CODOBIO - Continuous Downstream Processing of Biologics

The economic benefits of continuous PEG precipitation of antibodies compared to continuous capture by protein A affinity chromatography have been always lowered by the excess amount of material needed for the process. PEG is added as a concentrated stock solution and therefore liquid handling is increased during this step. To fully exploit the benefits of PEG precipitation, the precipitant must be added in solid form. We used an in-line feeding device with a screw conveyor delivery system for the continuous addition of PEG6000 in a powder form. The powder feeding device was connected to a tubular reactor where the precipitation occurs. Protein precipitation was continuously performed for 4 h. A yield of 76 % and 79 % with a purity of 98 % was achieved. The total cost of goods and the environmental footprint were compared with typical chromatography-based purification methods; batch and continuous periodic countercurrent protein A affinity chromatography with four columns. Solid PEG precipitation showed a remarkable reduction in water consumption and equipment size, reducing production costs by 45 % compared to liquid PEG and 53 % cheaper than Protein A periodic counter-current chromatography. Process mass intensity was reduced by 55 % and carbon emissions by 60 %. The reduction of water by the direct addition of PEG also impacted the environmental footprint and process costs. This is an attractive approach for a continuous capture step yielding an uninterrupted mass flow of the product and will pave the way for PEG precipitation as a capture step.

Protein solubility is a critical attribute in the development and production of monoclonal antibodies. Available solubility data refer to pure solutes which do not consider solvents and impurities which have a significant effect on solubility. Thus, solubility curves need to be determined experimentally. Previously established methods to determine the apparent solubility of proteins are based on manual assays which are time-consuming and labor-intensive. We present the design of simple and adaptable millidevices for fast solubility curve determination. Such a device in the form of a tubular reactor was manufactured from polymethylmethacrylate by laser cutting. The reactors had multiple injection points for the precipitant that allowed a controlled and precise addition of the precipitating agent at different concentrations. Hence, antibodies could be directly harvested at different concentrations of precipitating agents. The simple and flexible design allowed the number of pumps required to be reduced to only one for each solution and the distribution of the precipitating agent at different concentrations without valves. To demonstrate the wide applicability of the prototype in determining solubility curves, we used 2 industrially relevant precipitating agents, PEG6000 and ZnCl2, to measure the apparent solubilities of 4 antibodies and CaCl2 to measure the apparent solubility curves for dsDNA. In all cases, the data obtained were consistent between the device and manual assays with good reproducibility. This millidevice can be used for fast characterization of protein solutions such as solubility, degradation or stability of the antibodies under different conditions.

It is a big challenge in developing integrated continuous biomanufacturing processes when the parameters for design of the process cannot be accurately estimated from batch experiments. Process design is even more challenging if the outcome of one‐unit operation highly influences the performance of the subsequent one such as in harvesting of a precipitate by filtration. In case of protein precipitation, results from batch and continuous deviate and in addition the scale down is limited. Microfluidics suffer from poor mixing characteristics and therefore we developed milliscale devices to keep the mixing performance but on the expense of slightly larger scale. We have developed milliscale devices for the precipitation of antibodies in continuous tubular reactors to compare different dosage times in small scale. The reactors have multiple addition points for precipitant for a continuous, controlled and precise addition of the precipitating agent without valves. The designed devices have a narrow residence time distribution to achieve fast mixing, and a small volume that reduce time and cost for experiments tenfold. Milliscale devices were used to evaluate the most appropriate dosage time for protein precipitation, improving purity by a factor of 3 compared to single addition. The resulting filterability of precipitates in TFF and depth filtration was improved. We demonstrated that multiple additions were beneficial compared to single addition, reducing pressure and increasing filter capacity. Such devices can be used to determine and adjust the precipitation methodology to optimize floc formation and improving solid–liquid separation while reducing development time and cost. This article is protected by copyright. All rights reserved.

