RESUMO
Detergent-mediated virus inactivation (VI) provides a valuable orthogonal strategy for viral clearance in mammalian processes, in particular for next-generation continuous manufacturing. Furthermore, there exists an industry-wide need to replace the conventionally employed detergent Triton X-100 with eco-friendly alternatives. However, given Triton X-100 has been the gold standard for VI due its minimal impact on protein stability and high inactivation efficacy, inactivation by other eco-friendly detergents and its impact on protein stability is not well understood. In this study, the sugar-based detergent commonly used in membrane protein purification, n-dodecyl-ß- d-maltoside was found to be a promising alternative for VI. We investigated a panel of detergents to compare the relative VI efficacy, impact on therapeutic quality attributes, and clearance of the VI agent and other impurities through subsequent chromatographic steps. Detergent-mediated inactivation and protein stability showed comparable trends to low pH inactivation. Using experimental and modeling data, we found detergent-mediated product aggregation and its kinetics to be driven by extrinsic factors such as detergent and protein concentration. Detergent-mediated aggregation was also impacted by an initial aggregation level as well as intrinsic factors such as the protein sequence and detergent hydrophobicity, and critical micelle concentration. Knowledge gained here on factors driving product stability and VI provides valuable insight to design, standardize, and optimize conditions (concentration and duration of inactivation) for screening of detergent-mediated VI.
Assuntos
Produtos Biológicos , Inativação de Vírus , Animais , Detergentes/química , Cinética , Mamíferos , Octoxinol/química , Estabilidade ProteicaRESUMO
Process analytical technology (PAT) is a fast-growing field within bioprocessing that enables innovation in biological drug manufacturing. This study demonstrates novel PAT methods for monitoring multiple quality attributes simultaneously during the ultrafiltration and diafiltration (UF/DF) process operation, the final step of monoclonal antibody (mAb) purification. Size exclusion chromatography (SEC) methods were developed to measure excipients arginine, histidine, and high molecular weight (HMW) species using a liquid chromatography (LC) system with autosampler for both on-line and at-line PAT modes. The methods were applied in UF/DF studies for the comparison of single-use tangential flow filtration (TFF) cassettes to standard reusable cassettes to achieve very high concentration mAb drug substance (DS) in the order of 100-200 g/L. These case studies demonstrated that single-use TFF cassettes are a functionally equivalent, low-cost alternative to standard reusable cassettes, and that the on-line PAT measurement of purity and excipient concentration was comparable to orthogonal offline methods. These PAT applications using an on-line LC system equipped with onboard sample dilution can become a platform system for monitoring of multiple attributes over a wide dynamic range, a potentially valuable tool for biological drug development and manufacturing.
Assuntos
Anticorpos Monoclonais/biossíntese , Ultrafiltração , Arginina , Cromatografia Líquida de Alta Pressão , Excipientes/química , Histidina , Tecnologia , Ultrafiltração/instrumentaçãoRESUMO
To achieve the high protein concentrations required for subcutaneous administration of biologic therapeutics, numerous manufacturing process challenges are often encountered. From an operational perspective, high protein concentrations result in highly viscous solutions, which can cause pressure increases during ultrafiltration. This can also lead to low flux during ultrafiltration and sterile filtration, resulting in long processing times. In addition, there is a greater risk of product loss from the hold-up volumes during filtration operations. From a formulation perspective, higher protein concentrations present the risk of higher aggregation rates as the closer proximity of the constituent species results in stronger attractive intermolecular interactions and higher frequency of self-association events. There are also challenges in achieving pH and excipient concentration targets in the ultrafiltration/diafiltration (UF/DF) step due to volume exclusion and Donnan equilibrium effects, which are exacerbated at higher protein concentrations. This paper highlights strategies to address these challenges, including the use of viscosity-lowering excipients, appropriate selection of UF/DF cassettes with modified membranes and/or improved flow channel design, and increased understanding of pH and excipient behavior during UF/DF. Additional considerations for high-concentration drug substance manufacturing, such as appearance attributes, stability, and freezing and handling are also discussed. These strategies can be employed to overcome the manufacturing process challenges and streamline process development efforts for high-concentration drug substance manufacturing.
