Efficient MILP formulations for the optimal synthesis of chromatographic protein purification processes

The use of recombinant proteins has increased greatly in recent years, as well as the techniques used for their purification. The selection of an efficient process to purify proteins is a major bottleneck found when trying to scale up results obtained in the laboratory to a large-scale industrial process. One of the main challenges in the synthesis of downstream purification stages in biotechnological processes is the appropriate selection and sequencing of chromatographic steps. The objective of this work is to develop mixed integer linear programming models for the synthesis of protein purification processes. Models for each chromatographic technique rely on physicochemical data of a protein mixture, which contains the desired product and provide information on its potential purification. Formulations that are based on convex hull representations are proposed to calculate the minimum number of steps from a set of chromatographic techniques that must achieve a specified purity level and alternatively to maximize purity for a given number of steps. The proposed models are tested in several examples with experimental data and present time reductions of up to three orders of magnitude when compared to big-M formulations.     

MINLP models for the synthesis of optimal peptide tags and downstream protein processing

The development of systematic methods for the synthesis of downstream protein processing operations has seen growing interest in recent years, as purification is often the most complex and costly stage in biochemical production plants. The objective of the work presented here is to develop mathematical models based on mixed integer optimization techniques, which integrate the selection of optimal peptide purification tags into an established framework for the synthesis of protein purification processes. Peptide tags are comparatively short sequences of amino acids fused onto the protein product, capable of reducing the required purification steps. The methodology is illustrated through its application on two example protein mixtures involving up to 13 contaminants and a set of 11 candidate chromatographic steps. The results are indicative of the benefits resulting by the appropriate use of peptide tags in purification processes and provide a guideline for both optimal tag design and downstream process synthesis.     

Emergence of ideal membrane cascades for downstream processing

An algorithm is developed for describing ideal membrane cascades for fractionation of binary and pseudo-binary mixtures. It is shown that solvent management plays a key role in determining both purification and yield. Development of efficient diafilters is needed if membrane cascades are to achieve their full potential in competing with both chromatography and simulated moving bed operations in downstream processing of proteins. Such a replacement will also be important for fractionation of higher titers and larger substrates, such as plasmids, viruses, and even whole cells.     

Affinity ligands for industrial protein purification

Significant efforts are put into the design of large-scale purification processes of proteins due to great demands regarding cost efficiency and safety. In order to design an effective purification scheme the unit operations need to be reduced to a minimum. In this review we are discussing proteinaceous ligands as well as small synthetic mimics for use in affinity chromatography for large-scale applications. Different advantages as well as drawbacks of the two approaches are outlined.     

Optimal process synthesis for the production of multiple recombinant proteins

This paper presents a novel solution strategy for the synthesis of multiproduct and multihost protein production processes. There are several possible hosts that may express each of the products, and different downstream processing separation and purification tasks are needed, which in part depend on the host selection. Moreover, alternative unit operations may be available for some of these separation tasks. Finally, these processing units may be arranged in different configurations. A single mixed-integer optimization model represents the different decisions involved in synthesizing a plant for producing multiple proteins. The mathematical model optimizes the profit of the multiproduct plant and allows the decisions to be made simultaneously, namely, the choice of hosts, downstream operations, the configuration and size of units, as well as their scheduling. An example is solved for a plant that must produce four proteins for which there are alternative hosts for their expression (Escherichia coli, Chinese hamster ovary cells, and yeast that, depending on the product, may express it as an extracellular or intracellular protein) that require 15 stages with choices of unit operations as well as in or out of phase operations. Given the very large quantity of novel recombinant proteins for a number of novel therapeutic uses presently being approved or "in the pipeline", multiproduct and multihost recombinant protein production plants have recently been or are being built for the manufacture of these products. The strategy presented in this paper is of crucial value for the optimal utilization of such plants.     

Bromelain: an overview of industrial application and purification strategies

This review highlights the use of bromelain in various applications with up-to-date literature on the purification of bromelain from pineapple fruit and waste such as peel, core, crown, and leaves. Bromelain, a cysteine protease, has been exploited commercially in many applications in the food, beverage, tenderization, cosmetic, pharmaceutical, and textile industries. Researchers worldwide have been directing their interest to purification strategies by applying conventional and modern approaches, such as manipulating the pH, affinity, hydrophobicity, and temperature conditions in accord with the unique properties of bromelain. The amount of downstream processing will depend on its intended application in industries. The breakthrough of recombinant DNA technology has facilitated the large-scale production and purification of recombinant bromelain for novel applications in the future.     

