RESUMO
Generation of bispecific antibodies (BsAbs) having two unique Fab domains requires heterodimerization of the two heavy chains and pairing of each heavy chain with its cognate light chain. An alternative bispecific scaffold (Bipod) comprising an scFv and a Fab on a heterodimeric Fc eliminates the possibility of light chain mispairing. However, unpredictable levels of chain expression and scFv-induced aggregation can complicate purification and reduce the yield of desired Bipod. Here, we describe a high-throughput method for generation of Bipods based on protein A and CH1 domain affinity capture. This method exploits over-expression of the scFv chain to maximize heterodimer yield. Bipods purified by this method have purity suitable for cell-based functional assays and in vivo studies.
Assuntos
Anticorpos Biespecíficos/química , Fragmentos Fab das Imunoglobulinas/química , Engenharia de Proteínas/métodos , Anticorpos de Cadeia Única/química , Animais , Produtos Biológicos/uso terapêutico , Células CHO , Cricetulus , DNA/química , Dimerização , Avaliação Pré-Clínica de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Epitopos/química , Humanos , Imunoglobulina G/genética , Imunossupressores/uso terapêutico , Mutação , Neoplasias/terapia , Plasmídeos , Domínios ProteicosRESUMO
Prophylaxis with 2-4 times weekly dosing of factor (F)VIII or FIX is established as an efficacious and safe treatment in haemophilia. Although prophylaxis is not readily available for the inhibitor patient, recent studies have demonstrated a reduction in bleeding episodes in inhibitor patients treated with daily infusions of FVIIa. In order to develop a treatment option comparable to prophylaxis with FVIII or FIX we looked to PEGylation which is an established method for prolonging the circulatory half-life of proteins. However, due to the numerous interactions of FVIIa with the cell surface, TF, FIX and FX there are limited options for unspecific chemical modification of FVIIa without loss of activity. Consequently, we explored the GlycoPEGylationtrade mark technology for selective PEGylation of the two N-glycans in the FVIIa light chain and protease domain to generate seven specifically modified derivatives with PEG groups ranging from 2 to 40 kDa. These derivatives were evaluated in vitro for their ability to interact with small synthetic substrates as well as key molecules relevant to function in the coagulation pathway. The results demonstrate that modification of FVIIa using glycoPEGylation has only a very limited effect on the hydrolysis S-2288 and FX activation. However, the modification does to some extend alter the ability of FVIIa to interact with TF and more importantly, reduces the rate of ATIII inhibition by up to 50% which could allow for an extended active half-life in circulation.
Assuntos
Coagulantes/metabolismo , Fator VIIa/metabolismo , Animais , Antitrombina III/metabolismo , Coagulantes/antagonistas & inibidores , Coagulantes/síntese química , Fator VIIa/antagonistas & inibidores , Fator VIIa/síntese química , Fator Xa/metabolismo , Meia-Vida , Humanos , Hidrólise , Modelos Moleculares , Peso Molecular , Oligopeptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Tromboplastina/metabolismoRESUMO
The Fc effector functions of immunoglobulin G (IgG) antibodies are in part determined by structural features of carbohydrates linked to each of the paired gamma heavy chains in the antibody constant domain (C(H)2). One glycoform that has been shown to be advantageous is G2, where both arms of complex bi-antennary N-glycans terminate in galactose. In vitro treatment with glycosyltransferases can remodel heterogeneous IgG glycoforms, enabling preparation of IgG molecules with homogeneous glycan chains. Here we describe optimization of conditions for use of a soluble recombinant galactosyltransferase in vitro to remodel glycans of human serum IgG, and we demonstrate a scaled-up reaction in which >98% of neutral glycans attached to 1 kg IgG are converted to the G2 glycoform. Removal of glycosylation reagents from the product is achieved in one step by affinity chromatography on immobilized Protein A.
Assuntos
Anticorpos Monoclonais/química , Galactosiltransferases/química , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Oligossacarídeos de Cadeias Ramificadas/síntese química , Processamento de Proteína Pós-Traducional , Animais , Anticorpos Monoclonais/genética , Configuração de Carboidratos , Sequência de Carboidratos , Bovinos , Galactosiltransferases/genética , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/genética , Dados de Sequência Molecular , Oligossacarídeos de Cadeias Ramificadas/química , Polissacarídeos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
An improved method for the purification of dehydrin from soy (glycine max) is described. Acidic extraction of soy whey was followed by a three step chromatographic process: capture on copper charged Chelating Sepharose Big Beads, intermediate hydrophobic interaction chromatography on Source 15 PHE, and a polishing step on blue Sepharose. The 32-kDa native soy dehydrin was purified to a purity of greater than 98.5% with an overall recovery of 63%. When compared to a previously published purification procedure, recovery, time requirements, and sample preparation steps were improved. The developed method is readily scaleable. Preliminary results show that the process can be used for dehydrins from rosemary (Rosmarinum officinalis) and pea (Pisum sativum).