Assuntos
Anticorpos Monoclonais , Composição de Medicamentos/métodos , Infusões Subcutâneas , Ultrafiltração/métodos , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , ViscosidadeRESUMO
Technologies capable of monitoring product quality attributes and process parameters in real time are becoming popular due to the endorsement of regulatory agencies and also to support the agile development of biotherapeutic pipelines. The utility of vibrational spectroscopic techniques such as Fourier transform mid-infrared (Mid-IR) and multivariate data analysis (MVDA) models allows the prediction of multiple critical attributes simultaneously in real time. This study reports the use of Mid-IR and MVDA model sensors for monitoring of multiple attributes (excipients and protein concentrations) in real time (measurement frequency of every 40 s) at ultrafiltration and diafiltration (UF/DF) unit operation of biologics manufacturing. The platform features integration of fiber optic Mid-IR probe sensors to UF/DF set up at the bulk solution and through a flow cell at the retentate line followed by automated Mid-IR data piping into a process monitoring software platform with pre-loaded partial least square regression (PLS) chemometric models. Data visualization infrastructure is also built-in to the platform so that upon automated PLS prediction of excipients and protein concentrations, the results were projected in a graphical or numerical format in real time. The Mid-IR predicted concentrations of excipients and protein show excellent correlation with the offline measurements by traditional analytical methods. Absolute percent difference values between Mid-IR predicted results and offline reference assay results were ≤5% across all the excipients and the protein of interest; which shows a great promise as a reliable process analytical technology tool.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Espectroscopia de Infravermelho com Transformada de Fourier , UltrafiltraçãoRESUMO
Despite growing interest in light-driven ion pumps for use in optogenetics, current estimates of their transport rates span two orders of magnitude due to challenges in measuring slow transport processes and determining protein concentration and/or orientation in membranes in vitro. In this study, we report, to our knowledge, the first direct quantitative measurement of light-driven Cl- transport rates of the anion pump halorohodopsin from Natronomonas pharaonis (NpHR). We used light-interfaced voltage clamp measurements on NpHR-expressing oocytes to obtain a transport rate of 219 (± 98) Cl-/protein/s for a photon flux of 630 photons/protein/s. The measurement is consistent with the literature-reported quantum efficiency of â¼30% for NpHR, i.e., 0.3 isomerizations per photon absorbed. To reconcile our measurements with an earlier-reported 20 ms rate-limiting step, or 35 turnovers/protein/s, we conducted, to our knowledge, novel consecutive single-turnover flash experiments that demonstrate that under continuous illumination, NpHR bypasses this step in the photocycle.
Assuntos
Cloretos/metabolismo , Halorrodopsinas/metabolismo , Luz , Halobacteriaceae , Transporte de Íons/efeitos da radiação , CinéticaRESUMO
Membrane proteins (MPs), despite being critically important drug targets for the pharmaceutical industry, are difficult to study due to challenges in obtaining high yields of functional protein. Most current extraction efforts use specialized non-ionic detergents to solubilize and stabilize MPs, with MPs being concentrated by ultrafiltration (UF). However, many detergents are retained during the UF step, which can destabilize MPs and/or interfere with their characterization. Here, we studied the influence of detergent selection on the extraction and UF-based concentration of biomedically-relevant MPs, the light-driven sodium and chloride transporters, KR2 and halorhodopsin (pHR) which are also model proteins for more complex mammalian rhodopsins. We also designed a flat-bottomed centrifugal filter that can concentrate MPs with enhanced removal of free detergents by promoting concentration polarization (CP). We tested the performance of this new filter using four commonly employed MP detergents, octyl-ß-D maltoside (OM), decyl-ß-D maltoside (DM), dodecyl-ß-D maltoside (DDM) and octyl-ß-D glucoside (OG), over a range of detergent and salt concentrations. Detergent passage is significantly higher for the flat-bottomed filter achieving up to 2-fold greater sieving of detergent in DM-solubilized pHR system due to the high degree of CP. We observe more efficient, up to 5-fold higher extraction of KR2 in the presence of a longer 12-carbon alkyl chain detergent, DDM compared to a shorter 8-carbon detergent, OM. Assuming complete binding and elution of the extracted protein, DDM-based extraction of KR2 could lead to a potential 7-fold improvement in purification yields compared to conventional methods which yield â¼1 mg MP per liter of cell culture. However, the longer chain detergents like DDM form larger micelles that are difficult to remove by UF. Thus, there exists a trade-off between choosing a detergent that will enable efficient extraction of MP while showing easier removal during subsequent UF. The extraction efficiency and UF-based separation of detergent micelles provide insights for other applications involving detergent-mediated separation/extraction.
