Flow management strategies for a connected purification process.

This paper describes different flow management strategies for a connected purification process which includes two polishing steps, virus filtration and tangential flow filtration. Connecting these unit operations avoids introducing large intermediate product pool vessels in small manufacturing facilities. However, a connected-downstream process requires an elaborate control strategy enabling multiple unit operations to function as a single unit. The key strategy to enable the connected-downstream process is a robust management of flow disparities among unit operations. During a typical ultrafiltration step, product concentration increases as mass is added to the retentate tank, leading to a permeate flux decline. In a connected-downstream process, the inlet stream is directly connected to the prior unit operation and any decrease in permeate flow rate could cause a flow disparity. Four different flow management approaches are proposed to manage potential flow disparities and their advantages and challenges are discussed. Bench-scale results of these strategies are presented and evaluated.

Highland games: A benchmarking exercise in predicting biophysical and drug properties of monoclonal antibodies from amino acid sequences.

Biopharmaceutical product and process development do not yet take advantage of predictive computational modeling to nearly the degree seen in industries based on smaller molecules. To assess and advance progress in this area, spirited coopetition (mutually beneficial collaboration between competitors) was successfully used to motivate industrial scientists to develop, share, and compare data and methods which would normally have remained confidential. The first "Highland Games" competition was held in conjunction with the October 2018 Recovery of Biological Products Conference in Ashville, NC, with the goal of benchmarking and assessment of the ability to predict development-related properties of six antibodies from their amino acid sequences alone. Predictions included purification-influencing properties such as isoelectric point and protein A elution pH, and biophysical properties such as stability and viscosity at very high concentrations. Essential contributions were made by a large variety of individuals, including companies which consented to provide antibody amino acid sequences and test materials, volunteers who undertook the preparation and experimental characterization of these materials, and prediction teams who attempted to predict antibody properties from sequence alone. Best practices were identified and shared, and areas in which the community excels at making predictions were identified, as well as areas presenting opportunities for considerable improvement. Predictions of isoelectric point and protein A elution pH were especially good with all-prediction average errors of 0.2 and 1.6 pH unit, respectively, while predictions of some other properties were notably less good. This manuscript presents the events, methods, and results of the competition, and can serve as a tutorial and as a reference for in-house benchmarking by others. Organizations vary in their policies concerning disclosure of methods, but most managements were very cooperative with the Highland Games exercise, and considerable insight into common and best practices is available from the contributed methods. The accumulated data set will serve as a benchmarking tool for further development of in silico prediction tools.

Integrated use of ultra scale-down and financial modeling to identify optimal conditions for the precipitation and centrifugal recovery of milk proteins.

This article investigates the integrated application of ultra scale-down (USD) techniques and economic modeling as a means for identifying optimal bioprocess operating conditions. The benefits of the approach are illustrated for the recovery of lactoperoxidase (LPO) from bovine milk. In the process, milk is skimmed to deplete its lipid content, before being subjected to low pH incubation with acetic acid in order to precipitate the primary impurity (casein). Following removal of the solids by disk stack centrifugation, pH adjustment and filtration, cation exchange chromatography is used as a positive mode column step to bind the LPO before it is polished and freeze dried. An economic model of this process was used to identify where greatest product loss occurs and hence where the largest opportunity cost was being incurred. Scale-down analysis was used to characterize the influence of the critical steps, identified as precipitation and centrifugation, upon LPO recovery. A mathematical model was used to relate the centrifuge feed flowrate and discharge interval to the supernatant yield, and it was shown that increasing the centrifugal solids residence time achieved superior solids de-watering and so higher product yield, although this also increased the overall processing time. To resolve this conflict, scale-down data were used again in conjunction with an economic model to determine the most suitable conditions that maximized annual profit and minimized operating costs. The results demonstrate the power of combining USD data with models of economic and process performance in order to establish the best overall operating strategies for biopharmaceutical manufacture.

A methodical approach to ultra-scale-down of process sequences: application to casein removal from the milk of transgenic animals.

Scale-down models of individual operations are widely used in biopharmaceutical process development to obtain information about the performance of production-scale equipment on the basis of inexpensive and efficient laboratory-scale tests, for the purposes of validation or optimization or characterization studies. We have investigated the ability of scale-down models of whole process sequences to provide reliable information for process scale-up from laboratory- to pilot-scales of operation. Using the example of the recovery of a protein from transgenic milk, we have conducted an a priori scale-down analysis of a projected pilot-scale process sequence. A systematic approach was developed to ensure that all critical aspects of process behavior were included in the scale-down model, resulting in the creation of an accurate and reliable scale-down representation of the pilot-scale process. The data from scale-down process trials conducted at 70 and 200 mL scales of operation served to highlight crucial factors determining process performance, and proved reliable in predicting the performance of the pilot-scale process over a scaling factor of 1000.

The influence of major components on the direct chromatographic recovery of a protein from transgenic milk.

This work presents a systematic evaluation of the influence of lipids and casein on the performance of a chromatographic capture step for the recovery of a target protein from transgenic milk. Lactoperoxidase (LPO) was spiked at concentrations typical of those to be expected for transgenic proteins in commercial bovine milk and the dynamic adsorption of LPO to fixed beds of SP Sepharose FF studied in frontal analysis experiments. By removing successively selected components from whole milk, their individual influence on the dynamic adsorption behaviour of LPO could be studied. A mathematical model, fitted to the breakthrough curves of LPO, provided a quantitative measure of parameters describing mass transfer and adsorption in the column. A significant reduction in column capacity for LPO in the presence of milk or whey was recorded, which could be attributed to competing adsorption of alkaline earth metal ions to the cation exchange resin. While the high concentrations of lipids present in whole milk did strongly reduce the column permeability, no significant influence of either casein or low concentrations of lipids on the hydraulic properties of columns or on the adsorption of LPO could be detected. The results indicate that chromatography, which forms an essential part of all current large-scale processes for the recovery of proteins from transgenic milk, could potentially be moved further upstream. Alternatively, existing operations for the removal of lipid and casein could be re-designed so as to maximise product yields. This suggests that significant product losses during current pre-chromatography milk purification could be reduced or potentially even avoided.