Pressure-flow relationships for packed beds of compressible chromatography media at laboratory and production scale

Pressure drop across chromatography beds employing soft or semirigid media can be a significant problem in the operation of large-scale preparative chromatography columns. The shape or aspect ratio (length/diameter) of a packed bed has a significant effect on column pressure drop due to wall effects, which can result in unexpectedly high pressures in manufacturing. Two types of agarose-based media were packed in chromatography columns at various column aspect ratios, during which pressure drop, bed height, and flow rate were carefully monitored. Compression of the packed beds with increasing flow velocities was observed. An empirical model was developed to correlate pressure drop with the aspect ratio of the packed beds and the superficial velocity. Modeling employed the Blake-Kozeny equation in which empirical relationships were used to predict bed porosity as a function of aspect ratio and flow velocity. Model predictions were in good agreement with observed pressure drops of industrial scale chromatography columns. A protocol was developed to predict compression in industrial chromatography applications by a few laboratory experiments. The protocol is shown to be useful in the development of chromatographic methods and sizing of preparative columns.     

Improved process analytical technology for protein a chromatography using predictive principal component analysis tools

Protein A chromatography is widely employed for the capture and purification of antibodies and Fc‐fusion proteins. Due to the high cost of protein A resins, there is a significant economic driving force for using these chromatographic materials for a large number of cycles. The maintenance of column performance over the resin lifetime is also a significant concern in large‐scale manufacturing. In this work, several statistical methods are employed to develop a novel principal component analysis (PCA)‐based tool for predicting protein A chromatographic column performance over time. A method is developed to carry out detection of column integrity failures before their occurrence without the need for a separate integrity test. In addition, analysis of various transitions in the chromatograms was also employed to develop PCA‐based models to predict both subtle and general trends in real‐time protein A column yield decay. The developed approach has significant potential for facilitating timely and improved decisions in large‐scale chromatographic operations in line with the process analytical technology (PAT) guidance from the Food and Drug Administration (FDA). Biotechnol. Bioeng. 2011; 108:59–68. © 2010 Wiley Periodicals, Inc.     

Large scale demonstration of a process analytical technology application in bioprocessing: Use of on‐line high performance liquid chromatography for making real time pooling decisions for process chromatography

Process Analytical Technology (PAT) has been gaining a lot of momentum in the biopharmaceutical community because of the potential for continuous real time quality assurance resulting in improved operational control and compliance. In previous publications, we have demonstrated feasibility of applications involving use of high performance liquid chromatography (HPLC) and ultra performance liquid chromatography (UPLC) for real‐time pooling of process chromatography column. In this article we follow a similar approach to perform lab studies and create a model for a chromatography step of a different modality (hydrophobic interaction chromatography). It is seen that the predictions of the model compare well to actual experimental data, demonstrating the usefulness of the approach across the different modes of chromatography. Also, use of online HPLC when the step is scaled up to pilot scale (a 2294 fold scale‐up from a 3.4 mL column in the lab to a 7.8 L column in the pilot plant) and eventually to manufacturing scale (a 45930 fold scale‐up from a 3.4 mL column in the lab to a 158 L column in the manufacturing plant) is examined. Overall, the results confirm that for the application under consideration, online‐HPLC offers a feasible approach for analysis that can facilitate real‐time decisions for column pooling based on product quality attributes. The observations demonstrate that the proposed analytical scheme allows us to meet two of the key goals that have been outlined for PAT, i.e., “variability is managed by the process” and “product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions”. The application presented here can be extended to other modes of process chromatography and/or HPLC analysis. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Advective hydrogel membrane chromatography for monoclonal antibody purification in bioprocessing

Protein A chromatography is widely employed for the capture and purification of monoclonal antibodies (mAbs). Because of the high cost of protein A resins, there is a significant economic driving force to seek new downstream processing strategies. Membrane chromatography has emerged as a promising alternative to conventional resin based column chromatography. However, to date, the application has been limited to mostly ion exchange flow through (FT) mode. Recently, significant advances in Natrix hydrogel membrane has resulted in increased dynamic binding capacities for proteins, which makes membrane chromatography much more attractive for bind/elute operations. The dominantly advective mass transport property of the hydrogel membrane has also enabled Natrix membrane to be run at faster volumetric flow rates with high dynamic binding capacities. In this work, the potential of using Natrix weak cation exchange membrane as a mAb capture step is assessed. A series of cycle studies was also performed in the pilot scale device (> 30 cycles) with good reproducibility in terms of yield and product purities, suggesting potential for improved manufacturing flexibility and productivity. In addition, anion exchange (AEX) hydrogel membranes were also evaluated with multiple mAb programs in FT mode. Significantly higher binding capacity for impurities (support mAb loads up to 10Kg/L) and 40X faster processing speed were observed compared with traditional AEX column chromatography. A proposed protein A free mAb purification process platform could meet the demand of a downstream purification process with high purity, yield, and throughput. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:974–982, 2015     

