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1.
J Immunol Methods ; 386(1-2): 34-42, 2012 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-22982058

RESUMO

Previous studies have shown that glycoproteins expressed in wild-type Pichia pastoris bind to Dendritic cell-SIGN (DC-Specific Intercellular adhesion molecule-3 Grabbing Nonintegrin), a mannose-binding receptor found on dendritic cells in peripheral tissues which is involved in antigen presentation and the initiation of an immune response. However, the binding of DC-SIGN to glycoproteins purified from P. pastoris strains engineered to express humanized N- and O-linked glycans has not been tested to date. In this study, the binding of glycoproteins with specific high-mannose or human N- and O-linked glycan structures to DC-SIGN was tested. Proteins with humanized N-glycans including Man5 structures and O-glycans (up to as many as 24) with single mannose chain length showed DC-SIGN binding that was comparable to that measured for a CHO-produced IgG1 which lacks O-linked mannose. Glycoproteins with wild-type N-glycans and mannotriose and higher O-glycans bound to DC-SIGN in a manner that was strongly inhibited by either the use of enzymatic N-deglycosylation or sodium meta-periodate oxidation. Mannan purified from humanized P. pastoris also showed lower ability to inhibit DC-SIGN binding to glycoproteins with wild type fungal glycosylation than mannan purified from wild type strains. This study shows that humanized P. pastoris can produce glycoproteins that do not bind to DC-SIGN.


Assuntos
Moléculas de Adesão Celular/metabolismo , Glicoproteínas/metabolismo , Imunoglobulina G/metabolismo , Lectinas Tipo C/metabolismo , Pichia/genética , Polissacarídeos/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Células CHO , Cricetinae , Glicoproteínas/genética , Glicosilação , Humanos , Imunoglobulina G/genética , Manose/metabolismo , Ligação Proteica/genética , Engenharia de Proteínas
2.
MAbs ; 3(3): 289-98, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21487242

RESUMO

Mammalian cell culture systems are used predominantly for the production of therapeutic monoclonal antibody (mAb) products. A number of alternative platforms, such as Pichia engineered with a humanized N-linked glycosylation pathway, have recently been developed for the production of mAbs. The glycosylation profiles of mAbs produced in glycoengineered Pichia are similar to those of mAbs produced in mammalian systems. This report presents for the first time the comprehensive characterization of an anti-human epidermal growth factor receptor 2 (HER2) mAb produced in a glycoengineered Pichia, and a study comparing the anti-HER2 from Pichia, which had an amino acid sequence identical to trastuzumab, with trastuzumab. The comparative study covered a full spectrum of preclinical evaluation, including bioanalytical characterization, in vitro biological functions, in vivo anti-tumor efficacy and pharmacokinetics in both mice and non-human primates. Cell signaling and proliferation assays showed that anti-HER2 from Pichia had antagonist activities comparable to trastuzumab. However, Pichia-produced material showed a 5-fold increase in binding affinity to FcγIIIA and significantly enhanced antibody dependant cell-mediated cytotoxicity (ADCC) activity, presumably due to the lack of fucose on N-glycans. In a breast cancer xenograft mouse model, anti-HER2 was comparable to trastuzumab in tumor growth inhibition. Furthermore, comparable pharmacokinetic profiles were observed for anti-HER2 and trastuzumab in both mice and cynomolgus monkeys. We conclude that glycoengineered Pichia provides an alternative production platform for therapeutic mAbs and may be of particular interest for production of antibodies for which ADCC is part of the clinical mechanism of action.


Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais/imunologia , Pichia/genética , Receptor ErbB-2/imunologia , Proteínas Recombinantes/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Afinidade de Anticorpos/imunologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Área Sob a Curva , Ligação Competitiva/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Fucose/metabolismo , Engenharia Genética , Humanos , Macaca fascicularis , Camundongos , Camundongos Endogâmicos C57BL , Pichia/metabolismo , Polissacarídeos/metabolismo , Ligação Proteica/imunologia , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacologia , Trastuzumab , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Protein Expr Purif ; 76(1): 7-14, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21074617

RESUMO

A robust and scalable purification process was developed to quickly generate antibody of high purity and sufficient quantity from glycoengineered Pichia pastoris fermentation. Protein A affinity chromatography was used to capture the antibody from fermentation supernatant. A pH gradient elution was applied to the Protein A column to prevent antibody precipitation at low pH. Antibody from Protein A chromatography contained some product related impurities, which were the misassembling of cleaved heavy chain, heavy chain and light chain. It also had some process related impurities, including Protein A residues, endotoxin, host cell DNA and proteins. Cation exchange chromatography with optimal NaCl gradient at pH 4.5-6.0 efficiently removed these product and process related impurities. The antibody from glycoengineered P. pastoris was comparable to its commercial counterpart in heterotetramer folding, physical stability and binding affinity.