Assuntos
Detergentes , Proteínas de Membrana/isolamento & purificação , Ultrafiltração , Escherichia coli , Halorrodopsinas/isolamento & purificação , Micelas , Opsinas/isolamento & purificaçãoRESUMO
Membrane proteins (MPs) are of rapidly growing interest in the design of pharmaceutical products, novel sensors, and synthetic membranes. Ultrafiltration (UF) using commercially available centrifugal concentrators is typically employed for laboratory-scale concentration of low-yield MPs, but its use is accompanied by a concomitant increase in concentration of detergent micelles. We present a detailed analysis of the hydrodynamic processes that control detergent passage during ultrafiltration of MPs and propose methods to optimize detergent passage during protein concentration in larger-scale membrane processes. Experiments were conducted using nonionic detergents, octyl-ß-D glucoside (OG), and decyl-ß-D maltoside (DM) with the bacterial water channel protein, Aquaporin Z (AqpZ) and the light driven chloride pump, halorhodopsin (HR), respectively. The observed sieving coefficient (So ), a measure of detergent passage, was evaluated in both stirred cell and centrifugal systems. So for DM and OG increased with increasing filtrate flux and decreasing shear rates in the stirred cell, that is, with increasing concentration polarization (CP). Similar effects were observed during filtration of MP-detergent (MPD) micelles. However, lower transmission was observed in the centrifugal system for both detergent and MPD systems. This is attributed to free convection-induced shear and hence reduced CP along the membrane surface during centrifugal UF. Thus to concentrate MPs without retention of detergent, design of UF systems that promote CP is required. Biotechnol. Bioeng. 2016;113: 2122-2130. © 2016 Wiley Periodicals, Inc.
Assuntos
Centrifugação/instrumentação , Centrifugação/métodos , Detergentes/química , Proteínas de Membrana/isolamento & purificação , Ultrafiltração/instrumentação , Ultrafiltração/métodos , Desenho de Equipamento , Análise de Falha de EquipamentoRESUMO
Biologics manufacturing is capital and consumable intensive with need for advanced inventory planning to account for supply chain constraints. Early-stage process design and technology transfer are often challenging due to limited information on process variability regarding bioreactor titer, process yield, and product quality. Monte Carlo (MC) methods offer a stochastic modeling approach for process optimization where probabilities of occurrence for process inputs are incorporated into a deterministic model to simulate more likely scenarios for process outputs. In this study, we explore MC simulation-based design of a monoclonal antibody downstream manufacturing process. We demonstrate that this probabilistic approach offers more representative outcomes over the conventional worst-case approach where the theoretical minimum and maximum values of each process parameter are used without consideration for their probability of occurrence. Our work demonstrates case studies on more practically sizing unit operations to improve consumable utilization, thereby reducing manufacturing costs. We also used MC simulations to minimize process cadence by constraining the number of cycles per unit operation to fit facility preferences. By factoring in process uncertainty, we have implemented MC simulation-based facility fit analyses to efficiently plan for inventory when accounting for process constraints during technology transfer from lab-scale to clinical or commercial manufacturing.