Development of Vero Cell-Derived Inactivated Japanese Encephalitis Vaccine

We have established a manufacturing system for a Vero cell-derived inactivated Japanese encephalitis vaccine at a 500l scale. The production system involves expansion of Vero cells using microcarrier, followed by virus infection. Except for an additional purification step, the downstream purification processes are similar to those used for the current mouse brain-derived vaccine; cell removal, concentration and removal of low-molecular weight impurities by membrane filtration, formalin-inactivation, sucrose density gradient ultracentrifugation, and Sulfate-Cellulofine column chromatography are conducted. The antigen obtained from the manufacturing system was highly purified and its physico-chemical and immunological properties were comparable with those of antigen derived from mouse brains. Our system is very simple and could be easily scaled-up to allow vaccine production at a several thousand litre scale.     

Design and control of integrated chromatography column sequences

To increase the productivity in biopharmaceutical production, a natural step is to introduce integrated continuous biomanufacturing which leads to fewer buffer and storage tanks, smaller sizes of integrated unit operations, and full automation of the operation. The main contribution of this work is to illustrate a methodology for design and control of a downstream process based on integrated column sequences. For small scale production, for example, pre‐clinical studies, integrated column sequences can be implemented on a single chromatography system. This makes for a very efficient drug development platform. The proposed methodology is composed of four steps and is governed by a set of tools, that is presented, that makes the transition from batch separations to a complete integrated separation sequence as easy as possible. This methodology, its associated tools and the physical implementation is presented and illustrated on a case study where the target protein is separated from impurities through an integrated four column sequence. This article shows that the design and control of an integrated column sequence was successfully implemented for a tertiary protein separation problem. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:923–930, 2017     

State-of-the-art in downstream processing of monoclonal antibodies: Process trends in design and validation

Monoclonal antibodies (mAbs) are the most important family of biopharmaceutical compounds in terms of market share. At present, 30 mAbs have been approved and are now commercialized for therapeutic purposes. mAbs are typically produced by mammalian cell culture in bioreactors that range in scale of 1–20 m³. Regardless of scale, from laboratory to commercial settings, the recovery and purification of mAbs present important challenges. Depending on the scale, the particular product, and the type of production process (bioreactor operation, process time, complexity of the culture media, cell density, etc.), many possible downstream configurations are possible and have been used. In this contribution, we review each type of unit operation that forms a downstream train for mAb production. We provide information regarding typical operation settings and critical variables for centrifugation, ultrafiltration, affinity chromatography, ion exchange chromatography, and viral removal operations. In addition, we discuss some important considerations required for the formulation of drugs based on mAbs. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 899–916, 2012     

Bispecific antibody process development: Assembly and purification of knob and hole bispecific antibodies

Production of knob and hole dual light chain bispecific antibodies poses several unique challenges for development of a feasible industrial scale manufacturing process. We developed an efficient process for the assembly and purification of knob and hole dual light chain bispecific antibodies. Two distinct half‐antibodies targeting two different antigens were expressed separately in Escherichia coli cells and captured independently using Protein A chromatography. When combined, the knob and hole mutations in the CH3 domains promoted heterodimer formation. The hinge region disulfides were reduced and reoxidized to form the disulfide bridge between the two complementary half antibodies. Unreacted half antibodies, noncovalently linked homodimers, covalently linked homodimers, and noncovalently linked heterodimers are impurities closely related to the product of interest and are challenging to remove by standard processes. Characterization of the molecular properties of the half antibodies and high‐throughput screening predicted column chromatography performance and allowed for rapid development of downstream purification steps for removal of unique product‐related and process‐related impurities. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:397–404, 2018     

The effect of protein a cycle number on the performance and lifetime of an anion exchange polishing step