The trend in the biopharmaceutical industry is changing from batch process to continuous process. For continuous biomanufacturing, traceability of the material is required by regulatory authorities. The recent ICH draft guideline Q13 on continuous manufacturing of drug substances and drug products requests an “understanding of process dynamics as a function of input material attributes (e.g., potency, material flow properties), process conditions (e.g., mass flow rates) … One common approach is characterization of residence time distribution (RTD) for the individual unit operations and integrated system.” Thus, it is necessary to trace material through individual continuous unit operations and the integrated process. The RTD of a process is obtained experimentally by injecting a pulse of an inert tracer into the inlet and measuring the broadening of the injected pulse in the outlet. We investigated the RTD of three-column periodic counter-current chromatography (PCC) using staphylococcal protein A affinity chromatography, with a focus on how the material distributes over subsequent cycles. A fluorescent-labeled antibody was used as the inert tracer under high salt concentration. The tracer was injected once in each run but at different points of the loading phase. We then analyzed the outlet of the column. In the elution phase, regardless of the point of injection, we observed an even distribution of the tracer. In the loading phase, a constant exchange between the antibody in the solid phase and the liquid phase was observed, meaning that sending the outlet of one chromatography column into another column to improve resin utilization causes higher residence time in the system for some portion of the material.

Precipitation has gained interest as alternative to the costly protein A chromatography for monoclonal antibody purification. Traditional precipitation processes are based on direct addition of precipitant in a single dose, with limited control on co-precipitation of impurities and not considering batch-to-batch variations. We propose a gradual dosage of polyethylene glycol to prevent co-precipitation and control resulting floc size. We used focused beam reflectance measurement to demonstrate that the PEG6000 dosage time and the final concentration significantly changes the particle size distribution (PSD). We demonstrated that gradual and stepwise precipitant addition was superior to conventional batch PEG precipitation, improving product yield and purity by a factor of 4 for HCP removal, for samples pre-treated with CaCl2 and caprylic acid. We studied the 3D structure of the precipitates by fractal dimension and showed that precipitates exhibited different compactness and density depending on the dosage time, resulting in different filterability in tangential flow filtration and depth filtration. To switch from batch to continuous PEG addition, the 3D structure of precipitates needs to be considered due to its high impact on the resulting process performance. Focused beam reflectance measurement (FBRM) and fractal dimension can be used to adjust the precipitation methodology to improve the product quality attributes and inform about the design of further purification steps.

In recent years, there has been an increased interest in exploring the potential of micro-and mesoscale milling technologies for developing cost-effective microfluidic systems with high design flexibility and a rapid microfabrication process that does not require a cleanroom. Nevertheless, the number of current studies aiming to fully understand and establish the benefits of this technique in developing high-quality microsystems with simple integrability is still limited. In the first part of this study, we define a systematic and adaptable strategy for developing high-quality poly(methyl methacrylate) (PMMA)-based micromilled structures. A case study of the average surface roughness (Ra) minimization of a cuboid column is presented to better illustrate some of the developed strategies. In this example, the Ra of a cuboid column was reduced from 1.68 μm to 0.223 μm by implementing milling optimization and postprocessing steps. In the second part of this paper, new strategies for developing a 3D microsystem were introduced by using a specifically designed negative PMMA master mold for polydimethylsiloxane (PDMS) double-casting prototyping. The reported results in this study demonstrate the robustness of the proposed approach for developing microfluidic structures with high surface quality and structural integrability in a reasonable amount of time.

In the last decade, there has been a growing interest in developing microfluidic systems as new scale-down models for accelerated and cost-effective biopharmaceutical process development. Nonetheless, the research in this field is still in its infancy and requires further investigation to simplify and accelerate the microfabrication process. In addition, integration of different label-free sensors into the microcolumn systems has utmost importance to minimize result discrepancies during the scale-up process. In this study, we developed a simple, low-cost integrated microcolumn (26 μl). Micromilling technology was employed to define the geometry and shape of microfluidic structures using poly(methylmethacrylate) (PMMA). The design of PMMA microstructure was transferred to polydimethylsiloxane (PDMS), and interdigitated planar microelectrodes (IDE) were integrated into the system. To evaluate the scalability of the developed microcolumn column, column performance was assessed and compared with a conventional 1-ml prepacked column. Computational Fluid Dynamics (CFD) studies were performed for both columns to understand the differences between theoretical and experimental results regarding retention time and peak broadening. Despite obtaining an acceptable asymmetric factor for the microcolumn (1.03 ± 0.02), the reduced plate height value was still higher than the recommended range with the value of 4.14 ± 0.18. Nevertheless, the consistency and significant improvement of microcolumn efficiency compared to previous studies provide the possibility of developing robust simulation tools for transferring acquired experimental data for larger-scale units.