Modeling of purification operations in biotechnology: enabling process development, optimization, and scale-up

Process modeling involves the use of a set of mathematical equations to represent key physical phenomena involved in the process. An appropriately validated model can be used to predict process behavior with limited experimental data, identify critical ranges for process variables, and guide further process development. Although process modeling is extensively used in the chemical process industries, it has not been widely used in purification unit operations in biotechnology. Recent FDA guidelines encourage the use of process modeling during process development, along with multivariate statistical methods, detailed risk assessment, and other quantifiers of uncertainty. This paper will review recent advances in the modeling of key downstream unit operations: chromatography, filtration, and centrifugation. The focus will be on the application of modeling for industrial applications. Relevant papers presented at a session on this topic at the recent American Chemical Society National Meeting in San Francisco will also be reviewed.     

Membrane adsorbers as purification tools for monoclonal antibody purification

Downstream purification processes for monoclonal antibody production typically involve multiple steps; some of them are conventionally performed by bead-based column chromatography. Affinity chromatography with Protein A is the most selective method for protein purification and is conventionally used for the initial capturing step to facilitate rapid volume reduction as well as separation of the antibody. However, conventional affinity chromatography has some limitations that are inherent with the method, it exhibits slow intraparticle diffusion and high pressure drop within the column. Membrane-based separation processes can be used in order to overcome these mass transfer limitations. The ligand is immobilized in the membrane pores and the convective flow brings the solute molecules very close to the ligand and hence minimizes the diffusional limitations associated with the beads. Nonetheless, the adoption of this technology has been slow because membrane chromatography has been limited by a lower binding capacity than that of conventional columns, even though the high flux advantages provided by membrane adsorbers would lead to higher productivity. This review considers the use of membrane adsorbers as an alternative technology for capture and polishing steps for the purification of monoclonal antibodies. Promising industrial applications as well as new trends in research will be addressed.     

Downstream processing of plasmid DNA for gene therapy and DNA vaccine applications

Interest in producing large quantities of supercoiled plasmid DNA has recently increased as a result of the rapid evolution of gene therapy and DNA vaccines. Owing to the commercial interest in these approaches, the development of production and purification strategies for gene-therapy vectors has been performed in pharmaceutical companies within a confidential environment. Consequently, the information on large-scale plasmid purification is scarce and usually not available to the scientific community. This article reviews downstream operations for the large-scale purification of plasmid DNA, describing their principles and the strategy used to attain a final product that meets specifications.     

Bioseparation in antibody manufacturing: the good, the bad and the ugly

Improvements in upstream production have boosted productivity in the biomanufacturing industry, but this is leading to bottlenecks in downstream processing as current technology platforms reach their limits of throughput and scalability. Although chromatography remains an indispensible component of downstream processing due to its simplicity and high resolving power (The Good), there is virtually no economy of scale effect so more product translates almost linearly into greater production costs. Bind-and-elute processes (such as the initial capture step in antibody manufacturing) are volume-driven and therefore have knock-on effects that impact on the entire production facility since the space required for preparation, storage, and cleaning steps has to be similarly adapted (The Bad). During long-term operations with multiple cycles, thorough cleaning is necessary to prevent progressive fouling and microbial contamination (The Ugly). Innovative solutions are required, which may include revisiting simpler and less expensive separation technologies, the use of disposable modules, and the integration of improved processes that are scalable to cope with increased demands. Among the alternatives that have been put forward, membrane adsorbers are beginning to make a real impact on the industry, particularly for flow-through applications such as polishing and viral clearance.     

Optimal synthesis of protein purification processes

There has been an increasing interest in the development of systematic methods for the synthesis of purification steps for biotechnological products, which are often the most difficult and costly stages in a biochemical process. Chromatographic processes are extensively used in the purification of multicomponent biotechnological systems. One of the main challenges in the synthesis of purification processes is the appropriate selection and sequencing of chromatographic steps that are capable of producing the desired product at an acceptable cost and quality. This paper describes mathematical models and solution strategies based on mixed integer linear programming (MILP) for the synthesis of multistep purification processes. First, an optimization model is proposed that uses physicochemical data on a protein mixture, which contains the desired product, to select a sequence of operations with the minimum number of steps from a set of candidate chromatographic techniques that must achieve a specified purity level. Since several sequences that have the minimum number of steps may satisfy the purity level, it is possible to obtain the one that maximizes final purity. Then, a second model that may use the total number of steps obtained in the first model generates a solution with the maximum purity of the product. Whenever the sequence does not affect the final purity or more generally does not impact the objective function, alternative models that are of smaller size are developed for the optimal selection of steps. The models are tested in several examples, containing up to 13 contaminants and a set of 22 candidate high-resolution steps, generating sequences of six operations, and are compared to the current synthesis approaches.     

