Continuous Downstream Processing Research Articles

Continuous capture scale down model:A comparison of a continuous and a discrete approach.

In continuous pharmaceutical manufacturing, consisting of a perfused batch fermentation and integrated continuous downstream processing, the continuous capture is the linking unit operation. For the development of this unit operation, scale-down models (SDMs) are crucial, whereas discrete, noncontinuous SDMs are preferred over continuous SDM due to their simplistic nature, reduced material consumption, and shorter operation time. The results presented in this study show the suitability of a discrete SDM approach, compared to a continuous SDM for a continuous protein A purification step.

An integrated continuous downstream process with real-time control: A case study with periodic countercurrent chromatography and continuous virus inactivation.

Integrated continuous downstream processes with process analytical technology offer a promising opportunity to reduce production costs and increase process flexibility and adaptability. In this case study, an integrated continuous process was used to purify a recombinant protein on laboratory scale in a two-system setup that can be used as a general downstream setup offering multiproduct and multipurpose manufacturing capabilities. The process consisted of continuous solvent/detergent virus inactivation followed by periodic countercurrent chromatography in the capture step, and a final chromatographic polishing step. A real-time controller was implemented to ensure stable operation by adapting the downstream process to external changes. A concentration disturbance was introduced to test the controller. After the disturbance was applied, the product output recovered within 6 h, showing the effectiveness of the controller. In a comparison of the process with and without the controller, the product output per cycle increased by 27%, the resin utilization increased from 71.4% to 87.9%, and the specific buffer consumption was decreased by 21% with the controller, while maintaining a similar yield and purity as in the process without the disturbance. In addition, the integrated continuous process outperformed the batch process, increasing the productivity by 95% and the yield by 28%.

Review of Membrane Separation Models and Technologies: Processing Complex Food-Based Biomolecular Fractions

There is growing interest in the food industry to develop approaches for large-scale production of bioactive molecules through continuous downstream processing, especially from sustainable sources. Membrane-based separation technologies have the potential to reduce production costs while incorporating versatile multiproduct processing capabilities. This review describes advances in membrane technologies that may facilitate versatile and effective isolation of bioactive compounds. The benefits and drawbacks of pressure-driven membrane cascades, functionalized membranes and electromembrane separation technologies are highlighted, in the context of their applications in the food industry. Examples illustrate the separation of functional macromolecules (peptides, proteins, oligo/polysaccharides, plant secondary metabolites) from complex food-based streams. Theoretical and mechanistic models of membrane flux and fouling are also summarized. Overcoming existing challenges of these technologies will provide the food industry with several attractive options for bioprocessing operations.

Continuous downstream bioprocessing for intensified manufacture of biopharmaceuticals and antibodies

Continuous production delivers higher productivity and better quality than traditional batch-wise approaches. It intensifies production lowering capital cost and enables better control. Despite obvious advantages, continuous processing has not yet guided biomanufacturing to 21st Century Advanced Manufacturing status. Production relies primarily on batch-wise methods that have served the industry through its infancy. While great improvements have been achieved on the upstream side, downstream processing lacks development in continuous processing and is now the handbrake on modernisation. Nevertheless, there are hopeful advances. Research on continuous chromatographic purification for antibodies is maturing, and work has commenced on other unit operations and on process system integration. This exciting field of process intensification research is at a turning point, though considerably more research is needed. This review aims to summarize the latest developments and capabilities of continuous downstream processing applied in biopharmaceutical research and gives an overview of recent developments.

Process Development for 6-Aminopenicillanic Acid Production Using Lentikats-Encapsulated Escherichia coli Cells Expressing Penicillin V Acylase.

Penicillin V acylase (PVA, EC 3.5.1.11) hydrolyzes the side chain of phenoxymethylpenicillin (Pen V) and finds application in the manufacture of the pharmaceutical intermediate 6-aminopenicillanic acid (6-APA). Here, we report the scale-up of cultivation of Escherichia coli whole cells expressing a highly active PVA from Pectobacterium atrosepticum and their encapsulation in polyvinyl alcohol–poly(ethylene glycol) Lentikats hydrogels. A biocatalytic process for the hydrolysis of 2% (w/v) Pen V was set up in a 2 L reactor using the Lentikats-immobilized whole cells, with a customized setup to enable continuous downstream processing of the reaction products. The biocatalytic reaction afforded complete conversion of Pen V for 10 reaction cycles, with an overall 90% conversion up to 50 cycles. The bioprocess was further scaled up to the pilot-scale at 10 L, enabling complete conversion of Pen V to 6-APA for 10 cycles. The 6-APA and phenoxy acetic acid products were recovered from downstream processing with isolated yields of 85–90 and 87–92%, respectively. Immobilization in Lentikats beads improved the stability of the whole cells on storage, maintaining 90–100% activity and similar conversion efficiency after 3 months at 4 °C. The robust PVA biocatalyst can be employed in a continuous process to provide a sustainable route for bulk 6-APA production from Pen V.

