RESUMO
In the current environment of diverse product pipelines, rapidly fluctuating market demands and growing competition from biosimilars, biotechnology companies are increasingly driven to develop innovative solutions for highly flexible and cost-effective manufacturing. To address these challenging demands, integrated continuous processing, comprised of high-density perfusion cell culture and a directly coupled continuous capture step, can be used as a universal biomanufacturing platform. This study reports the first successful demonstration of the integration of a perfusion bioreactor and a four-column periodic counter-current chromatography (PCC) system for the continuous capture of candidate protein therapeutics. Two examples are presented: (1) a monoclonal antibody (model of a stable protein) and (2) a recombinant human enzyme (model of a highly complex, less stable protein). In both cases, high-density perfusion CHO cell cultures were operated at a quasi-steady state of 50-60 × 10(6) cells/mL for more than 60 days, achieving volumetric productivities much higher than current perfusion or fed-batch processes. The directly integrated and automated PCC system ran uninterrupted for 30 days without indications of time-based performance decline. The product quality observed for the continuous capture process was comparable to that for a batch-column operation. Furthermore, the integration of perfusion cell culture and PCC led to a dramatic decrease in the equipment footprint and elimination of several non-value-added unit operations, such as clarification and intermediate hold steps. These findings demonstrate the potential of integrated continuous bioprocessing as a universal platform for the manufacture of various kinds of therapeutic proteins.
Assuntos
Reatores Biológicos , Biotecnologia/instrumentação , Biotecnologia/métodos , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Proteínas Recombinantes/biossíntese , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Células CHO , Contagem de Células , Distribuição Contracorrente , Cricetinae , Cricetulus , Enzimas/biossíntese , Enzimas/química , Enzimas/isolamento & purificação , Enzimas/metabolismo , Humanos , Modelos Biológicos , Perfusão , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Hydrophobicity is often characterized macroscopically by the droplet contact angle. Molecular signatures of hydrophobicity have, however, remained elusive. Successful theories predict a drying transition leading to a vapor-like region near large hard-sphere solutes and interfaces. Adding attractions wets the interface with local density increasing with attractions. Here we present extensive molecular simulation studies of hydration of realistic surfaces with a wide range of chemistries from hydrophobic (-CF(3), -CH(3)) to hydrophilic (-OH, -CONH(2)). We show that the water density near weakly attractive hydrophobic surfaces (e.g., -CF(3)) can be bulk-like or larger, and provides a poor quantification of surface hydrophobicity. In contrast, the probability of cavity formation or the free energy of binding of hydrophobic solutes to interfaces correlates quantitatively with the macroscopic wetting properties and serves as an excellent signature of hydrophobicity. Specifically, the probability of cavity formation is enhanced in the vicinity of hydrophobic surfaces, and water-water correlations correspondingly display characteristics similar to those near a vapor-liquid interface. Hydrophilic surfaces suppress cavity formation and reduce the water-water correlation length. Our results suggest a potentially robust approach for characterizing hydrophobicity of more complex and heterogeneous surfaces of proteins and biomolecules, and other nanoscopic objects.
Assuntos
Interações Hidrofóbicas e Hidrofílicas , Modelos Químicos , Propriedades de Superfície , Molhabilidade , Simulação por Computador , Proteínas/química , Água/químicaRESUMO
An important consideration for integrated continuous biomanufacturing is that the downstream chromatography steps integrated with the bioreactor should maintain a low bioburden state throughout the entire duration of the operation. One potential strategy to achieve this is to start bioburden-free and functionally close the chromatography system. While chromatography skids themselves can be rendered bioburden-free, limitations exist in applying these methods to chromatography columns. The small column sizes used in continuous multicolumn chromatography enable gamma irradiation of disposable columns to render them bioburden-free. However, this approach has not been widely implemented, likely because gamma irradiation can negatively impact resin performance. Here, several protective mobile-phase modifiers were screened and shown to help chromatography resins retain naïve-like performance. Gamma irradiated columns were then integrated into perfusion bioreactors for continuous capture. Successful integrated continuous capture downstream of perfusion bioreactors for greater than 40 days using protein A, custom affinity, and non-affinity capture resins for multiple biologic modalities is demonstrated in development and commercial settings. No indications of time-based performance decline or bioburden growth have been observed. This strategy enables bioburden-free integrated continuous biomanufacturing operations and could allow full process closure and decreased environmental control requirements for facilities; thus, permitting simultaneous multi-product operations in a ballroom arrangement.
