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1.
J Sep Sci ; 2020 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-32187844

RESUMO

The baculovirus expression vector system is a very powerful tool to produce virus-like particles and gene-therapy vectors, but the removal of co-expressed baculovirus has been a major barrier for wider industrial use. We used chimeric HIV-1 gag influenza-HA VLPs produced in Tnms42 insect cells using the baculovirus insect cell expression vector system as model VLPs. A fast and simple purification method for these VLPs with direct capture and purification within one chromatography step was developed. The insect cell culture supernatant was treated with endonuclease and filtered, before it was directly loaded onto a polymer grafted anion exchanger and eluted by a linear salt gradient. A 4.3 log clearance of baculovirus from VLPs was achieved. The absence of the baculovirus capsid protein (vp39) in the product fraction was additionally shown by HPLC-MS. When considering a vaccination dose of 109 particles, 4200 doses can be purified per L pre-treated supernatant, meeting the requirements for vaccines with <10 ng dsDNA/dose and 3.4 µg protein/dose in a single step. The process is simple with a very low number of handling steps and has the characteristics to become a platform for purification of these types of VLPs. This article is protected by copyright. All rights reserved.

2.
Biotechnol Bioeng ; 2020 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-32017010

RESUMO

Continuous virus inactivation (VI) has received little attention in the efforts to realize fully continuous biomanufacturing in the future. Implementation of continuous VI must assure a specific minimum incubation time, typically 60 min. To guarantee the minimum incubation time, we implemented a packed bed continuous viral inactivation reactor (CVIR) with narrow residence time distribution (RTD) for low pH incubation. We show that the RTD does not broaden significantly over a wide range of linear flow velocities-which highlights the flexibility and robustness of the design. Prolonged exposure to acidic pH has no impact on bed stability, assuring constant RTD throughout long term operation. The suitability of the packed bed CVIR for low pH inactivation is shown with two industry-standard model viruses, that is xenotropic murine leukemia virus and pseudorabies virus. Controls at neutral pH showed no system-induced VI. At low pH, significant VI is observed, even after only 15 min. Based on the low pH inactivation kinetics, the continuous process is equivalent to traditional batch operation. This study establishes a concept for continuous low pH inactivation and, together with previous reports, highlights the versatility of the packed bed reactor for continuous VI, regardless of the inactivation method.

3.
J Chromatogr A ; : 460856, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31959462

RESUMO

A model-based approach for scaling up chromatographic capture step was developed. The purification of human basic fibroblast growth factor protein 2 (FGF2) from an E. coli homogenate on a cation exchange resin was selected as a case study. Non-ideal effects accompanying the capture operation were examined, including: reduction in the protein diffusivity in the presence of the homogenate, competitive adsorption between FGF2 and undefined impurities, and flow behavior in external column volumes. The viscosity of the homogenate was measured as a function of dilution degree and shear stress, and its contribution to the diffusivity reduction was quantified. A dynamic model was formulated which accounted for underlying kinetic and thermodynamic dependencies. The model parameters were determined for a lab scale system using a small 2-mL column. The model was successfully used to scale up the capture operation from the lab scale column to a preparative bench scale column of about 1 L volume.

4.
N Biotechnol ; 55: 98-107, 2020 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-31629875

RESUMO

A narrow residence time distribution (RTD) is highly desirable for continuous processes where a strict incubation time must be ensured, such as continuous virus inactivation. A narrow RTD also results in faster startup and shut down phases and limits the broadening of potential disturbances in continuous processes. A packed bed reactor with non-porous inert beads was developed to achieve narrow RTDs. The performance was defined as the ratio between the onset of the cumulative RTD and the median residence time (tx%/t50%). Laboratory-scale packed columns were used to study the influence of the column parameters on the RTD. A larger column with a void volume of 0.65 L and a length of 89 cm, packed with beads in a size range of 125 to 250 µm, achieved t0.5%/t50% >0.93 across flow rates from 0.1 to 9.8 mL/min. The RTD was significantly narrower than the RTDs of other reactor designs, such as the Coiled Flow Inverter and Jig in a Box. The pressure drop remained under 3 kPa for all tested flow rates. Fluorescent nanoparticles (30 and 200 nm) were used to mimic viruses. These two sizes showed less than 2% difference in terms of t1%/t50% and t0.01%/t50% scores. These results indicated that viruses travelled through the column at rates independent of size. This proposal of packed beds as incubation chambers for continuous virus inactivation is simple, scalable, and can be realized as single-use devices. Due to the low pressure drop, the system can be easily integrated into a fully continuous process.