Most mAb platform purification processes consist of an affinity capture step followed by one or two polishing steps. An understanding of the performance linkages between the unit operations can lead to robust manufacturing processes. In this study, a weak‐partitioning anion‐exchange chromatography polishing step used in a mAb purification process was characterized through high‐throughput screening (HTS) experiments, small‐scale experiments including a cycling study performed on qualified scale‐down models, and large‐scale manufacturing runs. When material from a Protein A column that had been cycled <10× was loaded on the AEX resin, early breakthrough of impurities and premature loss of capacity was observed. As the cycle number on the Protein A resin increased, the capacity of the subsequent AEX step increased. Different control strategies were considered for preventing impurity breakthrough and improving AEX resin lifetimes. Depth filtration of the Protein A peak pool significantly improved the AEX resin capacity, robustness, and lifetime. Further, the turbidity of the Protein A pool has the potential for use as an in‐process control parameter for monitoring the performance of the AEX step. Biotechnol. Bioeng. 2013; 110: 1142–1152. © 2012 Wiley Periodicals, Inc.     

Ultra scale‐down approach to correct dispersive and retentive effects in small‐scale columns when predicting larger scale elution profiles

Ultra scale‐down approaches represent valuable methods for chromatography development work in the biopharmaceutical sector, but for them to be of value, scale‐down mimics must predict large‐scale process performance accurately. For example, one application of a scale‐down model involves using it to predict large‐scale elution profiles correctly with respect to the size of a product peak and its position in a chromatogram relative to contaminants. Predicting large‐scale profiles from data generated by small laboratory columns is complicated, however, by differences in dispersion and retention volumes between the two scales of operation. Correcting for these effects would improve the accuracy of the scale‐down models when predicting outputs such as eluate volumes at larger scale and thus enable the efficient design and operation of subsequent steps. This paper describes a novel ultra scale‐down approach which uses empirical correlations derived from conductivity changes during operation of laboratory and pilot columns to correct chromatographic profiles for the differences in dispersion and retention. The methodology was tested by using 1 mL column data to predict elution profiles of a chimeric monoclonal antibody obtained from Protein A chromatography columns at 3 mL laboratory‐ and 18.3 L pilot‐scale. The predictions were then verified experimentally. Results showed that the empirical corrections enabled accurate estimations of the characteristics of larger‐scale elution profiles. These data then provide the justification to adjust small‐scale conditions to achieve an eluate volume and product concentration which is consistent with that obtained at large‐scale and which can then be used for subsequent ultra scale‐down operations. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Methods for protecting silica sorbents used in high-performance liquid chromatography from strongly adsorbed impurities during purification of human recombinant insulin

One of the main stages of human recombinant insulin (HRI) production is the hormone purification by means of reversed phase high-performance liquid chromatography (RP HPLC). The optimization of this stage determines the increase of the total manufacturing yield. Therefore, the cost of the sorbent used in HPLC influences the cost of the manufacturing product, i.e. HRI substance. However, resolution between HRI and its admixtures decreases with time. The reason for this is the sorbent contamination with strongly adsorbed impurities (SAI) which are accumulated during elution. In the following research several methods for sorbent protection are studied. The opinion that SAI are mainly high-molecular weight compounds was examined using gel filtration. Different sorbent types were tested for the use in guard column. The results obtained were applied and improved at preparative level.     

Towards robust cell culture processes — Unraveling the impact of media preparation by spectroscopic online monitoring

Biopharmaceutical manufacturing processes can be affected by variability in cell culture media, e.g. caused by raw material impurities. Although efforts have been made in industry and academia to characterize cell culture media and raw materials with advanced analytics, the process of industrial cell culture media preparation itself has not been reported so far. Within this publication, we first compare mid‐infrared and two‐dimensional fluorescence spectroscopy with respect to their suitability as online monitoring tools during cell culture media preparation, followed by a thorough assessment of the impact of preparation parameters on media quality. Through the application of spectroscopic methods, we can show that media variability and its corresponding root cause can be detected online during the preparation process. This methodology is a powerful tool to avoid batch failure and is a valuable technology for media troubleshooting activities. Moreover, in a design of experiments approach, including additional liquid chromatography–mass spectrometry analytics, it is shown that variable preparation parameters such as temperature, power input and preparation time can have a strong impact on the physico‐chemical composition of the media. The effect on cell culture process performance and product quality in subsequent fed‐batch processes was also investigated. The presented results reveal the need for online spectroscopic methods during the preparation process and show that media variability can already be introduced by variation in media preparation parameters, with a potential impact on scale‐up to a commercial manufacturing process.     