Assuntos
Anticorpos Monoclonais/biossíntese , Pichia/metabolismo , Proteínas Recombinantes/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Organismos Geneticamente Modificados , Pichia/genética , Proteínas Recombinantes/isolamento & purificação
4.
Protein Expr Purif ; 74(1): 9-15, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20447459

RESUMO

Glycoengineered Pichia pastoris provides a unique platform for screening monoclonal antibody (mAb) leads and high expressing strains. A simple, economic, and high-throughput purification for mAb from P. pastoris fermentation has been developed that can be easily operated in various commercially available liquid handlers. The method includes the use of STREAMLINE rProtein A in a 96-well platform and demonstrates good linear alignment and reproducibility in a wide concentration range. The antibody titers measured by the method have less than 15% variation in comparison to spiking titers. The mAb titer and quality obtained from this method are comparable to that from conventional column chromatography. The method can process hundreds of expression screening samples in a day, not only to accurately determine titers, but also to generate milligram quantities of mAb for quality assessment, including purity, folding, glycosylation, and antigen binding affinity.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Fermentação , Microbiologia Industrial/métodos , Pichia/genética , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Cromatografia Líquida de Alta Pressão/economia , Cromatografia Líquida de Alta Pressão/métodos , Expressão Gênica , Glicosilação , Imunoglobulina G/genética , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/metabolismo , Microbiologia Industrial/economia , Reprodutibilidade dos Testes , Proteína Estafilocócica A/metabolismo
5.
Methods Mol Biol ; 534: 213-23, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19277549

RESUMO

The production of recombinant therapeutic glycoproteins is an active area of research and drug development. Typically, improvements in therapeutic glycoprotein efficacy have focused on engineering additional N-glycosylation sites into the primary amino acid sequence or attempting to control a particular glycoform profile on a protein through process improvements. Recently, a number of alternative expression systems have appeared that are challenging the dominance of mammalian cell culture. Our laboratory has focused on the re-engineering of the secretory pathway in the yeast Pichia pastoris to perform glycosylation reactions that mimic processing of N-glycans in humans. We have demonstrated that human antibodies with specific human N-glycan structures can be produced in glycoengineered lines of Pichia pastoris and that antibody-mediated effector functions can be optimized by generating specific glycoforms. In this chapter we provide detailed protocols for the analysis of glycosylation on intact glycoproteins by MALDI-TOF and site specific N-glycan occupancy on digested glycoprotein using ESI-MS.


Assuntos
Glicoproteínas/metabolismo , Glicosilação , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Cromatografia Líquida de Alta Pressão/métodos , Glicoproteínas/química , Humanos , Proteínas Recombinantes/química
6.
J Biotechnol ; 139(4): 318-25, 2009 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-19162096

RESUMO

The growing antibody market and the pressure to improve productivity as well as reduce cost of production have fueled the development of alternative expression systems. The therapeutic function of many antibodies is influenced by N-linked glycosylation, which is affected by a combination of the expression host and culture conditions. This paper reports the generation of a glycoengineered Pichia pastoris strain capable of producing more than 1 g l(-1) of a functional monoclonal antibody in a robust, scalable and portable cultivation process with uniform N-linked glycans of the type Man(5)GlcNAc(2). N-linked glycan uniformity and volumetric productivity have been maintained across a range of cultivation process conditions including pH (5.5-7.5), temperature (16-24 degrees C), dissolved oxygen concentration (0.85-3.40 mg l(-1)) and specific methanol feed rate (9-19 mg g(-1) h(-1)) as well as across different cultivation scales (0.5, 3.0, 15 and 40 l). Compared to a marketed CHO-produced therapeutic antibody, the glycoengineered yeast-produced antibody has similar motilities on SDS-PAGE, comparable size exclusion chromatograms (SEC) and antigen binding affinities. This paper provides proof of concept that glycoengineered yeast can be used to produce functional full-length monoclonal antibodies at commercially viable productivities.