5.
Biotechnol Prog ; : e2928, 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31622530

RESUMO

Antibodies of the IgG2 subclass were captured from the clarified cell culture fluid either by protein A chromatography or by polyethylene glycol precipitation. The captured intermediates were stored as neutralized eluates (protein A chromatography) or in solid form as polyethylene glycol precipitates over a period of 13 months at three temperatures, -20°C, 5°C, and room temperature to compare the capture technologies in regard of the resulting product storability. Monomer content, high molecular mass impurities product loss and changes in the composition of the charge variants were determined at six time points during the storage. At the beginning and end of the study, samples were additionally tested by differential scanning calorimetry, differential scanning fluorimetry, and circular dichroism to determine structural alterations occurring during storage. Protein A purified material was highly stable at all tested temperatures in regard of monomer content and product losses. A transient, acidic isoform was formed during the chromatography step which re-converted to the main charged variant upon storage within a matter of days. Precipitated antibodies could be stored at -20 or 5°C for 3 months without product losses but afterwards recovery yields dropped to 65%. At room temperature, the precipitated antibody was not stable and degraded within 3 months.

6.
Vaccine ; 37(47): 7070-7080, 2019 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-31300289

RESUMO

Polymer-grafted chromatography media, especially ion exchangers, are high performance materials for protein purification. However, due to the pore size limitation, conventional chromatography beads are usually not considered for the downstream processing of large biomolecules such as virus-like particles (VLPs). Contrariwise, since the outer surface of the chromatography beads provides satisfactory binding capacity for VLPs and impurities of smaller size can bind inside of the beads, conventional porous beads should be considered for VLP capture and purification. We used HIV-1 gag VLPs with a diameter of 100-200 nm as a model to demonstrate that polymer-grafted anion exchangers are suitable for the purification of bionanoparticles. The equilibrium binding capacity was 1 × 1013 part/mL resin. Moderate salt concentration up to 100 mM NaCl did not affect binding, allowing direct loading of cell culture supernatant onto the column for purification. Dynamic binding capacity at 10% breakthrough, when loading cell culture supernatant, was approximately 6 × 1011 part/mL column; only 1-log lower than for monoliths. Endonuclease treatment of the cell culture supernatant did not increase the dynamic binding capacity, suggesting that dsDNA does not compete for the binding sites of VLPs. Nevertheless, due to simultaneous elution of particles and dsDNA, endonuclease treatment is required to reduce dsDNA contamination in the product. Proteomic analysis revealed that HIV-1 gag VLPs contain different host cell proteins in their cargo. This cargo is composed of conserved proteins and other proteins that vary from one particle population to another, as well as from batch to batch. This process allowed the separation of different particle populations. HIV-1 gag VLPs were directly captured and purified from cell culture supernatant with a total particle recovery in the elution of about 35%. Columns packed with beads can be scaled to practically any dimension and therefore a tailored design of the process is possible.