Modeling the Residence Time Distribution of Integrated Continuous Bioprocesses.

In response to the biopharmaceutical industry advancing from traditional batch operation to continuous operation, the Food and Drug Administration (FDA) has published a draft for continuous integrated biomanufacturing. This draft outlines the most important rules for establishing continuous integration. One of these rules is a thorough understanding of mass flows in the process. A computer simulation framework is developed for modeling the residence time distribution (RTD) of integrated continuous downstream processes based on a unit-by-unit modeling approach in which unit operations are simulated one-by-one across the entire processing time, and then combined into an integrated RTD model. The framework allows for easy addition or replacement of new unit operations, as well as quick adjustment of process parameters during evaluation of the RTD model. With this RTD model, the start-up phase to reach steady state can be accelerated, the effects of process disturbances at any stage of the process can be calculated, and virtual tracking of a section of the inlet material throughout the process is possible. A hypothetical biomanufacturing process for an antibody was chosen for showcasing the RTD modeling approach.

Viral clearance capacity by continuous Protein A chromatography step using Sequential MultiColumn Chromatography

In response to the strong demand of biological protein therapeutics, such as monoclonal antibodies (MAbs), continuous downstream process was developed to deliver these molecules while maintaining desired product consistency and quality attributes, and providing manufacturing efficiency and flexibility. Viral safety is a critical quality attribute for biopharmaceuticals, such as MAbs. Evaluation of the viral clearance by the downstream process is a key component of risk mitigation. Protein A chromatography is typically used as an initial capture step for MAbs and efficient for the removal of process-related impurities like Host Cell Proteins (HCP). This step can also contribute to the clearance of potential viral contaminants.Murine Minute Virus (MMV)-spiking experiments were performed at small scale to investigate the impact on the viral clearance efficiency of the way the Protein A chromatography step is carried out, whether in batch or multicolumn mode. Protein A chromatography step using Novasep Sequential MultiColumn Chromatography (SMCC) technology demonstrated no statistical difference in the viral reduction with reduction factor (RF) of 3.7 log10 (vs. RF of 3.8 log10 for batch). The experiments showed also similar viral distribution over the purification cycles and columns.Data confirmed that the viral clearance capacity by the continuous Protein A chromatography step using SMCC technology is maintained and efficient.

The effect of pH on antibody retention in multimodal cation exchange chromatographic systems

The present paper builds upon previous work on mAb domain contributions to multimodal (MM) chromatography by examining how pH can impact mAb surface properties and retention in these systems. Linear salt gradient experiments were carried out between pH 5–7 for several mAbs with different pI and surface hydrophobicities in four different MM CEX resins at two ligand densities. mAb retention showed an inverse, non-linear correlation with pH. Changing pH affected the elution order, creating unique windows of selectivity in each of the MM CEX resins. One mAb showed a pH-dependent spectrum of domain contributions, demonstrating that pH can be used to tune the relative importance of the (Fab)2 and Fc domains for some mAbs in MM systems. Positive, negative, and hydrophobic patches were calculated between pH 5–7 for the mAbs. Visualizing these patches on the protein surface demonstrated that each mAb showed a unique distribution of surface charge and hydrophobicity that changed with pH. The sum of patch areas was tracked across this pH range to quantitatively understand how pH impacted these important surface properties. The quantitative analysis then was narrowed to consider only patches in the CDR loops, which were hypothesized to be an important interaction site for some mAbs in these systems. Interestingly, differences in the titration of CDR loop patches for each mAb were shown to be a result of Histidine titrations and patches in this region were qualitatively correlated with experimental trends including the observed elution order reversals. These results indicate that pH potentially can be employed as a lever for the strategic design of multimodal steps to create flow through, bind and elute, or weak partitioning operations with important implications for the design of integrated and/or continuous downstream purification processes. Furthermore, the ability to tune domain contributions in MM separations using pH creates intriguing possibilities for current downstream challenges such as the removal of product-related impurities, as well as the purification of bispecific mAbs.

Continuous Formulation Approaches of Amorphous Solid Dispersions: Significance of Powder Flow Properties and Feeding Performance.

