM foi aplicada em uma coluna de afinidade a metal IMAC Co2+ em pH 5,0 e pH 6,0 O recolhido de todas as frações foram analisadas pelo ensaio de Bradford, gel de poliacrilamida e turbidimetria. Resultados e discussão: O novo corte da fração 2 da Sepharose 4 FF não se apresentou superior em relação ao rendimento de IgM, porem o dobro de imunoglobulinas totais foram recuperadas, na coluna ANX Sepharose 4 FF foi recuperada 77% da IgM na fração 350mM-1, porém nesta fração contém apenas 60% de imunoglobulinas (IgM e IgA), necessitando de uma nova etapa de purificação, e IgG e a Albumina não se ligam fortemente a coluna, eluiram no flowthrough, permitindo uma boa separação dessas proteínas. A fração 350mM foi aplicada na coluna de afinidade IMAC Co2+ para aumento da pureza de igM em pH 5,0 e 6,0, a recuperação de IgM no flowthroug são parecidos, porém no gel de poliacrilamida do pH 6,0 é possível analisar que as proteínas de alta massa se ligam mais fortemente a coluna que no pH 5,0, e em ensaios quantitativos o fator de purificação do pH 6,0 é superior ao pH 5,0.Conclusão: O novo corte da fração 2 da coluna Sepharose 4FF não foi eficiente para a diminuição de proteínas contaminantes e rendimento de IgM, a coluna ANX Sepharose FF é eficiente na separação de IgG e albumina da IgM mesmo com maior carga de proteínas, a coluna de afinidade a metal IMAC Co2+ não se mostrou efetiva na separação total de IgM de proteínas contaminantes mesmo em diferentes pH’s empregados, sendo necessário desenvolvimento de novas estratégias e estudos para o aumento de pureza das imunoglobulinas.
ABSTRACT
Los anticuerpos constituyen un componente fundamental del sistema inmune, permitiendo el reconocimiento con alta especificidad y posterior destrucción de moléculas extrañas. Los anticuerpos monoclonales, producidos por la tecnología del hibridoma, presentan desventajas para su uso en terapia humana debido a su origen en una especie diferente. La ingeniería genética posibilitó la utilización de los anticuerpos monoclonales para terapias humanas, generando los anticuerpos recombinantes terapéuticos. Así, los anticuerpos recombinantes se han transformado en un importante grupo de fármacos; con decenas de ellos aprobados para terapia humana y cientos en desarrollo. Se utilizan con éxito como tratamiento para un amplio rango de patologías, tales como cáncer, autoinmunidad e infecciones, siendo desde hace años el biofármaco con mayores ventas. Inicialmente todos los anticuerpos recombinantes terapéuticos presentaban la estructura convencional de los anticuerpos. Sin embargo, más recientemente, se han generado nuevos diseños que no poseen las características estructurales naturales, como los anticuerpos de simple cadena y bi-específicos. Debido al desarrollo y éxito de la tecnología de anticuerpos recombinantes, se espera un aumento constante en el número de anticuerpos terapéuticos contra nuevos blancos, además de la generación de nuevas estructuras, usos y estrategias terapéuticas. En esta revisión, nos centraremos en las características estructurales y los nuevos formatos de anticuerpos, así como su aplicación clínica en el tratamiento de diversas patologías. Además analizaremos los nuevos formatos de anticuerpos que se encuentran en el mercado y la aparición de los anticuerpos biosimilares.
Antibodies are a key component of the immune system, acting in the highly specific recognition and subsequent destruction of foreign molecules. Monoclonal antibodies produced by hybridoma technology have disadvantages for use in human therapy becauseof its origin in a different species. Genetic engineering enabled the use of monoclonalantibodies for human therapies, generating recombinant therapeutic antibodies. Thus, the recombinant antibodies have become an important group of drugs; dozens of them are approved for human therapy and there are hundreds in development. They are successfully used as a treatment for a wide range of pathologies, such as cancer, autoimmunity and infections, being the biopharmaceutical with higher sales. Initially therapeutic recombinant antibodies showed the conventional structure of the antibodies. However, more recently, new designs that do not have natural structural features havebeen generated such as single chain formats and bi-specific antibodies. Due to development and success of recombinant antibody technology, a steady increase in the number of newtherapeutic drugs against new targets is expected in addition to the generation of new structures, uses and therapeutic strategies. In this review, we will focus on their structural features and clinical application in the treatment of various pathologies. We will also discuss new formats of antibodies and the emergence of biosimilar antibodies.
Subject(s)
Humans , Antibodies/therapeutic use , Immunoglobulins , Antibodies, MonoclonalABSTRACT
The diagnosis of Celiac Disease (CD) relies on the concordance of pathological, serological, genetic and clinical features. For this reason, the diagnosis of CD is often a challenge. Seronegative celiac disease (SNCD) is defined by the negativity of anti-tissue transglutaminase antibodies in the presence of a positive histology on duodenal biopsy samples, i.e. inflammatory infiltrate of intra-epithelial lymphocytes (IELs > 25/100 enterocytes), mild villous atrophy and uneven brush border associated to human leukocyte antigen (HLA) haplotype DQ2 and/or DQ8. SNCD is characterized by mucosal deposits of tissue transglutaminase (tTG)/anti-tTG immuno-complexes. These may counteract the passage of anti-tTG into the bloodstream, thus explaining seronegativity. Another reason for seronegativity may be found in an incomplete maturation of plasma cells with a consequent failure of antibodies production. This condition often characterizes immunoglobulin deficiencies, and, indeed, SNCD is common in subjects with immunoglobulin deficiencies. The management of SNCD still remains debated. The treatment option for SNCD may be represented by gluten free diet (GFD), but the usefulness and appropriateness of prescribing GFD are controversial. Some evidences support its use only in SNCD subjects showing CD clear clinical picture and compatible HLA status. The choice of GFD administration could be linked to an investigation able to diagnose SNCD in no doubt even if a reliable test is not currently available. On these bases, a test helping the diagnosis of SNCD is justifiable and desirable.