Online integrity monitoring in the Protein A step of mAb Production Processes—increasing reliability and process robustness

The purification of recombinant proteins and antibodies using large packed‐bed columns is a key component in most biotechnology purification processes. Because of its efficiency and established practice in the industry, column chromatography is a state of the art technology with a proven capability for removal of impurities, viral clearance, and process efficiency. In general, the validation and monitoring of chromatographic operations—especially of critical process parameters—is required to ensure robust product quality and compliance with health authority expectations. One key aspect of chromatography that needs to be monitored is the integrity of the packed bed, since this is often critical to achieving sufficient separation of protein species. Identification of potential column integrity issues before they occur is important for both product quality and economic efficiency. In this article, we examine how transition analysis techniques can be utilized to monitor column integrity. A case study on the application of this method during a large scale Protein A capture step in an antibody purification process shows how it can assist with improving process knowledge and increasing the efficiency of manufacturing operations. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:383–390, 2014     

Separation of plasmid DNA from protein and bacterial lipopolysaccharides using displacement chromatography

The preparation of plasmid DNA at large scale constitutes a pressing problem in bioseparation. This paper describes a first investigation of displacement chromatography as a means to separate plasmid DNA (4.7 kb) from E. coli lipopolysaccharides and protein (holo transferrin), respectively. Displacement chromatography has advantages in this regard, since the substance mixture is resolved into rectangular zones of the individual components rather than into peaks. Thus a higher total concentration can be maintained in the pooled product fractions. Hydroxyapatite (type I and II) and anion exchange stationary phases were included in the experiments. In addition to a conventional anion exchange column packed with porous particles, the recently introduced continuous bed UNOTM anion exchange column was investigated. No DNA purification was possible with either hydroxyapatite material. Conventional particle based columns in general were not suited to the separation of any two substances varying considerably in molecular mass, e.g. plasmid DNA and standard protein. Presumably, the direct competition for the binding sites, which is essential in displacement chromatography, was restricted by the size dependency of the accessible stationary phase surface area in this case. Better results were obtained with the continuous bed column, in which the adsorptive surface coincides with the walls of the flow through pores. As a result the accessible surface does not vary as much with the size of the interacting molecules as for the conventional stationary phase materials. Sharper transitions were also observed between substance zones recovered from the UNOTM column. The steric mass action model was used to aid method development in case of the anion exchange approach. While further research in obviously necessary, displacement chromatography on continuous bed columns has been shown to be capable of separating plasmid DNA from typical impurities. This revised version was published online in July 2006 with corrections to the Cover Date.     

Negative chromatography: Progress,applications and future perspectives

In negative chromatography, the impurities bind on the adsorbent, and the product is allowed to flow through the chromatographic column. Negative chromatography is an alternative to positive chromatography under certain circumstances and has been used to purify various biomolecules. For this review, a detailed survey of the performance of reported studies on negative chromatography was conducted. The applications of negative chromatography in the capture and intermediate purification steps for biomolecules (e.g., plasmid DNA, antibodies, enzymes, hemoglobin, virus particles and cells) are reviewed. The negative chromatographic adsorbents adsorb the impurities through surface charge, hydrophobic interaction at specific sites on the surface, hydrophobic interaction, hydrogen bonding and functional groups. Examples of applications of negative chromatography according to the type of chromatography matrix used are summarized and discussed. In addition, the effects of operating conditions (initial protein concentration, buffer ions, pH and salt concentration) are discussed, and the criteria for choosing negative or positive chromatography are summarized. The literature survey showed that there will be future limitations and challenges ahead in implementation of negative chromatography. Possible solutions to the limitations and challenges of negative chromatography and future trends for developing negative chromatography are discussed.     

Allogeneic cell therapy bioprocess economics and optimization: Single‐use cell expansion technologies

For allogeneic cell therapies to reach their therapeutic potential, challenges related to achieving scalable and robust manufacturing processes will need to be addressed. A particular challenge is producing lot‐sizes capable of meeting commercial demands of up to 10⁹ cells/dose for large patient numbers due to the current limitations of expansion technologies. This article describes the application of a decisional tool to identify the most cost‐effective expansion technologies for different scales of production as well as current gaps in the technology capabilities for allogeneic cell therapy manufacture. The tool integrates bioprocess economics with optimization to assess the economic competitiveness of planar and microcarrier‐based cell expansion technologies. Visualization methods were used to identify the production scales where planar technologies will cease to be cost‐effective and where microcarrier‐based bioreactors become the only option. The tool outputs also predict that for the industry to be sustainable for high demand scenarios, significant increases will likely be needed in the performance capabilities of microcarrier‐based systems. These data are presented using a technology S‐curve as well as windows of operation to identify the combination of cell productivities and scale of single‐use bioreactors required to meet future lot sizes. The modeling insights can be used to identify where future R&D investment should be focused to improve the performance of the most promising technologies so that they become a robust and scalable option that enables the cell therapy industry reach commercially relevant lot sizes. The tool outputs can facilitate decision‐making very early on in development and be used to predict, and better manage, the risk of process changes needed as products proceed through the development pathway. Biotechnol. Bioeng. 2014;111: 69–83. © 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Purification of Arsenazo III,a Ca2+-sensitive dye