7.
J Sep Sci ; 42(16): 2640-2649, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31169979

RESUMO

At-line static light scattering and fluorescence monitoring allows direct in-process tracking of fluorescent virus-like particles. We have demonstrated this by coupling at-line multi-angle light scattering and fluorescence detectors to the downstream processing of enveloped virus-like particles. Since light scattering intensity is directly proportional to particle concentration, our strategy allowed a swift identification of product containing fractions and rapid process development. Virus-like particles containing the Human Immunodeficiency Virus-1 Gag protein fused to the Green Fluorescence protein were produced in Human Embryonic Kidney 293 cells by transient transfection. A single-column anion-exchange chromatography method was used for direct capture and purification. The majority of host-cell protein impurities passed through the column without binding. Virus-like particles bound to the column were eluted by linear or step salt gradients. Particles recovered in the step gradient purification were characterized by nanoparticle tracking analysis, size exclusion chromatography coupled to multi-angle light scattering and fluorescence detectors and transmission electron microscopy. A total recovery of 66% for the fluorescent particles was obtained with a 50% yield in the main product peak. Virus-like particles were concentrated 17-fold to final a concentration of 4.45 × 1010 particles/mL. Simple buffers and operation make this process suitable for large scale purposes.


Assuntos
Luz , Vírion/isolamento & purificação , Produtos do Gene gag do Vírus da Imunodeficiência Humana/isolamento & purificação , Células Cultivadas , Cromatografia , Células HEK293 , Humanos , Nanopartículas/química , Espalhamento de Radiação , Vírion/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química
8.
J Chromatogr A ; 1599: 55-65, 2019 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-31036361

RESUMO

Peak broadening in small columns is dominated by spreading in the extra column volume and not by hydrodynamic dispersion or mass transfer resistances. Computational fluid dynamics (CFD) permits to study the influence of these effects separately. Here, peak broadening of three single component solutes - silica nanoparticles, acetone, and lysozyme - was experimentally determined for two different columns (100 mm × 8 mm inner diameter and 10 mm × 5 mm inner diameter) under non-binding conditions. A mass transfer model between mobile and stationary phases as well as a hydrodynamic dispersion model were implemented in the CFD environment STAR-CCM+®. The mass transfer model combines a model of external mass transfer with a model of pore diffusion. The model was validated with experiments performed on the larger column. We find that extra column volume plays an important role in peak broadening of the silica nanoparticles pulse in that column; it is less important for acetone and is weakly pronounced for lysozyme. Hydrodynamic dispersion plays the dominant role at low and medium flow rates for acetone because we are in a regime of 1-10 ReSc. Mass transfer is important for high flow rates of acetone and for all flow rates of lysozyme. Then, peak broadening was predicted in the smaller column with the packed bed parameters taken from larger column. The scalability of the prepacked columns is demonstrated for acetone and silica nanoparticles by excellent agreement with the experimental data. In contrast to the larger column, peak broadening in the smaller column is dominated by extra column volume for all solutes. Peak broadening of lysozyme is controlled only at high flow rates by mass transfer and overrides extra column volume and hydrodynamic dispersion. CFD simulations with implemented mass transfer models successfully model peak broadening in chromatography columns taking all broadening effects into consideration and therefore are a valuable tool for scale up and scale down. Our simulations underscore the importance of extra column volume.


Assuntos
Cromatografia , Modelos Químicos , Acetona/química , Simulação por Computador , Difusão , Muramidase/química , Dióxido de Silício/química
9.
Biotechnol J ; 14(7): e1800521, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30945440

RESUMO

Regulatory recommendations for quality by design instead of quality by testing raise increasing interest in new sensor technologies. An online monitoring system for downstream processes is developed, which is based on an array of online detectors. Besides standard detectors (UV, pH, and conductivity), our chromatographic workstation is equipped with a fluorescence and a mid-infrared spectrometer, a light scattering, and a refractive index detector. The combination of these sensors enables the prediction of specific protein concentration and various purity attributes, such as high molecular weight impurities, DNA and host cell protein content during the elution phase of a chromatographic antibody capture process. Prediction models solely based on online signals are set up providing real-time predictions. No mechanistic models or information about the chromatographic runs is used. These predictions allow online pooling decisions replacing time- and labor-intensive laboratory measurements. Different process variations, such as changes in the column load or elution buffer, are introduced to test the predictive power of the models. Extrapolation of the models worked well when the column load is changed, whereas model adjustment is necessary when the elution conditions are changed considerably.


