A fast, automated and accurate millidevice for determination of the apparent solubility of proteins and impurities and different industrially relevant precipitating agents.

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.

BACKGROUND The development of integrated continuous biomanufacturing processes faces a significant challenge when the parameters for the design of the process cannot be accurately estimated from those of 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 the case of protein precipitation, results from the batch and continuous experiements deviate and their scale-down is limited. Microfluidics suffer from poor mixing characteristics. Thus, milliscale devices were developed to maintain mixing performance but at the expense of a slightly larger scale. RESULTS Milliscale devices were developed 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 the 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 reduces the time and cost for experiments tenfold. Milliscale devices were used to evaluate the most appropriate dosage time for protein precipitation, thus improving purity by a factor of 3 compared to single addition. The resulting filterability of precipitates in tangential flow filtration and depth filtration was improved. CONCLUSION Results demonstrated that multiple additions were more beneficial than single addition because they help to reduce pressure and increase filter capacity. Such devices can be used to determine and adjust the precipitation methodology to optimize floc formation and improve solid-liquid separation while reducing development time and cost. (c) 2022 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI).

Most of the existing production capacity is based on fed‐batch bioreactors. Thanks to the development of more efficient cell lines and the development of high‐performance culture media, cell productivity dramatically increased. In a manufacturing perspective, it is necessary to clear as quickly as possible the protein A capture step to respect the manufacturing agenda. This article describes the methodology applied for the design of a multicolumn chromatography process with the objective of purifying as quickly as possible 1,000 and 15,000 L fed‐batch bioreactors. Several recent and reference protein A resins are compared based on characteristic values obtained from breakthrough curves. The importance and relevance of resin parameters are explained, and purposely simple indicators are proposed to quickly evaluate the potential of each candidate. Based on simulation data, the optimum BioSC systems associated with each resin are then compared. The quality of the elution delivered by each resin is also compared to complete the assessment. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:941–953, 2017

An integrated experimental and modeling approach for the design of sequential multi-column chromatography (SMCC) is presented to maximize productivity in bioprocessing. The approach consists of three steps: (1) single-column model development and validation, (2) multi-column model development and validation, and (3) productivity optimization. The integrated use of process experimentation and modeling enables sufficient process understanding to be gained during process development such that the optimal SMCC design is found even with limited time and materials. The application of the approach is demonstrated by determining the optimal SMCC design that maximizes the capture of human IgG by a silica-based protein A adsorbent named AbSolute. For this example, the optimum productivity was found to increase from 2.9kgL−1day−1 for batch operation to 4.0kgL−1day−1 for SMCC operation with three columns. A second case study considering a hypothetical adsorbent of larger particle size and slower mass transfer is also presented, to further demonstrate the applicability of the integrated approach. The case studies clearly illustrate the capabilities of the integrated approach in quickly determining the optimal design and operation for an SMCC arrangement and with minimal, carefully targeted, experimentation.

An integrated experimental and modeling approach for the design of high productivity protein A chromatography is presented to maximize productivity in bioproduct manufacture. The approach consists of four steps: (1) small-scale experimentation, (2) model parameter estimation, (3) productivity optimization and (4) model validation with process verification. The integrated use of process experimentation and modeling enables fewer experiments to be performed, and thus minimizes the time and materials required in order to gain process understanding, which is of key importance during process development. The application of the approach is demonstrated for the capture of antibody by a novel silica-based high performance protein A adsorbent named AbSolute. In the example, a series of pulse injections and breakthrough experiments were performed to develop a lumped parameter model, which was then used to find the best design that optimizes the productivity of a batch protein A chromatographic process for human IgG capture. An optimum productivity of 2.9 kg L-1 day(-1) for a column of 5 mm diameter and 8.5 cm length was predicted, and subsequently verified experimentally, completing the whole process design approach in only 75 person-hours (or approximately 2 weeks). (C) 2012 Elsevier B.V. All rights reserved.

Chiral preparative chromatography has long been disregarded as a potential means of production of chiral compounds at industrial scale. However, with the development of scalable chromatographic technologies including continuous processes, preparative chiral stationary phases (CSPs), and process development methodologies, it has now emerged as one of the most time-efficient and cost-effective means of producing large volumes of enantiomerically pure compounds. In this chapter, all aspects of industrial chiral chromatography are covered. This includes its principles and its differences with analytical chromatography; modern preparative CSPs, and method development; the main preparative chromatography techniques including batch processes such as high performance liquid chromatography (HPLC) and superficial fluid chromatography (SFC), continuous processes such as simulated moving bed and Varicol as well as solvent recycling technologies; current large-scale process development methodologies; and the successful integration of chiral chromatography in a global chiral synthesis through various industrial examples.

Productivity and buffer consumption of protein A chromatography are important process attributes for antibody capture, driven by industry's need to handle increasing upstream titer and market demand economically. In this work, a model-based optimization approach is presented which evaluates the productivity and buffer consumption of protein A chromatography for antibody capture. The approach offers great potential time and costs savings during process development compared with the traditional experimental approach. To demonstrate the application of the proposed model-based optimization approach, the approach is applied to a case study of antibody capture by a novel industrial silica-based protein A adsorbent.

A comprehensive description of a new process--the GSSR (Gradient with Steady State Recycle) process--for center-cut separation by solvent-gradient chromatography is provided, highlighting its versatility, flexibility, and ease of operation. The GSSR process is particularly suited for ternary separation of bioproducts: it provides three main fractions or cuts, with a target product contained in the intermediate fraction. The process comprises a multicolumn, open-loop system, with cyclic steady state operation, that simulates a solvent gradient moving countercurrently with respect to the solid phase. However, the feed is always injected into the same column and the product always collected from the same column as in a batch process; moreover, both steps occur only once per cycle. The GSSR process was experimentally validated in a pilot unit, using the purification of a crude peptide mixture by reversed phase as a proof of concept; the crude mixture is roughly 50% pure and some of its impurities have isocratic retention times very close to that of the target peptide. Experimental results are reported in terms of cyclic steady-state profiles and process performance indicators, which include product purity and yield. A simplified model-based approach, which uses only a few key components of the crude mixture, is employed to assist in the explanation of the process operation. By dynamically adjusting the switching interval while the process is running, to correctly position the composition profile with respect to the outlet ports, pure product satisfying the target specifications--98% purity and 95% recovery--was obtained under stable operation in the pilot unit.

This chapter contains sections titled: Introduction Basic Principles of Chromatography Process Optimization to Reduce Eluent Consumption Use of a Green Solvent: Supercritical Carbon Dioxide Solvent Recycling Technologies Application Examples Conclusion: An Environmentally Friendly Solution for Each Separation Acknowledgment References