Preparation and formulation of amorphous solid dispersions (ASDs) are becoming more and more popular in the pharmaceutical field because the dissolution of poorly water-soluble drugs can be effectively improved this way, which can lead to increased bioavailability in many cases. During downstream processing of ASDs, technologists need to keep in mind both traditional challenges and the newest trends. In the last decade, the pharmaceutical industry began to display considerable interest in continuous processing, which can be explained with their potential advantages such as smaller footprint, easier scale-up, and more consistent product, better quality and quality assurance. Continuous downstream processing of drug-loaded ASDs opens new ways for automatic operation. Therefore, the formulation of poorly water-soluble drugs may be more effective and safe. However, developments can be challenging due to the poor flowability and feeding properties of ASDs. Consequently, this review pays special attention to these characteristics since the feeding of the components greatly influences the content uniformity in the final dosage form. The main purpose of this paper is to summarize the most important steps of the possible ASD-based continuous downstream processes in order to give a clear overview of current course lines and future perspectives.

Economics and ecology: Modelling of continuous primary recovery and capture scenarios for recombinant antibody production

With the maturation of antibody production technologies, both economic optimization and ecological aspects have become important. Continuous downstream processing is a way to reduce the environmental footprint and improve process economics. We compared different primary recovery, capture, and fermentation methods for two output-based antibody production scales: 50 kg/year and 1000 kg/year. In addition, a fixed fermentation volume case of 1000 L was analysed in terms of total cost of goods and process mass intensity as a measure of the environmental footprint. In our scenario, a significant amount of water can be saved in downstream processing when single use equipment is utilized. The overall economic and ecological impact is governed by the product titre in our perfusion (1 g/L) and fed-batch (4 g/L). A low titre in fermentation with similar downstream purification leads to higher process mass intensity and cost of goods due to the higher media demand upstream. The economic perspective for continuous integrated biomanufacturing is very attractive, but environmental consequences should not be neglected. Here, we have shown that perfusion has a higher environmental footprint in the form of water consumption compared to fed-batch. As general guidance to improve process economics, we recommend reducing water consumption.

The Rocky Road From Fed-Batch to Continuous Processing With E. coli.

Escherichia coli still serves as a beloved workhorse for the production of many biopharmaceuticals as it fulfills essential criteria, such as having fast doubling times, exhibiting a low risk of contamination, and being easy to upscale. Most industrial processes in E. coli are carried out in fed-batch mode. However, recent trends show that the biotech industry is moving toward time-independent processing, trying to improve the space–time yield, and especially targeting constant quality attributes. In the 1950s, the term “chemostat” was introduced for the first time by Novick and Szilard, who followed up on the previous work performed by Monod. Chemostat processing resulted in a major hype 10 years after its official introduction. However, enthusiasm decreased as experiments suffered from genetic instabilities and physiology issues. Major improvements in strain engineering and the usage of tunable promotor systems facilitated chemostat processes. In addition, critical process parameters have been identified, and the effects they have on diverse quality attributes are understood in much more depth, thereby easing process control. By pooling the knowledge gained throughout the recent years, new applications, such as parallelization, cascade processing, and population controls, are applied nowadays. However, to control the highly heterogeneous cultivation broth to achieve stable productivity throughout long-term cultivations is still tricky. Within this review, we discuss the current state of E. coli fed-batch process understanding and its tech transfer potential within continuous processing. Furthermore, the achievements in the continuous upstream applications of E. coli and the continuous downstream processing of intracellular proteins will be discussed.

Optimization of factors influencing enzyme activity and product selectivity and the role of proton transfer in the catalytic mechanism of patchoulol synthase.

The patchoulol synthase (PTS) from Pogostemon cablin is a versatile sesquiterpene synthase and produces more than 20 valuable sesquiterpenes by conversion of the natural substrate farnesyl pyrophosphate (FPP). PTS has the potential to be used as a biocatalyst for the production of valuable sesquiterpenes such as (-)-patchoulol. The objective of the present study is to develop an efficient biotransformation and to characterize the biocatalytic mechanism of the PTS in detail. For this purpose, soluble PTS was prepared using an optimized cultivation protocol and continuous downstream process with a purity of 98%. The PTS biotransformation was then optimized regarding buffer composition, pH-value, and temperature for biotransformation as well as functional and kinetic properties to improve productivity. For the bioconversion of FPP, the highest enzyme activity was reached with the 2-(N-morphlino)ethanesulfonic acid (MES) buffer containing 10% (v/v) glycerol and 10 mM MgCl2 at pH 6.4 and 34°C. The PTS showed an unusual substrate inhibition for sesquiterpene synthases indicating an intermediate sesquiterpene formed in the active center. Deuteration experiments were used to gain further insights into the biocatalytic mechanism described in literature. Thus it could be shown that a second substrate binding site must be responsible for substrate inhibition and that further protonation and deprotonation steps are involved in the reaction mechanism.