Optimization of a high-throughput CTAB-based protocol for the extraction of qPCR-grade DNA from rumen fluid, plant and bacterial pure cultures

The quality and yield of extracted DNA are critical for the majority of downstream applications in molecular biology. Moreover, molecular techniques such as quantitative real-time PCR (qPCR) are becoming increasingly widespread; thus, validation and cross-laboratory comparison of data require standardization of upstream experimental procedures. DNA extraction methods depend on the type and size of starting material(s) used. As such, the extraction of template DNA is arguably the most significant variable when cross-comparing data from different laboratories. Here, we describe a reliable, inexpensive and rapid method of DNA purification that is equally applicable to small or large scale or high-throughput purification of DNA. The protocol relies on a CTAB-based buffer for cell lysis and further purification of DNA with phenol : chloroform : isoamyl alcohol. The protocol has been used successfully for DNA purification from rumen fluid and plant cells. Moreover, after slight alterations, the same protocol was used for large-scale extraction of DNA from pure cultures of Gram-positive and Gram-negative bacteria. The yield of the DNA obtained with this method exceeded that from the same samples using commercial kits, and the quality was confirmed by successful qPCR applications.     

Process and economic evaluation for monoclonal antibody purification using a membrane-only process

In recent years, the market for therapeutic monoclonal antibodies (mAb) has grown exponentially, and with this there has been a desire to reduce the costs associated with production and purification of these high-value biological products. A typical mAb purification process involves three adsorption/chromatography steps [protein A, ion exchange (IEX), and hydrophobic interaction (HIC)], along with ultrafiltration, nanofiltration, and microfiltration. With the development of membrane adsorption/chromatography as a viable alternative to traditional pack bed systems, the opportunity exists to complete the entire downstream purification process using only membrane operations. In this study, the process simulation tool SuperPro Designer was used to evaluate the application of recently developed ultra-high capacity electrospun nanofibrous adsorption membranes as a replacement for conventional chromatographic media in the downstream mAb production process. The simulation showed that nanofibrous adsorption membranes in place of the three packed bed chromatography steps reduced the required volume of protein A, IEX, and HIC adsorptive medium by 25, 80, and 80%, respectively. In addition, the membrane-only process reduced the downstream processing time by 50%, decreased the number of labor hours associated with the purification steps by 40%, generated 40% less aqueous waste, and reduced the overall downstream process operating expenses per unit product by 23%. There were also significant savings in facility construction costs and the price of fixed equipment required for separations. With these savings not only is the membrane-only process economically competitive with the traditional packed bed operations, but it offers the possibility of moving toward more disposable process.     

Development of an activated carbon filtration step and high throughput screening method to remove host cell proteins from a recombinant enzyme process

An increasing number of non-mAb recombinant proteins are being developed today. These biotherapeutics provide greater purification challenges where multiple polishing steps may be required to meet final purity specifications or the process steps may require extensive optimization. Recent studies have shown that activated carbon can be employed in downstream purification processes to selectively separate host cell proteins (HCPs) from monoclonal antibodies (mAb). However, the use of activated carbon as a unit operation in a cGMP purification process is relatively new. As such, the goal of this work is to provide guidance on development approaches, insight into operating parameters and solution conditions that can impact HCP removal, as well as further investigate the mechanism of removal by using mass spectrometry. In this work, activated carbon was evaluated to remove HCPs in the downstream purification process of a recombinant enzyme. Impact of process placement, flux (or residence time), and mass loading on HCP removal was investigated. Feasibility of high throughput screening (HTS) using loose activated carbon was assessed to reduce the amount of therapeutic protein needed and enable testing of a larger number of solution conditions. Finally, mass spectrometry was used to determine the population of HCPs removed by activated carbon. Our work demonstrates that activated carbon can be used effectively in downstream processes of biopharmaceuticals to remove HCPs (up to a 3 log₁₀ reduction) and that an HTS format can be implemented to reduce material demands by up to 23x and allow for process optimization of this adsorbent for purification purposes.     

Summary of recombinant human serum albumin development.

The methylotrophic yeast Pichia pastoris is well known as a host strain for the production of a variety of heterologous proteins. We have used P. pastoris for the production of recombinant human serum albumin (rHSA), for which we have developed efficient and specialized downstream processes. Results from structural analysis suggest that purified rHSA possesses an identical conformation to plasma derived human albumin (pdHA) and no difference from pdHA has been observed in neo-antigenicity. Host-cell-derived impurities (i.e. Pichia yeast component, DNA and mannan) have been evaluated in the purification process as well as in the drug substance and relevant specifications established. The efficacy and safety of rHSA have been tested in clinical studies and no difference from pdHA has been found in comparative study. Such studies have confirmed rHSA to have high efficacy with little or no adverse reaction.     

