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Novel Affinity-Based Technologies for Targeting Distinct Biological Systems
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O desenvolvimento de estratégias de purificação baseadas em afinidade foi impulsionado pelas vantagens teóricas que esta modalidade oferece para o bioprocessamento de PMTAs (produtos medicinais de terapia avançada), podendo resolver problemas atuais da indústria. A cromatografia de afinidade permitiu que a bioprodução de anticorpos monoclonais (mAbs) atingisse novos patamares, provocando a indústria a desenvolver estas tecnologias para outras biomoléculas, como vetores virais. O foco deste trabalho foi a caracterização/otimização de duas estratégias baseadas em afinidade para a purificação de diferentes biomoléculas, mAbs e vetores lentivirais (LVs). A primeira estratégia, focada em cromatografia, visou o uso de diferentes ligandos de afinidade (Z6Alk-Cys e Petasis-Ugi), imobilizados em diferentes tipos de meios cromatográficos. O ligando com melhor desempenho foi o Z6Alk-Cys imobilizado em esferas de agarose, onde uma recuperação de 83% (rendimento total de 31%) de IgG adsorvido foi obtida. O mesmo ligando foi imobilizado em membranas, porém, nenhuma recuperação foi conseguida. A outra estratégia foi um método não cromatográfico de purificação de LV, baseado em separação de fase líquida através de ultracentrifugação ou filtração de fluxo tangencial (TFF). A separação ocorre devido à presença de um copolipeptídeo termorresponsivo com afinidade para este alvo. Dois lotes desta solução de copolipeptídeo foram avaliados. Uma taxa de recuperação de TU (unidades de transdução) de 100% foi alcançada em escala de bancada e à escala de 200 mL (TFF), um rendimento total de 49,0 ± 25,5% foi obtido. A necessidade de um entendimento mais profundo da termorresponsividade deste copolipeptídeo ficou clara antes de prosseguir com o aumento de escala, tendo esta sido conseguida através de DLS e SEC-HPLC, cujos resultados também estão apresentados. O foco deste trabalho foram tecnologias promissoras baseadas em afinidade e também as adversidades enfrentadas durante o seu desenvolvimento, mostrando que otimização ainda é necessária para seu potencial uso comercial.
The development of affinity-based purification strategies was supported by the theoretical advantages this modality poses for ATMPs (advanced therapeutic medicinal products) bioprocessing, which could tackle current industry problems. Affinity chromatography allowed the bioproduction of monoclonal antibodies (mAbs) to reach new heights and the industry has moved towards developing these technologies for other biomolecules, such as viral vectors. This work was focused on process optimization and technology characterization for two affinity-based strategies for different biomolecules, mAbs and lentiviral vectors (LVs). The first strategy was chromatography-focused, using two different affinity ligands (Z6Alk-Cys and Petasis-Ugi) for mAbs, immobilized in different types of chromatographic media. The best performing ligand was Z6Alk-Cys immobilized in agarose beads, where a bound IgG recovery rate of 83% (overall yield of 31%) was obtained. The same ligand was immobilized in novel membrane constructs based on fibers electrospun in a polyamide mesh, however, no recovery was obtained. The other strategy was a non-chromatographic LV purification method, based on liquid phase separation through ultracentrifugation or tangential flow filtration (TFF). Separation occurs due to the presence of a thermo-responsive copolypeptide with affinity to this target. Two batches of this copolypeptide solution were evaluated. A TU (transducing unit) recovery rate of 100% was achieved at benchtop scale and at 200 mL TFF scale, an overall yield of 49 ± 25.5% was obtained. The need for deeper understanding of this copolypeptide thermo-responsiveness was made clear before proceeding with scale-up, this was addressed by DLS and SEC-HPLC analysis which results are also reported. This work highlights promising affinity-based technologies and the adversities faced during their development, showing optimization is still needed for their potential commercial use.
The development of affinity-based purification strategies was supported by the theoretical advantages this modality poses for ATMPs (advanced therapeutic medicinal products) bioprocessing, which could tackle current industry problems. Affinity chromatography allowed the bioproduction of monoclonal antibodies (mAbs) to reach new heights and the industry has moved towards developing these technologies for other biomolecules, such as viral vectors. This work was focused on process optimization and technology characterization for two affinity-based strategies for different biomolecules, mAbs and lentiviral vectors (LVs). The first strategy was chromatography-focused, using two different affinity ligands (Z6Alk-Cys and Petasis-Ugi) for mAbs, immobilized in different types of chromatographic media. The best performing ligand was Z6Alk-Cys immobilized in agarose beads, where a bound IgG recovery rate of 83% (overall yield of 31%) was obtained. The same ligand was immobilized in novel membrane constructs based on fibers electrospun in a polyamide mesh, however, no recovery was obtained. The other strategy was a non-chromatographic LV purification method, based on liquid phase separation through ultracentrifugation or tangential flow filtration (TFF). Separation occurs due to the presence of a thermo-responsive copolypeptide with affinity to this target. Two batches of this copolypeptide solution were evaluated. A TU (transducing unit) recovery rate of 100% was achieved at benchtop scale and at 200 mL TFF scale, an overall yield of 49 ± 25.5% was obtained. The need for deeper understanding of this copolypeptide thermo-responsiveness was made clear before proceeding with scale-up, this was addressed by DLS and SEC-HPLC analysis which results are also reported. This work highlights promising affinity-based technologies and the adversities faced during their development, showing optimization is still needed for their potential commercial use.
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Palavras-chave
Affinity purification Chromatography Liquid-phase separation