Parameter estimation for scale-up of downstream operations from microtiter plates (MTPs) is mostly done empirically because engineering correlations between microplates and stirred tank reactors (STRs) are not yet available. It is challenging to change the operation mode from shaken MTPs to large-scale STRs. For the scale-up of STRs, volumetric power input is well-established although it is unclear if this parameter can be used to transfer the operations from MTPs. We determine the volumetric power input in MTPs via the temperature increase caused by the motion of the liquid. The hydrodynamics in MTPs are studied with computational fluid dynamics (CFD). Mixing is investigated in 96-, 24- and six-well MTPs to cover different geometries, filling volumes, shaking diameters and shaking frequencies. All CFD simulations are validated by experimental results, which now allows prediction of the volumetric power input and hydrodynamics at various conditions in MTPs without the need for further experiments. We provide a map of the power input achievable in MTPs. Based on this map, from knowing about large-scale conditions, adequate microscale conditions can be adjusted for process development. This enables the direct scale-up of downstream unit operations from MTPs to STRs. This article is protected by copyright. All rights reserved.

Traditional chromatographic purification steps consist of long loading steps at a constant residence time, especially during early stages of the downstream process. The breakthrough behavior of the product is usually well defined. This information can be used to optimize the loading phase by starting the loading phase at a low residence time and gradually increasing the residence time to boost productivity and resin utilization. Process modeling of the loading of lysozyme on a Toyopearl SP-650C stationary phase, a strong cation exchanger, revealed optimal residence time gradients. These gradients result in a faster breakthrough in the time domain, while having the same breakthrough as constant residence time loading, in the volume domain. Modeling also revealed that a delayed start of the gradient further increases productivity. Eventually, several mathematical optimizations were employed to ensure the optimality of the idea. We confirmed the process model results in lab experiments using Lysozyme breakthrough in ion-exchange chromatography and monoclonal antibody breakthrough in protein A affinity chromatography. Productivity of the loading step could be increased by 68 % from 40.8 to 68.7 g L⁻¹ h⁻¹ resin for protein A chromatography while retaining the same breakthrough behavior and better manufacturability of instable proteins. Consequently, the bound fraction of protein is maintained above 99%. This affords similar productivity gains as multicolumn chromatography while employing an easy-to-implement process model that can be used on conventional systems.

BACKGROUND A major improvement in biomanufacturing will arise with the transition from batch processing to continuous processing. Two important challenges to address in this change are batch definition and the ability to trace raw material through the process. RESULTS We used an established simulation of a process train to compare the conventional batch definition based on a fixed time to a new batch definition method based on the greatest common divisor (GCD) of the time period of the unit operations. We successfully demonstrated that, by using the new concept based on GCD, we will have a constant periodic concentration of product. With this basis, we can define batches in a continuous process, which will lead to higher control over the process, and we will be able to trace the material through the process. CONCLUSION We achieved better control over the process using the batch definition based on the GCD method. In comparison to collecting the outlet products over arbitrary hours or days, collecting the product based on a section using the GCD method meets the criteria for knowing the residence-time distribution of the process, as advised by regulatory authorities. This method can be used in a continuous process or a hybrid process in which there are only a few continuous unit operations along with batch process operations. This article is protected by copyright. All rights reserved.