Assuntos
Anticorpos Monoclonais/análise , Anticorpos Monoclonais/isolamento & purificação , Cromatografia Líquida de Alta Pressão/métodos , Espectrofotometria Infravermelho/métodos , Animais , Anticorpos Monoclonais/química , Células CHO , Cricetinae , Cricetulus , Modelos Estatísticos
10.
Biotechnol J ; 14(8): e1800632, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30945463

RESUMO

Protein A affinity chromatography is a core unit operation in antibody manufacturing. Nevertheless, there is not enough understanding of in-column antibody adsorption in the Protein A capture step. This work aims to investigate in situ the establishment of an antibody (trastuzumab) layer during Protein A chromatography both in terms of energetic contributions and uptake kinetics. Flow microcalorimetry is employed as a technique with an in situ operating detector, which provides an understanding of the thermodynamics of the adsorption process. In addition, the antibody uptake rate is also investigated in order to establish a correlation between its diffusion on the stationary phase and the associated thermodynamics. Two resins with different particle size, intraparticle porosity, and a Protein A ligand structure are studied: the synthetically engineered B-domain tetrameric MabSelect SuRe and the synthetically engineered C-domain hexameric TOYOPEARL AF-rProtein A HC. The uptake rate follows a pore diffusion model at low equilibrium time, showing a slower diffusivity after a certain time because of the heterogeneous binding nature of these two resins. In addition, the microcalorimetric studies show that adsorption enthalpy is highly favourable at low isotherm concentrations and evolves toward an equilibrium with increasing surface concentration. These data suggest that the relationship between adsorption enthalpy and the establishment of the antibody layer in the Protein A chain is consistent with heterogeneous adsorption.


Assuntos
Anticorpos/metabolismo , Proteína Estafilocócica A/metabolismo , Resinas de Troca de Ânions , Sítios de Ligação , Calorimetria , Cromatografia de Afinidade/métodos , Cinética , Ligantes , Trastuzumab/metabolismo
11.
Biotechnol Bioeng ; 116(8): 1999-2009, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30934111

RESUMO

Process analytical technology combines understanding and control of the process with real-time monitoring of critical quality and performance attributes. The goal is to ensure the quality of the final product. Currently, chromatographic processes in biopharmaceutical production are predominantly monitored with UV/Vis absorbance and a direct correlation with purity and quantity is limited. In this study, a chromatographic workstation was equipped with additional online sensors, such as multi-angle light scattering, refractive index, attenuated total reflection Fourier-transform infrared, and fluorescence spectroscopy. Models to predict quantity, host cell proteins (HCP), and double-stranded DNA (dsDNA) content simultaneously were developed and exemplified by a cation exchange capture step for fibroblast growth factor 2 expressed in Escherichia coliOnline data and corresponding offline data for product quantity and co-eluting impurities, such as dsDNA and HCP, were analyzed using boosted structured additive regression. Different sensor combinations were used to achieve the best prediction performance for each quality attribute. Quantity can be adequately predicted by applying a small predictor set of the typical chromatographic workstation sensor signals with a test error of 0.85 mg/ml (range in training data: 0.1-28 mg/ml). For HCP and dsDNA additional fluorescence and/or attenuated total reflection Fourier-transform infrared spectral information was important to achieve prediction errors of 200 (2-6579 ppm) and 340 ppm (8-3773 ppm), respectively.