Assuntos
Anticorpos Monoclonais , Cromatografia , Reatores Biológicos , Perfusão , Proteína Estafilocócica ARESUMO
We use high resolution dynamical structure factor S(q,omega) data measured with inelastic x-ray scattering to reconstruct the Green's function of water, which describes its density response to a point charge, and provides a fundamental comparative model for solvation behavior at molecular time scales and length scales. Good agreement is found with simulations, scattering and spectroscopic experiments. These results suggest that a moving point charge will modify its hydration structure, evolving from a spherical closed shell to a steady-state cylindrical hydration "sleeve".
RESUMO
A parallel batch screening technique was employed to identify chemically selective displacers which exhibited exclusive separation behavior for the protein pair alpha-chymotrypsin/ribonuclease A on a strong cation exchange resin. Two selective displacers, 1-(4-chlorobenzyl)piperidin-3-aminesulfate and N'1'-(4-methyl-quinolin-2-yl)-ethane-1,2-diamine dinitrate, and one non-selective displacer, spermidine, were selected as model systems to investigate the mechanism of chemically selective displacement chromatography. Saturation transfer difference (STD) NMR was used to directly evaluate displacer-protein binding. The results indicated that while binding occurred between the two chemically selective displacers and the more hydrophobic protein, alpha-chymotrypsin, no binding was observed with ribonuclease A. Further, the non-selective displacer, spermidine, was not observed to bind to either protein. Importantly, the binding event was observed to occur primarily on the aromatic portion of the selective displacers. Extensive molecular dynamic simulations of protein-displacer-water solution were also carried out. The MD results corroborated the NMR findings demonstrating that the binding of selective displacers occurred primarily on hydrophobic surface patches of alpha-chymotrypsin, while no significant long term binding to ribonuclease A was observed. The non-selective displacer did not show significant binding to either of the proteins. MD simulations also indicated that the charged amine group of the selective displacers in the bound state was primarily oriented towards the solvent, potentially facilitating their interaction with a resin surface. These results directly confirm that selective binding between a protein and displacer is the mechanism by which chemically selective displacement occurs. This opens up many possibilities for future molecular design of selective displacers for a range of applications.
Assuntos
Cromatografia Líquida/métodos , Quimotripsina/isolamento & purificação , Ribonuclease Pancreático/isolamento & purificação , Quimotripsina/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Piperidinas/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Quinolinas/metabolismo , Ribonuclease Pancreático/metabolismo , Espermidina/metabolismoRESUMO
We present an extensive molecular simulation study of the behavior of a flexible hydrophobic 25-mer polymer at interfaces presenting a range of chemistries from hydrophobic (-CH(3)) to hydrophilic (-CONH(2)). We quantify the free energy of adsorption, conformational equilibria, and translational and conformational dynamics of the polymer at these diverse interfaces. Water-mediated interactions drive the polymer to adsorb strongly at a hydrophobic interface and repel it from hydrophilic ones. At hydrophilic surfaces, van der Waals interactions between the polymer and the surface mitigate this water-mediated repulsion, leading to weak adsorption of the polymer. Although the polymer is strongly adsorbed to hydrophobic surfaces, it is also most dynamic there. Translational diffusion and conformational dynamics are faster at hydrophobic surfaces compared to those at hydrophilic ones. In bulk water, the polymer collapses into compact globular shapes, whereas the thermodynamic stability of folded polymers is significantly lowered at hydrophobic surfaces. The polymer spreads into pancake-like 2D conformations at hydrophobic surfaces and gradually beads up into globular shapes as the surface is made more hydrophilic. Interestingly, the binding thermodynamics and dynamics correlate with macroscopic droplet contact angles that characterize the wetting properties of the different interfaces.