A fluidized-bed-riser adsorption system for continuous bioproduct recovery from crude feedstock

In this work, a novel technique for continuous purification of biologics from a crude feedstock is demonstrated with equipment referred to as Fluidized Bed Adsorption System (FBRAS). The development and validation of such unit operations were performed utilizing lysozyme as a model protein and Relisorb™ SP405/EB as a carrier. The performance of FBRAS to carry out combined clarification and purification was evaluated by capturing of antifungal peptides directly from the lysed broth. The novel technique reduced the number of process unit operations from six to three without having an impact on purity. Overall productivity increased by 250% in comparison to the existing downstream processing routine.     

Pool‐less processing to streamline downstream purification of monoclonal antibodies

With cell culture titers and productivity increasing in the last few years, pressure has been placed on downstream purification to look at alternative strategies to meet the demand of biotech products with high dose requirements. Even when the upstream process is not continuous (perfusion based), adopting a more productive and/or continuous downstream process can be of significant advantage. Due to the recent trend in exploring continuous processing options for biomolecules, several enabling technologies have been assessed at Biogen. In this paper, we evaluate the capability of one of these technologies to streamline and improve our downstream mAb purification platform. Current conventional downstream polishing steps at Biogen are operated in flow‐through mode to achieve higher loadings while maintaining good selectivity. As titers increase, this would result in larger columns and larger intermediate product pool holding tanks. A semicontinuous downstream process linking the second and third chromatography steps in tandem can reduce/eliminate intermediate holding tanks, reduce overall processing time, and combine unit operations to reduce validation burdens. A pool‐less processing technology utilizing inline adjustment functionality was evaluated to address facility fit challenges for three high titer mAbs. Two different configurations of polishing steps were examined: (i) anion exchange and hydrophobic interaction and (ii) anion exchange and mixed mode chromatography. Initial laboratory scale proof of concept studies showed comparable performance between the batch purification process and the pool‐less process configuration.     

Review of computational fluid dynamics applications in biotechnology processes

Computational fluid dynamics (CFD) is well established as a tool of choice for solving problems that involve one or more of the following phenomena: flow of fluids, heat transfer,mass transfer, and chemical reaction. Unit operations that are commonly utilized in biotechnology processes are often complex and as such would greatly benefit from application of CFD. The thirst for deeper process and product understanding that has arisen out of initiatives such as quality by design provides further impetus toward usefulness of CFD for problems that may otherwise require extensive experimentation. Not surprisingly, there has been increasing interest in applying CFD toward a variety of applications in biotechnology processing in the last decade. In this article, we will review applications in the major unit operations involved with processing of biotechnology products. These include fermentation,centrifugation, chromatography, ultrafiltration, microfiltration, and freeze drying. We feel that the future applications of CFD in biotechnology processing will focus on establishing CFD as a tool of choice for providing process understanding that can be then used to guide more efficient and effective experimentation. This article puts special emphasis on the work done in the last 10 years.     

The use of affinity adsorbents in expanded bed adsorption

The potential for the use of affinity ligands in expanded bed adsorption (EBA) procedures is reviewed. The use of affinity ligands in EBA may improve its use in direct recovery operations, as the enhanced selectivity of the adsorbent permits selective capture of the target from complex feedstocks and high degrees of purification. The properties of ligands suitable for use in EBA processes are identified and illustrated with examples. In addition to its use in the recovery of soluble products, such as proteins and nucleic acids, from particulate feedstocks, EBA can also be used to recover particulate entities, such as cells and packaged DNA (viruses and phages), from feedstocks. Affinity ligands coupled to appropriate chosen support materials will be required for such processes in order to achieve the necessary selectivity for the required particulate entity. The latter point is illustrated by the use of proteinaceous ligands immobilized to perfluorocarbon emulsions to achieve separations of microbial cells.     

Extension of the selection of protein chromatography and the rate model to affinity chromatography

The rational selection of optimal protein purification sequences, as well as mathematical models that simulate and allow optimization of chromatographic protein purification processes have been developed for purification procedures such as ion-exchange, hydrophobic interaction and gel filtration chromatography. This paper investigates the extension of such analysis to affinity chromatography both in the selection of chromatographic processes and in the use of the rate model for mathematical modelling and simulation. Two affinity systems were used: Blue Sepharose and Protein A. The extension of the theory developed previously for ion-exchange and HIC chromatography to affinity separations is analyzed in this paper. For the selection of operations two algorithms are used. In the first, the value of η, which corresponds to the efficiency (resolution) of the actual chromatography and, Σ, which determines the amount of a particular contaminant eliminated after each separation step, which determines the purity, have to be determined. It was found that the value of both these parameters is not generic for affinity separations but will depend on the type of affinity system used and will have to be determined on a case by case basis. With Blue Sepharose a salt gradient was used and with Protein A, a pH gradient. Parameters were determined with individual proteins and simulations of the protein mixtures were done. This approach allows investigation of chromatographic protein purification in a holistic manner that includes ion-exchange, HIC, gel filtration and affinity separations for the first time.