Assuntos
Reatores Biológicos , Transferência de Tecnologia , Método de Monte Carlo , Simulação por Computador , Anticorpos MonoclonaisRESUMO
The removal of viruses by filtration is a critical unit operation to ensure the overall safety of monoclonal antibody (mAb) products. Many mAbs show very low filtrate flux during virus removal filtration, although there are still significant uncertainties regarding both the mechanisms and antibody properties that determine the filtration behavior. Experiments were performed with three highly purified mAbs through three different commercial virus filters (Viresolve Pro, Viresolve NFP, and Pegasus SV4) with different pore structures and chemistries. The flux decline observed during mAb filtration was largely reversible, even under conditions where the filtrate flux with the mAb was more than 100-fold smaller than the corresponding buffer flux. The extent of flux decline was highly correlated with the hydrodynamic diameter of the mAb as determined by dynamic light scattering (DLS). The mAb with the lowest filtrate flux for all three membranes showed the largest attractive intermolecular interactions and the greatest hydrophobicity, with the latter determined by binding to a butyl resin in an analytical hydrophobic interaction chromatography (HIC) column. These results strongly suggest that the flux behavior is dominated by reversible self-association of the mAbs, providing important insights into the design of more effective virus filtration processes and in the early identification of problematic mAbs/solution conditions.
Assuntos
Anticorpos Monoclonais , Vírus , Anticorpos Monoclonais/química , Filtração , Vírus/química , Hidrodinâmica , Interações Hidrofóbicas e HidrofílicasRESUMO
BACKGROUND: Virus inactivation is a critical operation in therapeutic protein manufacturing. Low pH buffers are a widely used strategy to ensure robust enveloped virus clearance. However, the choice of model virus can give varying results in viral clearance studies. Pseudorabies virus (SuHV) or herpes simplex virus-1 (HSV-1) are frequently chosen as model viruses to demonstrate the inactivation for the herpes family. RESULTS: In this study, SuHV, HSV-1, and equine arteritis virus (EAV) were used to compare the inactivation susceptibility at pH 4.0 and 4°C. SuHV and HSV-1 are from the same family, and EAV was chosen as a small, enveloped virus. Glycine, acetate, and citrate buffers at pH 4.0 and varying buffer strengths were studied. The inactivation susceptibility was found to be in the order of SuHV > HSV > EAV. The buffer effectiveness was found to be in the order of citrate > acetate > glycine. The smaller virus, EAV, remained stable and infectious in all the buffer types and compositions studied. CONCLUSION: The variation in inactivation susceptibility of herpes viruses indicated that SuHV and HSV cannot be interchangeably used as a virus model for inactivation studies. Smaller viruses might remain adventitiously infective at moderately low pH.
Assuntos
Herpesvirus Humano 1 , Vírus , Animais , Cavalos , Concentração de Íons de Hidrogênio , Inativação de VírusRESUMO
Virus removal filtration is a critical step in the manufacture of monoclonal antibody products, providing a robust size-based removal of both enveloped and non-enveloped viruses. Many monoclonal antibodies show very large reductions in filtrate flux during virus filtration, with the mechanisms governing this behavior and its dependence on the properties of the virus filter and antibody remaining largely unknown. Experiments were performed using the highly asymmetric Viresolve® Pro and the relatively homogeneous Pegasus™ SV4 virus filters using a highly purified monoclonal antibody. The filtrate flux for a 4 g/L antibody solution through the Viresolve® Pro decreased by about 10-fold when the filter was oriented with the skin side down but by more than 1000-fold when the asymmetric filter orientation was reversed and used with the skin side up. The very large flux decline observed with the skin side up could be eliminated by placing a large pore size prefilter directly on top of the virus filter; this improvement in filtrate flux was not seen when the prefilter was used inline or as a batch prefiltration step. The increase in flux due to the prefilter was not related to the removal of large protein aggregates or to an alteration in the extent of concentration polarization. Instead, the prefilter appears to transiently disrupt reversible associations of the antibodies caused by strong intermolecular attractions. These results provide important insights into the role of membrane morphology and antibody properties on the filtrate flux during virus filtration.