12.
Biotechnol J ; 14(8): e1800646, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30810288

RESUMO

Continuous virus inactivation (VI) remains one of the missing pieces while the biopharma industry moves toward continuous manufacturing. The challenges of adapting VI to the continuous operation are two-fold: 1) achieving fluid homogeneity and 2) a narrow residence time distribution (RTD) for fluid incubation. To address these challenges, a dynamic active in-line mixer and a packed-bed continuous virus inactivation reactor (CVIR) are implemented, which act as a narrow RTD incubation chamber. The developed concept is applied using solvent/detergent (S/D) treatment for inactivation of two commonly used model viruses. The in-line mixer is characterized and enables mixing of the viscous S/D chemicals to ±1.0% of the target concentration in a small dead volume. The reactor's RTD is characterized and additional control experiments confirm that the VI is due to the S/D action and not induced by system components. The CVIR setup achieves steady state rapidly before two reactor volumes and the logarithmic reduction values of the continuous inactivation process are identical to those obtained by the traditional batch operation. The packed-bed reactor for continuous VI unites fully continuous processing with very low-pressure drop and scalability.


Assuntos
Biotecnologia/instrumentação , Biotecnologia/métodos , Solventes , Inativação de Vírus , Animais , Vírus da Diarreia Viral Bovina/patogenicidade , Desenho de Equipamento , Cinética , Vírus da Leucemia Murina/patogenicidade
13.
Prep Biochem Biotechnol ; 49(1): 1-20, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30735098

RESUMO

Hydrophobic interaction chromatography is a very popular chromatography method for purification of proteins and plasmids in all scales from analytical to industrial manufacturing. Despite this frequent use, the complex interaction mechanism and the thermodynamic aspects of adsorption in hydrophobic interaction chromatography are still not well understood. Calorimetric methods such as isothermal titration calorimetry and flow calorimetry can help to gain a deeper understanding of the adsorption strength, the influence of salt type and temperature. They can be used to study conformational changes of proteins, which are often associated with the adsorption in hydrophobic interaction chromatography. This review offers a detailed introduction into the thermodynamic fundamentals of adsorption in hydrophobic interaction chromatography with a special focus on the potential applications of isothermal titration calorimetry and flow calorimetry for studying specific problems and relationships of the adsorption behavior of proteins and its various influencing factors. Models for characterizing conformational changes upon adsorption are presented together with methods for assessing this problem for different proteins and stationary phases. All of this knowledge can contribute greatly to forming a sound basis for method development, process optimization and finding modelling strategies in hydrophobic interaction chromatography.


Assuntos
Calorimetria/métodos , Cromatografia Líquida/métodos , Proteínas/química , Adsorção , Interações Hidrofóbicas e Hidrofílicas , Termodinâmica
14.
Biotechnol Bioeng ; 116(5): 1053-1065, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30636284

RESUMO

Continuous precipitation is a new unit operation for the continuous capture of antibodies. The capture step is based on continuous precipitation with PEG6000 and Zn++ in a tubular reactor integrated with a two-stage continuous tangential flow filtration unit. The precipitate cannot be separated with centrifugation, because a highly compressed sediment results in poor resolubilization. We developed a new two-stage tangential flow microfiltration method, where part of the concentrated retentate of the first stage was directly fed to the second stage, together with the wash buffer. Thus, the precipitate was concentrated and washed in a continuous process. We obtained 97% antibody purity, a 95% process yield during continuous operation, and a fivefold reduction in pre-existing high-molecular-weight impurities. For other unit operations, surge tanks are often required, due to interruptions in the product mass flow out of the unit operation (e.g., the bind/elute mode in periodic counter-current chromatography). Our setup required no surge tanks; thus, it provided a truly continuous antibody capture operation with uninterrupted product mass flow. Continuous virus inactivation and other flow-through unit operations can be readily integrated downstream of the capture step to create truly continuous, integrated, downstream antibody processing without the need for hold tanks.