Assuntos
Polímeros/química , Interações Hidrofóbicas e Hidrofílicas , Modelos Químicos , Simulação de Dinâmica Molecular , Termodinâmica , MolhabilidadeRESUMO
Studies of folding-unfolding of hydrophobic polymers in water provide an excellent starting point to probe manybody hydrophobic interactions in the context of realistic self-assembly processes. Such studies in bulk water have highlighted the similarities between thermodynamics of polymer collapse and of protein folding, and emphasized the role of hydration-water structure, density, and fluctuations-in the folding kinetics. Hydrophobic polymers are interfacially active-that is, they prefer locations at aqueous interfaces relative to bulk water-consistent with their low solubility. How does the presence of a hydrophobic solid surface or an essentially hydrophobic vapor-water interface affect the structural, thermodynamic, and kinetic aspects of polymer folding? Using extensive molecular dynamics simulations, we show that the large hydrophobic driving force for polymer collapse in bulk water is reduced at a solid alkane-water interface and further reduced at a vapor-water interface. As a result, at the solid-water interface, folded structures are marginally stable, whereas the vapor-liquid interface unfolds polymers completely. Structural sampling is also significantly affected by the interface. For example, at the solid-water interface, polymer conformations are quasi-2- dimensional, with folded states being pancake-like structures. At the vapor-water interface, the hydrophobic polymer is significantly excluded from the water phase and freely samples a broad range of compact to extended structures. Interestingly, although the driving force for folding is considerably lower, kinetics of folding are faster at both interfaces, highlighting the role of enhanced water fluctuations and dynamics at a hydrophobic interface.
Assuntos
Interações Hidrofóbicas e Hidrofílicas , Conformação Molecular , Polímeros/química , Cinética , Modelos Moleculares , Temperatura , Termodinâmica , Água/químicaRESUMO
The biotechnology industry primarily uses batch technologies to manufacture recombinant proteins. The natural evolution of other industries has shown that transitioning from batch to continuous processing can yield significant benefits. A quantitative understanding of these benefits is critical to guide the implementation of continuous processing. In this manuscript, we use process economic modeling and Monte Carlo simulations to evaluate an integrated continuous biomanufacturing (ICB) platform and conduct risk-based valuation to generate a probabilistic range of net-present values (NPVs). For a specific ten-year product portfolio, the ICB platform reduces average cost by 55% compared to conventional batch processing, considering both capital and operating expenses. The model predicts that these savings can further increase by an additional 25% in situations with higher-than-expected product demand showing the upward potential of the ICB platform. The ICB platform achieves these savings and corresponding flexibility mainly due to process intensification in both upstream and downstream unit operations. This study demonstrates the promise of continuous bioprocessing while also establishing a novel framework to quantify financial benefits of other platform process technologies.
Assuntos
Indústria Farmacêutica/economia , Modelos Teóricos , Proteínas Recombinantes/economia , Reatores Biológicos/economia , Biotecnologia/economia , Comércio , Método de Monte Carlo , RiscoRESUMO
Downstream sample purification for quality attribute analysis is a significant bottleneck in process development for non-antibody biologics. Multi-step chromatography process train purifications are typically required prior to many critical analytical tests. This prerequisite leads to limited throughput, long lead times to obtain purified product, and significant resource requirements. In this work, immunoaffinity purification technology has been leveraged to achieve single-step affinity purification of two different enzyme biotherapeutics (Fabrazyme® [agalsidase beta] and Enzyme 2) with polyclonal and monoclonal antibodies, respectively, as ligands. Target molecules were rapidly isolated from cell culture harvest in sufficient purity to enable analysis of critical quality attributes (CQAs). Most importantly, this is the first study that demonstrates the application of predictive analytics techniques to predict critical quality attributes of a commercial biologic. The data obtained using the affinity columns were used to generate appropriate models to predict quality attributes that would be obtained after traditional multi-step purification trains. These models empower process development decision-making with drug substance-equivalent product quality information without generation of actual drug substance. Optimization was performed to ensure maximum target recovery and minimal target protein degradation. The methodologies developed for Fabrazyme were successfully reapplied for Enzyme 2, indicating platform opportunities. The impact of the technology is significant, including reductions in time and personnel requirements, rapid product purification, and substantially increased throughput. Applications are discussed, including upstream and downstream process development support to achieve the principles of Quality by Design (QbD) as well as integration with bioprocesses as a process analytical technology (PAT).
