Cell-Free Synthesis Meets Antibody Production: A Review

Abstract

Engineered antibodies are key players in therapy, diagnostics and research. In addition to full size immunoglobulin gamma (IgG) molecules, smaller formats of recombinant antibodies, such as single-chain variable fragments (scFv) and antigen binding fragments (Fab), have emerged as promising alternatives since they possess different advantageous properties. Cell-based production technologies of antibodies and antibody fragments are well-established, allowing researchers to design and manufacture highly specific molecular recognition tools. However, as these technologies are accompanied by the drawbacks of being rather time-consuming and cost-intensive, efficient and powerful cell-free protein synthesis systems have been developed over the last decade as alternatives. So far, prokaryotic cell-free systems have been the focus of interest. Recently, eukaryotic in vitro translation systems have enriched the antibody production pipeline, as these systems are able to mimic the natural pathway of antibody synthesis in eukaryotic cells. This review aims to overview and summarize the advances made in the production of antibodies and antibody fragments in cell-free systems.

Establishing a high yielding streptomyces‐based cell‐free protein synthesis system

“…CFPS systems help address a growing need for simple, inexpensive, and efficient protein production technologies. So far, they have been widely utilized for manufacturing a wide variety of active protein products that include therapeutic vaccines (Kanter et al, 2007;Yang et al, 2005), antibodies (Min et al, 2016;Stech and Kubick, 2015), viruslike particles (Bundy et al, 2008;Lu et al, 2015), membrane proteins (Henrich et al, 2015;Sachse et al, 2014), metalloproteins (Boyer et al, 2008;Kwon et al, 2013;Li et al, 2016) and proteins harboring non-standard amino acids (Hong et al, 2014(Hong et al, , 2015. CFPS has also been applied for the rapid prototyping of biological circuits and metabolic pathways (Garamella et al, 2016;Karim and Jewett, 2016;Sun et al, 2014;Takahashi et al, 2015), biosynthesis of natural products (Goering et al, 2017) as well as designing of paperbased diagnostics (Pardee et al, 2014(Pardee et al, , 2016a.…”

ChemInform Abstract: Cell‐Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems

“…[167] As the incorporation of ncAAs into proteins in prokaryotic cellfree systems has resulted in many successful modificationo f proteins characteristics, this method was appliedt oe ukaryotic cell-free systems. Incorporation of an ncAA into as ingle-chain ChemBioChem 2015, 16,2420 -2431 www.chembiochem.org antibodyf ragment and into the potassium channel KcsA was achieved in an insect-cell free system [168,101,103] In addition, structuralc hanges to flavin-mononucleotide-binding protein were achieved by the incorporationo fn cAAs into aw heat germ cell-free system. [169] The incorporation of ncAAs bearing af luorophore has been employed to analyze protein-protein interactions, ligand-bindings tudies by FRET,a nd structural conformational changes.…”

Produção da proteína recombinante Interferon-β1b na plataforma se síntese proteica livre de célula

“…No entanto, uma das maiores desvantagens do sistema cell-free em organismo procarioto é a deficiência em organelas que realizem modificações pós-traducionais. As células de ovário de hamster chinês (CHO) são as células mamíferas mais frequentemente usadas para produção de proteínas terapêuticas complexas no sistema baseado em célula, como os anticorpos monoclonais e proteínas de membrana (STECH, 2015;THORING et al, 2017). Células humanas também têm sido estudadas para preparo de lisados (BRÖDEL e KUBICK, 2014).…”

“…T7 RNA polimerase(SALEHI et al, 2016;ZAWADA et al, 2011). Ajuste no potencial redox pode ser feito com a proporção de glutationa oxidada e reduzida, isomerase de dissulfeto pode ser adicionado para auxiliar na formação de ponte dissulfeto, chaperonas para facilitar a produção de fragmentos de anticorpos de cadeia única (scFv), fragmentos ligadores de antígeno (Fab) e anticorpos inteiros(JIN e HONG, 2018;STECH et al, 2015;MATSUDA et al, 2013;KANTER, 2007). Adição de um detergente não iônico à mistura de incubação pode ser útil na prevenção da agregação do produto e melhorar a solubilidade e a atividade específica das proteínas; porém é preciso cuidado na escolha já que este pode interferir nos processos downstream (KWON e JEWETT, 2015).…”

“…Em relação à extensão do tempo de reação, pesquisadores buscam novos formatos além da tradicional batelada. Formatos como fluxo continuo e troca contínua permitem que substratos sejam reabastecidos para a continuidade da síntese e a retirada dos co-produtos que possam inibir a produção da proteína e cessar a reação(PEÑALBER-JOHNSTONE et al, 2017;THORING;TRAN, 2018;STECH et al, 2015;HONG et al, 2015).Com isso, produções em sistemas cell-free puderam aumentar o rendimento de 20 a 40 vezes em comparação com o formato em batelada (STECH et al, 2017). A plataforma CFPS vêm sendo otimizada cada vez mais, com melhores rendimentos, melhor performance, menores custos e cada vez mais competitivo em relação à expressão baseada em células.…”