15.
J Chromatogr A ; 1591: 79-86, 2019 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-30661762

RESUMO

Pre-packed chromatography columns are routinely used in downstream process development and scale-down studies. In recent years they have also been widely adopted for large scale, cGMP manufacturing of biopharmaceuticals. Despite columns being qualified at their point of manufacture before release for sale, the suitability of pre-packed chromatography columns for protein separations at different scales has not yet been demonstrated. In this study, we demonstrated that the performance results obtained with small scale columns (0.5 cm diameter × 5 cm length, 1 mL column volume) are scalable to production sized columns (60 cm diameter × 20 cm length, 57 L column volume). The columns were characterized with acetone and blue dextran pulses to determine the packing density and packed bed consistency. Chromatography performance was evaluated with breakthrough curves including capacity measurements and with separation of a ternary protein mixture (lysozyme, cytochrome C and RNase A) with a step gradient. The equilibrium binding capacity and dynamic binding capacity were equivalent for all columns. The step gradient separation of the ternary protein mixture displayed similar peak profiles when normalized in respect to column volume and the eluted protein pools had the same purities for all scales. Scalable performance of pre-packed columns is demonstrated but as with conventionally packed columns the influence of extra column volume and system configurations, especially buffer mixing, must be taken into account when comparing separations at different scales.


Assuntos
Cromatografia/métodos , Indústrias , Acetona/análise , Citocromos c/metabolismo , Condutividade Elétrica , Muramidase/metabolismo , Ligação Proteica , Ribonuclease Pancreático/metabolismo
16.
J Chromatogr A ; 1588: 77-84, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30616980

RESUMO

Separation of enveloped virus-like particles from other extracellular vesicles is a challenging separation problem due to the similarity of these bionanoparticles. Without simple and scalable methods for purification and analytics, it is difficult to gain deeper insight into their biological function. A two-step chromatographic purification method was developed. In the first step, virus-like particles and extracellular vesicles were collected and separated from smaller impurities in a flow-through mode. Benzonase® treated HEK 293 cell culture supernatant was directly loaded onto a column packed with core-shell beads. The collected flow-through was further purified using heparin affinity chromatography. In heparin affinity chromatography 54% of the total particle load were found in the flow-through, and 15% of the particles were eluted during the salt linear gradient. The particle characterization, especially particle size distribution and mass spectrometry data, suggests that extracellular vesicles dominate the flow-through fraction and HIV-1 gag VLPs are enriched in the elution peak. This is in part in contradiction to other protocols where the extracellular vesicles are recovered by binding to heparin affinity chromatography. The developed method is easily scalable to pilot and process scale and allows a fast accomplishment of this separation within one day.


Assuntos
Técnicas de Química Analítica/métodos , Cromatografia de Afinidade , Vesículas Extracelulares/química , Heparina/química , Vírion/isolamento & purificação , Células HEK293 , HIV-1/isolamento & purificação , Humanos
17.
Biotechnol Bioeng ; 116(1): 76-86, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30252938

RESUMO

Staphylococcal protein A chromatography is an established core technology for monoclonal antibody purification and capture in the downstream processing. MabSelect SuRe involves a tetrameric chain of a recombinant form of the B domain of staphylococcal protein A, called the Z-domain. Little is known about the stoichiometry, binding orientation, or preferred binding. We analyzed small-angle X-ray scattering data of the antibody-protein A complex immobilized in an industrial highly relevant chromatographic resin at different antibody concentrations. From scattering data, we computed the normalized radial density distributions. We designed three-dimensional (3D) models with protein data bank crystallographic structures of an IgG1 (the isoform of trastuzumab, used here; Protein Data Bank: 1HZH) and the staphylococcal protein A B domain (the native form of the recombinant structure contained in MabSelect SuRe resin; Protein Data Bank: 1BDD). We computed different binding conformations for different antibody to protein A stoichiometries (1:1, 2:1, and 3:1) and compared the normalized radial density distributions computed from 3D models with those obtained from the experimental data. In the linear range of the isotherm we favor a 1:1 ratio, with the antibody binding to the outer domains in the protein A chain at very low and high concentrations. In the saturation region, a 2:1 ratio is more likely to occur. A 3:1 stoichiometry is excluded because of steric effects.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Cromatografia de Afinidade/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteína Estafilocócica A/química , Proteína Estafilocócica A/metabolismo , Imunoglobulina G/química , Imunoglobulina G/isolamento & purificação , Ligação Proteica , Conformação Proteica , Espalhamento a Baixo Ângulo
18.
Protein Expr Purif ; 153: 70-82, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30130579