Assuntos
Anticorpos Monoclonais , Vírus , Anticorpos Monoclonais/química , Filtração/métodos , Membranas Artificiais , Vírus/químicaRESUMO
Viral surrogates to screen for virus inactivation (VI) can be a faster, cheaper and safer alternative to third-party testing of pathogenic BSL2 (Biosafety level 2) model viruses. Although the bacteriophage surrogate, Ø6, has been used to assess low pH BSL2 VI, it has not been used for evaluation of detergent-mediated VI. Furthermore, Ø6 is typically assayed through host cell infectivity which introduces the risk of cross-contaminating other cell lines in the facility. To circumvent contamination, we developed an in-house RT-qPCR (Reverse transcriptase quantitative polymerase chain reaction) assay for selective detection of active Ø6 from a population of live and dead phage. The RT-qPCR assay was used to evaluate Ø6 inactivation in cell culture fluid of monoclonal antibody and fusion protein. Complementary Ø6 infectivity was also conducted at a third-party testing facility. The Ø6 RT-qPCR and infectivity data was modeled against VI of three BSL2 viruses, X- MuLV, A- MuLV and HSV-1 in corresponding therapeutics. Both Ø6 methods demonstrate that any VI agent showing Ø6 clearance of a minimum of 2.5 logs would demonstrate complete BSL2 VI of ≥ 4.0 logs. Compared to BSL2 virus testing, this in-house Ø6 RT-qPCR tool can screen VI agents at 5% the cost and a turnaround time of 2 to 3 days vs. 4 to 7 months.
Assuntos
Inativação de Vírus , Vírus , Vírus da Leucemia Murina , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: Therapeutic protein manufacturing would benefit by having an arsenal of ways to inactivate viruses. There have been many publications on the virus inactivation ability of arginine at pH 4.0, but the mechanism of this inactivation is unknown. This study explored how virus structure and solution conditions enhance virus inactivation by arginine and leads to a better understanding of the mechanism of virus inactivation by arginine. RESULTS: Large diameter viruses from the Herpesviridae family (SuHV-1, HSV-1) with loosely packed lipids were highly inactivated by arginine, whereas small diameter, enveloped viruses (equine arteritis virus (EAV) and bovine viral diarrhea virus (BVDV)) with tightly packed lipids were negligibly inactivated by arginine. To increase the inactivation of viruses resistant to arginine, arginine-derivatives and arginine peptides were tested. Derivates and peptides demonstrated that a greater capacity for clustering and added hydrophobicity enhanced virus inactivation. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) detected increases in virus size after arginine exposure, supporting the mechanism of lipid expansion. CONCLUSIONS: Arginine most likely interacts with the lipid membrane to cause inactivation. This is shown by larger viruses being more sensitive to inactivation and expansion of the viral size. The enhancement of arginine inactivation when increased hydrophobic molecules are present or arginine is clustered demonstrates a potential mechanism of how arginine interacts with the lipid membrane.
Assuntos
Vírus da Diarreia Viral Bovina , Vírus , Animais , Arginina , Cavalos , Inativação de VírusRESUMO
We report a simple and direct fluorimetric vesicle-based method for measuring the transport rate of the light-driven ions pumps as specifically applied to the chloride pump, halorhodopsin, from Natronomonas pharaonis (pHR). Previous measurements were cell-based and methods to determine average single channel permeability challenging. We used a water-in-oil emulsion method for directional pHR reconstitution into two different types of vesicles: lipid vesicles and asymmetric lipid-block copolymer vesicles. We then used stopped-flow experiments combined with fluorescence correlation spectroscopy to determine per protein Cl- transport rates. We obtained a Cl- transport rate of 442 (±17.7) Cl-/protein/s in egg phosphatidyl choline (PC) lipid vesicles and 413 (±26) Cl-/protein/s in hybrid block copolymer/lipid (BCP/PC) vesicles with polybutadine-polyethylene oxide (PB12PEO8) on the outer leaflet and PC in the inner leaflet at a photon flux of 1450 photons/protein/s. Normalizing to a per photon basis, this corresponds to 0.30 (±0.07) Cl-/photon and 0.28 (±0.04) Cl-/photon for pure PC and BCP/PC hybrid vesicles respectively, both of which are in agreement with recently reported turnover of ~500 Cl-/protein/s from flash photolysis experiments and with voltage-clamp measurements of 0.35 (±0.16) Cl-/photon in pHR-expressing oocytes as well as with a pHR quantum efficiency of ~30%.