Assuntos
Anticorpos Monoclonais/biossíntese , Imunoglobulina G/biossíntese , Pichia/genética , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/isolamento & purificação , Afinidade de Anticorpos , Sítios de Ligação de Anticorpos , Reatores Biológicos , Células Cultivadas , Engenharia Genética , Instabilidade Genômica , Glicosilação , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina G/genética , Imunoglobulina G/isolamento & purificação , Metanol/química , Oxigênio/química , Pichia/metabolismo , Temperatura
7.
MAbs ; 1(6): 572-9, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-20073128

RESUMO

The Fc region of an antibody mediates effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and plays a key role in the in vivo half-life of an antibody. In designing antibody therapeutics, it is sometimes desirable that the antibody has altered Fc-mediated properties. In the case of a "benign blocker" antibody, it is often desirable to diminish or abolish the ADCC and CDC functions while retaining its PK profile. Here, we report a novel engineered IgG isotype, IgG2m4, with reduced Fc functionality. IgG2m4 is based on the IgG2 isotype with four key amino acid residue changes derived from IgG4 (H268Q, V309L, A330S and P331S). An IgG2m4 antibody has an overall reduction in complement and Fc gamma receptor binding in in vitro binding analyses while maintaining the normal in vivo serum half-life in rhesus.


Assuntos
Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Complemento C1q/metabolismo , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/metabolismo , Engenharia de Proteínas , Receptores de IgG/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Citotoxicidade Celular Dependente de Anticorpos/genética , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Complemento C1q/genética , Complemento C1q/imunologia , Cricetinae , Meia-Vida , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , Ligação Proteica/imunologia , Receptores de IgG/genética , Receptores de IgG/imunologia
8.
Glycoconj J ; 25(6): 581-93, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18365311

RESUMO

Traditional production of therapeutic glycoproteins relies on mammalian cell culture technology. Glycoproteins produced by mammalian cells invariably display N-glycan heterogeneity resulting in a mixture of glycoforms the composition of which varies from production batch to production batch. However, extent and type of N-glycosylation has a profound impact on the therapeutic properties of many commercially relevant therapeutic proteins making control of N-glycosylation an emerging field of high importance. We have employed a combinatorial library approach to generate glycoengineered Pichia pastoris strains capable of displaying defined human-like N-linked glycans at high uniformity. The availability of these strains allows us to elucidate the relationship between specific N-linked glycans and the function of glycoproteins. The aim of this study was to utilize this novel technology platform and produce two human-like N-linked glycoforms of recombinant human lactoferrin (rhLF), sialylated and non-sialylated, and to evaluate the effects of terminal N-glycan structures on in vitro secondary humoral immune responses. Lactoferrin is considered an important first line defense protein involved in protection against various microbial infections. Here, it is established that glycoengineered P. pastoris strains are bioprocess compatible. Analytical protein and glycan data are presented to demonstrate the capability of glycoengineered P. pastoris to produce fully humanized, active and immunologically compatible rhLF. In addition, the biological activity of the rhLF glycoforms produced was tested in vitro revealing the importance of N-acetylneuraminic (sialic) acid as a terminal sugar in propagation of proper immune responses.


Assuntos
Lactoferrina/metabolismo , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Feminino , Expressão Gênica , Engenharia Genética/métodos , Glicoproteínas/química , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Glicosilação , Humanos , Lactoferrina/química , Lactoferrina/genética , Masculino , Espectrometria de Massas , Dados de Sequência Molecular , Pichia/genética , Proteínas Recombinantes/química , Alinhamento de Sequência , Ovinos , Ácidos Siálicos/química , Ácidos Siálicos/imunologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
9.
Nat Biotechnol ; 24(2): 210-5, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16429149

RESUMO

As the fastest growing class of therapeutic proteins, monoclonal antibodies (mAbs) represent a major potential drug class. Human antibodies are glycosylated in their native state and all clinically approved mAbs are produced by mammalian cell lines, which secrete mAbs with glycosylation structures that are similar, but not identical, to their human counterparts. Glycosylation of mAbs influences their interaction with immune effector cells that kill antibody-targeted cells. Here we demonstrate that human antibodies with specific human N-glycan structures can be produced in glycoengineered lines of the yeast Pichia pastoris and that antibody-mediated effector functions can be optimized by generating specific glycoforms. Glycoengineered P. pastoris provides a general platform for producing recombinant antibodies with human N-glycosylation.


Assuntos
Anticorpos Monoclonais/biossíntese , Melhoramento Genético/métodos , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , Pichia/genética , Pichia/metabolismo , Engenharia de Proteínas/métodos , Anticorpos Monoclonais/genética , Glicosilação , Humanos , Proteínas Recombinantes/biossíntese
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