Commercial preparations of Arsenazo III. (o-(1,8 dihydroxy-3,6-disulfonaphthylene-2,7-bisazo)-bisbenzenearsonic acid), a dye useful for detection of micromolar concentrations of ionized Ca²⁺, have been found to be grossly and variably contaminated with colored impurities and Ca²⁺. In this paper, methods are described for detection of the colored impurities using DEAE-cellulose thinlayer chromatography in semiorganic solvents, along with methods for purification of the dye using DEAE-cellulose column chromatography and Chelex-100 ion-exchange resin. The impurities from one commercial lot are shown to exhibit significant absorbances over the range 400–725 nm; some of the impurities exhibit spectral shifts in the presence of Ca²⁺, implying Ca²⁺ binding. Thus, use of unpurified dye to quantitate ionized Ca²⁺ might yield data which vary with the source and batch of dye. The chromatographically pure Arsenazo III obtained from column chromatography is characterized with respect to Na⁺, Ca²⁺, and dye content.     

Packing of large‐scale chromatography columns with irregularly shaped glass based resins using a stop‐flow method

Rigid chromatography resins, such as controlled pore glass based adsorbents, offer the advantage of high permeability and a linear pressure‐flow relationship irrespective of column diameter which improves process time and maximizes productivity. However, the rigidity and irregularly shaped nature of these resins often present challenges in achieving consistent and uniform packed beds as formation of bridges between resin particles can hinder bed consolidation. The standard flow‐pack method when applied to irregularly shaped particles does not yield well‐consolidated packed beds, resulting in formation of a head space and increased band broadening during operation. Vibration packing methods requiring the use of pneumatically driven vibrators are recommended to achieve full packed bed consolidation but limitations in manufacturing facilities and equipment may prevent the implementation of such devices. The stop‐flow packing method was developed as an improvement over the flow‐pack method to overcome these limitations and to improve bed consolidation without the use of vibrating devices. Transition analysis of large‐scale columns packed using the stop‐flow method over multiple cycles has shown a two‐ to three‐fold reduction of change in bed integrity values as compared to a flow‐packed bed demonstrating an improvement in packed bed stability in terms of the height equivalent to a theoretical plate (HETP) and peak asymmetry (A_s). © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1319–1325, 2014     

再生水用于地下回灌过程中有机物的迁移和去除

人工地下水回灌是解决我国水资源短缺的有效途径之一.在实验室利用土壤柱系统,考查了回灌过程中有机物的迁移和去除.二级处理出水经活性碳吸附后进入土壤柱系统,土壤柱系统对TOC、UV-254和BOD₅的去除率分别为44%、34.36%和95%,并且大部分有机物是在土壤表层0～0.5 m被去除的.进水TOC浓度为9～11 mg·L^-1时,回灌过程中TOC浓度随土壤柱深度的变化符合指数方程：C=10e^-0.6934h(R²=0.8697). 用该方程可以较好地预测土壤柱出水中的TOC浓度.     

Purification of recombinant brain derived neurotrophic factor using reversed phase displacement chromatography

This work investigates the utility of RPLC displacement chromatography for the purification of recombinant brain derived neurotrophic factor (rHu-BDNF) from its variants and E. coli. protein (ECP) impurities. The closely associated variants (six in total) differ by one amino acid from the native BDNF and thus pose a challenging separation problem. Several operational parameters were investigated to study their effects on the yield of the displacement process. The results indicated that the concentration of trifluoroacetic acid (TFA) in the buffer was a key factor in achieving the desired purification. Displacement chromatography on an analytical scale column resulted in extremely high purity and yield in a single chromatographic step. The process was successfully scaled-up with respect to particle and column diameter. The production rate of a pilot scale RPLC displacement process was shown to be 23 times higher than the combined production rates of the current preparative ion exchange and hydrophobic interaction gradient elution steps that are used to remove variant and ECP impurities, respectively.