Assuntos
Cloretos/metabolismo , Halorrodopsinas/química , Transporte de Íons/genética , Lipossomos/química , Cloretos/química , Cloretos/efeitos da radiação , Halobacteriaceae/química , Halobacteriaceae/genética , Halorrodopsinas/genética , Cinética , Luz , Lipossomos/metabolismo , Lipossomos/efeitos da radiaçãoRESUMO
This study develops procedures to rapidly screen conditions for purification of membrane proteins (MPs) using 96-well plates containing nickel-functionalized membranes. In addition to their application in the pharmaceutical industry, MPs are important components of new sensors, synthetic membranes, and bioelectronic devices. However, purification of MPs is challenging due to their hydrophobic exterior, which requires stabilization in amphipathic detergent micelles. We examined the extent of extraction of the light-driven sodium transporter, Krokinobacter eikastus rhodopsin 2 (KR2) heterologously expressed in Escherichia coli using different salts and maltoside-based detergents. The extraction was followed by subsequent affinity purification in membranes functionalized with Ni2+ -nitrilotriacetate complexes that bind the His-tagged KR2. We also employed a hydrophobic chelator to separate detergent micelles from the aqueous phase as an initial isolation step prior to affinity purification. Unlike conventional resin-based capture, which can take a full day or more, the membrane-based screening of purification conditions takes only a few hours, and its scale-up involves changing from a 96-well format to a larger membrane module. The novelty of the method lies in utilizing membrane-based ultracentrifugation-free purification of MPs from cell membrane fragments; the optimized purification conditions from the screening method can potentially be applied to large-scale/conventional resin-based purification of MPs.
Assuntos
Membrana Celular/química , Proteínas de Membrana/isolamento & purificação , Membranas Artificiais , Rodopsina/genética , Membrana Celular/genética , Cromatografia de Afinidade , Detergentes/química , Escherichia coli/genética , Flavobacteriaceae/genética , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/química , Níquel/química , Rodopsina/químicaRESUMO
The original version of this Article contained an error in the spelling of the author Woochul Song, which was incorrectly given as Woochul C. Song. This has been corrected in both the PDF and HTML versions of the Article.
RESUMO
Synthetic polymer membranes, critical to diverse energy-efficient separations, are subject to permeability-selectivity trade-offs that decrease their overall efficacy. These trade-offs are due to structural variations (e.g., broad pore size distributions) in both nonporous membranes used for Angstrom-scale separations and porous membranes used for nano to micron-scale separations. Biological membranes utilize well-defined Angstrom-scale pores to provide exceptional transport properties and can be used as inspiration to overcome this trade-off. Here, we present a comprehensive demonstration of such a bioinspired approach based on pillar[5]arene artificial water channels, resulting in artificial water channel-based block copolymer membranes. These membranes have a sharp selectivity profile with a molecular weight cutoff of ~ 500 Da, a size range challenging to achieve with current membranes, while achieving a large improvement in permeability (~65 L m-2 h-1 bar-1 compared with 4-7 L m-2 h-1 bar-1) over similarly rated commercial membranes.
Assuntos
Membranas Artificiais , Simulação de Dinâmica Molecular , Polímeros/química , Água/química , Aquaporinas/química , Simulação por Computador , Detergentes/química , Bicamadas Lipídicas/química , Lipossomos/química , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Peso Molecular , Permeabilidade , Porosidade , Sais/químicaRESUMO
Membrane protein and membrane protein-mimic functionalized materials are rapidly gaining interest across a wide range of applications, including drug screening, DNA sequencing, drug delivery, sensors, water desalination, and bioelectronics. In these applications, material performance is highly dependent on activity-per-protein and protein packing density in bilayer and bilayer-like structures collectively known as biomimetic membranes. However, a clear understanding of, and accurate tools to study these properties of biomimetic membranes does not exist. This paper presents methods to evaluate membrane protein compatibility with biomimetic membrane materials. The methods utilized provide average single protein activity, and for the first time, provide experimentally quantifiable measures of the chemical and physical compatibility between proteins (and their mimics) and membrane materials. Water transport proteins, rhodopsins, and artificial water channels are reconstituted into the full range of current biomimetic membrane matrices to evaluate the proposed platform. Compatibility measurement results show that both biological and artificial water channels tested largely preserve their single protein water transport rates in biomimetic membranes, while their reconstitution density is variable, leading to different overall membrane permeabilities. It is also shown that membrane protein insertion efficiency inversely correlates with both chemical and physical hydrophobicity mismatch between membrane protein and the membrane matrix.