RESUMO

A two-step purification process for human basic fibroblast growth factor (FGF-2) from clarified E. coli homogenate has been developed in which the impurity level after the second step is below the limit of quantification. Endotoxin content is cleared to 0.02 EU/µg FGF-2 and the overall yield is 67%. The performance of the cation exchanger Carboxymethyl-Sepharose Fast Flow (CM-SFF) was compared to the affinity resin Heparin-SFF regarding the impurity profile and product quality in the elution peak. The CM-SFF eluate was further purified using hydrophobic interaction resin Toyopearl-Hexyl-650C. The relative amounts of target product, host cell proteins (HCPs), dsDNA, endotoxin, monomer content, and high molecular weight impurities differed along the elution peak depending on the applied method. The bioactive monomer (>99%) was obtained with a yield of 48% for CM-SFF and 68% for Heparin-SFF. A half-load reduction in CM-SFF increased the yield up to 67% without deterioration of the impurity content. Assuming a dose of 400 µg FGF-2, endotoxin was reduced to 188 EU/dose, dsDNA <10 ng/dose, and HCP <2 ppm/dose using the cation exchanger. In the pooled eluate fractions, dsDNA was removed 4-fold (291 ng/mL) and endotoxin 14-fold (0.47 EU/µg FGF-2) more efficiently by CM-SFF than by affinity chromatography. In contrast, HCP clearance was 3-fold (13 ppm) more efficient with Heparin-SFF than CM-SFF. In contrast to process monitoring by UV280nm or SDS-PAGE, this characterization is the basis for a Process Analytical Technology attempt when correlated with online monitored signals, as it enables knowledge-based pooling according to defined quality criteria.


Assuntos
Cromatografia de Afinidade/métodos , Cromatografia por Troca Iônica/métodos , Endotoxinas/isolamento & purificação , Fator 2 de Crescimento de Fibroblastos/isolamento & purificação , Animais , Sobrevivência Celular/efeitos dos fármacos , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Heparina/química , Humanos , Camundongos , Células NIH 3T3 , Polímeros/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Sefarose/química
19.
Biotechnol J ; 14(2): e1800278, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30207077

RESUMO

To realize continuous integrated manufacturing of biopharmaceuticals, all steps of production have to be operated in a continuous mode. Virus inactivation is a crucial step in this process, and due to standard procedures and a fixed residence time, it is particularly challenging to realize. Two articles in this issue present possible solutions to this problem.


Assuntos
Reatores Biológicos , Inativação de Vírus
20.
Biotechnol J ; 14(4): e1800340, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30315690

RESUMO

Biosimilars are increasing in economic importance. Just how similar a biosimilar needs to be to gain market approval is currently still decided on a per case basis. The authors try to shed light on one often cited critical quality attribute of monoclonal antibodies, namely charge heterogeneity. Using high resolution electrophoretic and chromatographic methods, the authors are able to separate and quantify the charge variant content of infliximab originator and three biosimilars. Additionally the authors quantified and compared the antigen binding affinity in an SPR based binding assay and analyzed the glycosylation pattern of all four of these infliximab biosimilar products. Even though the analytical methods did not show full similarity between originator and some biosimilars, all of the biosimilars have gained approval based on their clinical comparability. The authors would therefore argue, that analytical comparison is not always a good predictor for clinical interchangeability. Any future regulatory framework for the approval of biosimilars should reflect that the parameters chosen for analytical comparability have to be chosen carefully.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Medicamentos Biossimilares/química , Infliximab/imunologia , Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Antígenos/uso terapêutico , Medicamentos Biossimilares/uso terapêutico , Glicosilação , Humanos , Infliximab/uso terapêutico
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