Integrated system for temperature-controlled fast protein liquid chromatography. III. Continuous downstream processing of monoclonal antibodies

Three different applications of travelling heating zone reactor (THZR) chromatography for the downstream processing of monoclonal antibodies (mAbs) are described. mAb containing feedstocks were applied to a fixed bed of the thermoresponsive rProtein A matrix, Byzen Pro™, contained in a bespoke column (held at 15 °C) fitted with a travelling heating (42 °C) device encircling a narrow section of the column. For the demonstration of continuous concentration, uninterrupted loading of 1.0 g/L mAb in a pH 8 binding buffer was synchronized with 5 repeated movements of the heating zone along the column’s full length at a velocity of 0.1 mm/s. Elution of mAbs was induced solely by the travelling heating zone’s action, each full movement generating a sharp concentrated elution peak accompanied by a small transient mAb concentration-dependent dip in conductivity. Quasi-steady-state operation occurred from the third elution onwards, delivering a mean mAb concentration of 4.9 g/L and process yield >93%. Quasi-continuous separation of the target mAb (1.41 g/L) from bovine serum albumin, BSA (1.0 g/L), was achieved by cyclically alternating the feeding of the mAb + BSA feedstock, with that of the binding buffer alone; supply of the latter was timed to coincide with movement of the heating zone. Accurate coordination of the heating zone’s travel and switching from feed to buffer permitted quasi-steady-state collection (elutions 3–6) of sharp peaks of mAb in high purity (98.7%) and yield (88.7%) in 4.5–fold concentrated form, with BSA exiting in the flow through fractions between successive mAb elution peaks. Fully automated THZR-mediated quasi-continuous buffer exchange of 1.34 g/L mAb from a phosphate buffer pH 8 into a HEPES buffer pH 8 of slightly lower conductivity was performed over a 19 h period by carefully timed switching from one feed solution to the other and back again, whilst synchronising movement of the heating zone with feeding of the exchange buffer. Quasi-steady-state operation (elutions 2–9) resulted in an average eluted mAb yield of 94.5% and concentration of 4.8 g/L. Triggering movement of the heating zone slightly ahead of the switch from mAb feed to exchange buffer permitted the positioning of mAb elution peaks in 9 mL volume segments with the lowest recorded conductivity. Measurements of buffer exchange performance conducted with two ‘protein-free’ systems demonstrated that compared to tangential flow filtration in diafiltration mode, which represents the ‘state-of-the-art’ technology for buffer exchange, the THZR chromatography based approach affords a >60% saving in minimum volume of exchange buffer required to remove 99.9% of the original buffer. Combined far and near UV circular dichroism, intrinsic fluorescence and thermal melting experiments showed that, unlike conventional Protein A/G affinity chromatography, the conditions for THZR Protein A chromatography respect maintenance of a favourable structural profile for mAbs.

Continuous precipitation for monoclonal antibody capture using countercurrent washing by microfiltration.

There is renewed interest in the possibility of using precipitation for initial capture of high-value therapeutic proteins as part of an integrated continuous downstream process. Precipitation is greatly facilitated by the high product titers now achieved in most cell culture processes, in sharp contrast to chromatographic processes whose performance is reduced at high titers. The current study used a combination of reversible cross-linking (zinc chloride, ZnCl2 ) and volume exclusion (polyethylene glycol) agents to precipitate a monoclonal antibody product directly from harvested cell culture fluid using a continuous tubular precipitation reactor. The precipitates were then dewatered and continuously washed using tangential flow filtration, with a countercurrent-staged configuration used to reduce the amount of wash buffer required and increase host cell protein removal. Long-term operation was achieved by operating the membrane modules below the critical filtrate flux to avoid fouling. Experimental results demonstrate the feasibility of this fully continuous integrated precipitation process at bench scale, with design calculations used to explore the key factors affecting the performance of this system for initial antibody capture.

Optimization of integrated chromatography sequences for purification of biopharmaceuticals.

With continued development of integrated and continuous downstream purification processes, tuning and optimization become increasingly complicated with additional parameters and codependent variables over the sequence. This article offers a novel perspective of nonlinear optimization of integrated sequences with regard to individual column sizes, flow rates, and scheduling. The problem setup itself is a versatile tool to be used in downstream design which is demonstrated in two case studies: a four‐column integrated sequence and a continuously loaded twin‐capture setup with five columns.