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Cromatografia de Afinidade/métodos , Isoenzimas/química , Proteínas/isolamento & purificação , alfa-Galactosidase/química , Anticorpos Monoclonais/imunologia , Técnicas de Cultura de Células , Humanos , Isoenzimas/metabolismo , Proteínas/química , Proteólise , alfa-Galactosidase/metabolismoRESUMO
Integrated and continuous processing of recombinant proteins offers several advantages over batch or semi-batch processing used traditionally in the biotechnology industry. This paper presents a theoretical and practical approach for designing a periodic counter-current chromatography (PCC) operation as a continuous capture purification step that is integrated with a perfusion cell culture process. The constraints for continuous and optimal PCC operation govern the selection of residence time and number of columns. The flexibility available in PCC design for selection of these parameters is dictated by the binding characteristics of the target protein on the capture resin. Using an empirical model for the protein breakthrough curve, analytical solutions to determine these conditions were derived and verified with experimental results for three different proteins: two relatively unstable proteins (recombinant enzymes) and a relatively stable protein (monoclonal antibody). The advantages of a continuous downstream capture step are highlighted for the three case studies in comparison with the existing batch chromatography processes. The use of PCC leads to improvements in process economics due to higher resin capacity utilization and correspondingly lower buffer consumption. Furthermore, integrated and continuous bioprocessing results in a smaller facility footprint by elimination of harvest hold vessels and clarification, as well as by reducing the capture column size by one to two orders of magnitude.
Assuntos
Biotecnologia/métodos , Distribuição Contracorrente/métodos , Proteínas Recombinantes/isolamento & purificação , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Células CHO , Distribuição Contracorrente/instrumentação , Cricetinae , Cricetulus , Enzimas/química , Enzimas/isolamento & purificação , Proteínas Recombinantes/química , Projetos de PesquisaRESUMO
Macroscopic characterizations of hydrophobicity (e.g., contact angle measurements) do not extend to the surfaces of proteins and nanoparticles. Molecular measures of hydrophobicity of such surfaces need to account for the behavior of hydration water. Theory and state-of-the-art simulations suggest that water density fluctuations provide such a measure; fluctuations are enhanced near hydrophobic surfaces and quenched with increasing surface hydrophilicity. Fluctuations affect conformational equilibria and dynamics of molecules at interfaces. Enhanced fluctuations are reflected in enhanced cavity formation, more favorable binding of hydrophobic solutes, increased compressibility of hydration water, and enhanced water-water correlations at hydrophobic surfaces. These density fluctuation-based measures can be used to develop practical methods to map the hydrophobicity/philicity of heterogeneous surfaces including those of proteins. They highlight that the hydrophobicity of a group is context dependent and is significantly affected by its environment (e.g., chemistry and topography) and especially by confinement. The ability to include information about hydration water in mapping hydrophobicity is expected to significantly impact our understanding of protein-protein interactions as well as improve drug design and discovery methods and bioseparation processes.
Assuntos
Proteínas/química , Água/química , Bases de Dados Factuais , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína , Temperatura , MolhabilidadeRESUMO
Guanidinium chloride (GdmCl) is a widely used chemical denaturant that unfolds proteins. Its effects on hydrophobic interactions are, however, not fully understood. We quantify the effects of GdmCl on various manifestations of hydrophobicity--from solvation and interactions of small solutes to folding-unfolding of hydrophobic polymers--in water and in concentrated GdmCl solutions. For comparison, we also perform similar calculations in solutions of NaCl and CsCl in water. Like NaCl and CsCl, GdmCl increases the surface tension of water, decreases the solubility of small hydrophobic solutes, and enhances the strength of hydrophobic interactions at the pair level. However, unlike NaCl and CsCl, GdmCl destabilizes folded states of hydrophobic polymers. We show that Gdm(+) ions preferentially coat the hydrophobic polymer, and it is the direct van der Waals interaction between Gdm(+) ions and the polymer that contributes to the destabilization of folded states. Interestingly, the temperature dependence of the free energy of unfolding of the hydrophobic polymer in water is protein-like, with signatures of both heat and cold denaturation. Addition of GdmCl shifts the cold denaturation temperature higher, into the experimentally accessible region. Finally, translational as well as conformational dynamics of the polymer are slower in GdmCl and correlate with dynamics of water molecules in solution.
Assuntos
Guanidina/química , Polímeros/química , Interações Hidrofóbicas e Hidrofílicas , Soluções , Temperatura , Termodinâmica , Água/químicaRESUMO
We quantify the strength of interfacial thermal coupling at water-solid interfaces over a broad range of surface chemistries from hydrophobic to hydrophilic using molecular simulations. We show that the Kapitza conductance is proportional to the work of adhesion-a wetting property of that interface-enabling the use of thermal transport measurements as probes of the molecular environment and bonding at an interface. Excellent agreement with experiments on similar systems [Z. B. Ge, Phys. Rev. Lett. 96, 186101 (2006)10.1103/PhysRevLett.96.186101] highlights the convergence of simulation and experiments on these complex nanoscopic systems.