Recent advances in process analytical technologies and modelling techniques present opportunities to improve industrial chromatography control strategies to enhance process robustness, increase productivity and move towards real-time release testing. This paper provides a critical overview of batch and continuous industrial chromatography control systems for therapeutic protein purification. Firstly, the limitations of conventional industrial fractionation control strategies using in-line UV spectroscopy and on-line HPLC are outlined. Following this, an evaluation of monitoring and control techniques showing promise within research, process development and manufacturing is provided. These novel control strategies combine rapid in-line data capture (e.g. NIR, MALS and variable pathlength UV) with enhanced process understanding obtained from mechanistic and empirical modelling techniques. Finally, a summary of the future states of industrial chromatography control systems is proposed, including strategies to control buffer formulation, product fractionation, column switching and column fouling. The implementation of these control systems improves process capabilities to fulfil product quality criteria as processes are scaled, transferred and operated, thus fast tracking the delivery of new medicines to market.

Continuous manufacturing is an indicator of a maturing industry, as can be seen by the example of petrochemical industry. Patent expiry promotes a price competition between manufacturing companies, and more efficient and cheaper processes are needed to achieve lower production costs. Over the last decade, continuous biomanufacturing has had significant breakthroughs, with regulatory agencies encouraging industry to implement this processing mode. Process Development is resource and time consuming and, although it is increasingly becoming less expensive and faster through High‐Throughput Process Development (HTPD) implementation, reliable HTPD technology for integrated and continuous biomanufacturing is still lacking and is considered to be an emerging field. Therefore, this paper aims to illustrate the major gaps in HTPD and to discuss the major needs and possible solutions to achieve an end‐to‐end Integrated Continuous Biomanufacturing, as discussed in the context of the 2019 ICB Conference. The current HTPD state‐of‐the‐art for several unit operations is discussed, as well as the emerging technologies which will expedite a shift to a continuous biomanufacturing. This article is protected by copyright. All rights reserved.

Process development in the biotech industry leads to investments around hundred‐millions of dollars. It is important to mitigate costs without neglecting the quality of process development. Biopharmaceutical process development is important for companies to develop new processes and be first to market, improve a pre‐established process, or start manufacturing a product available by patent expiry (biosimilars). Laboratory automation enables methodical and standardized process development. Miniaturization and parallelization empower laboratories to screen several experimental conditions and define operating windows for purification processes, improving process robustness. Together, they allow for fast and accurate process development at a fraction of time and cost. The most widely used High‐Throughput Screening technique is a liquid‐handling station and microfluidics is taking its first steps in process development. Both are attractive scale‐down tools for the characterization of bioprocesses and allow to perform thousands of experiments per day. High‐Throughput Process Development (HTPD) has helped to achieve major breakthroughs in process optimization, both for Upstream and Downstream Processing. Continuous processing is the next step in process development which leads to cost reduction, higher productivity and better quality control; the integration of upstream and downstream processes is seen as a major challenge. In this review, we will focus on the state‐of‐the‐art of miniaturized techniques for process development in biotechnology industry and how automation and miniaturization drive process development. A comparison between liquid‐handling stations and microfluidics is made and an indication is given of which tools are still lacking for HTPD in the context of Integrated Continuous Biomanufacturing. This article is protected by copyright. All rights reserved.

The global pollution caused by plastics and microplastics is stimulating intense research towards more environmentally friendly materials, preserving the remarkable application characteristics of the currently available polymers. Among these, polyhydroxyalkanoates (PHAs) have been hailed as the solution to replace conventional, oil-based plastics. Given their biodegradable nature and mechanical properties, their use can be envisioned in a wide range of applications reducing the environmental footprint. Several types of processes have been proposed for their production, which can be grouped in three main classes: (i) microbiological, (ii) enzymatic and (iii) chemical processes. Given the significant amount of literature available on this topic, this review aims to critically analyse what has been proposed so far in each of these classes, with specific reference to their potential to provide bioplastics that can actually replace the currently available materials. A comparison is made, based on the following aspects: achievable molecular structures (such as molecular weight and composition distributions), raw-material and production costs and availability of large-scale production technologies. Finally, some considerations and ideas on what should be further investigated and implemented to realize the economically sustainable production of PHA are brought forward.