Optimization of continuous purification of recombinant patchoulol synthase from Escherichia coli with membrane adsorbers.

The natural production of patchouli oil in developing countries cannot meet the increasing demand any more. This leads to socioecological consequences, such as the use of arable land, which is actually intended for food. Hence, the world market price increased up to $150/kg. An alternative is the biotechnological production of patchouli oil using a multiproduct sesquiterpene synthase, the patchoulol synthase (PTS). Here, we report the optimization of recombinant PTS purification from Escherichia coli lysate using continuous immobilized metal affinity chromatography. First, the purification conditions of the batch process were optimized in regard to optimal buffer composition and optimized chromatographic conditions. The best purification result was achieved with Co2+ -immobilized metal affinity chromatography (Sartobind® IDA 75) with a triethanolamine buffer at pH 7, 0.5 M NaCl, 10% [vol/vol] glycerol, 5 mM MgCl2 and 250 mM imidazole for product elution. This optimized method was then transferred to a continuous chromatography system using three membrane adsorber units (surface of 75 cm2 each). Within 1.5 hr in total, 4.55 mg PTS with a final purity of 98% and recovery of 68% could be gained. The purified enzyme was used to produce 126 mg/L (-)-patchoulol from farnesyl pyrophosphate. Here, for the first time bioactive PTS was successfully purified using membrane adsorbers in a continuous downstream process.

Connected flow-through chromatography processes as continuous downstream processing of proteins

Continuous manufacturing is expected to increase the productivity of protein drug production. However, it is not easy to build the continuous process especially for downstream processing as many unit operations (chromatography and membrane filtration) are involved. An operation method known as flow-through chromatography (FTC) is considered to be an efficient method for separating two components as the flow is continuous. In FTC, a target protein is eluted from the chromatography column without adsorption whereas contaminants are strongly bound. Since at least two different modes of chromatography are needed in order to remove contaminants, two FTC columns have to be connected in order to build the continuous process. This is not an easy task since the mobile phase properties (pH, salt, buffer ions) are different for the two columns. In this paper, we investigated how connected FTC columns can remove impurities efficiently from the cell culture broth containing monoclonal antibody. It was found that the sequence (activated carbon - anion exchange chromatography - cation exchange chromatography) is most efficient when the mobile phase pH and conductivity were properly chosen.

Cyclic operation of a semi-batch reactor for the hydroformylation of long-chain olefins and integration in a continuous production process

The transition to renewable feedstocks in chemicals production requires innovative processes which are able to exploit the special properties of the feed material while still maintaining high process performance. Flexible semi-batch processes offer the advantage of dynamic adaptation to changing process requirements but suffer in terms of automation and production capacity where continuous processes excel. Combining the flexibility of a semi-batch reactor with the reliability of a continuous downstream process is the motivation for the application of a repeatedly operated semi-batch reactor (RSBR) concept to the hydroformylation of 1-dodecene in a n-decane/DMF thermomorphic multiphase system (TMS). In order to predict the dynamic process behavior, a detailed dynamic process model is introduced and compared to a corresponding steady-state model. In addition, the RSBR concept is embedded in a miniplant process to prove its feasibility and convergence to a cyclic steady-state experimentally. Finally, the collected experimental data is compared to the results from the dynamic process model indicating accurate predictions of the integral process behavior.

Progression of continuous downstream processing of monoclonal antibodies: Current trends and challenges.

Rapid advances in intensifying upstream processes for biologics production have left downstream processing as a bottleneck in the manufacturing scheme. Biomanufacturers are pursuing continuous downstream process development to increase efficiency and flexibility, reduce footprint and cost of goods, and improve product consistency and quality. Even after successful laboratory trials, the implementation of a continuous process at manufacturing scale is not easy to achieve. This paper reviews specific challenges in converting each downstream unit operation to a continuous mode. Key elements of developing practical strategies for overcoming these challenges are detailed. These include equipment valve complexity, favorable column aspect ratio, protein-A resin selection, quantitative assessment of chromatogram peak size and shape, holistic process characterization approach, and a customized process economic evaluation. Overall, this study provides a comprehensive review of current trends and the path forward for implementing continuous downstream processing at the manufacturing scale.

Current state of the art in continuous bioprocessing.

There is an upsurge of interest in continuous bioprocessing, but currently continuous downstream bioprocessing has not been implemented to generate clinical material. This review focusses on the current state of the art of continuous downstream processing, highlighting the key advantages over traditional batch manufacturing. This allows the identification of scenarios where continuous downstream processing may be critical for commercial manufacturing success.