An impurity characterization based approach for the rapid development of integrated downstream purification processes

In this study, we describe a new approach for the characterization of process‐related impurities along with an in silico tool to generate orthogonal, integrated downstream purification processes for biological products. A one‐time characterization of process‐related impurities from product expression in Pichia pastoris was first carried out using linear salt and pH gradients on a library of multimodal, salt‐tolerant, and hydrophobic charge induction chromatographic resins. The Reversed‐phase ultra‐performance liquid chromatography (UPLC) analysis of the fractions from these gradients was then used to generate large data sets of impurity profiles. A retention database of the biological product was also generated using the same linear salt and pH gradients on these resins, without fraction collection. The resulting two data sets were then analyzed using an in silico tool, which incorporated integrated manufacturing constraints to generate and rank potential three‐step purification sequences based on their predicted purification performance as well as whole‐process “orthogonality” for impurity removal. Highly ranked sequences were further examined to identify templates for process development. The efficacy of this approach was successfully demonstrated for the rapid development of robust integrated processes for human growth hormone and granulocyte‐colony stimulating factor.     

A combined screening and in silico strategy for the rapid design of integrated downstream processes for process and product-related impurity removal

Integrated designs of chromatographic processes for purification of biopharmaceuticals provides potential gains in operational efficiency and reductions of costs and material requirements. We describe a combined method using screening and in silico algorithms for ranking chromatographic steps to rapidly design orthogonally selective integrated processes for purifying protein therapeutics from both process- and product-related impurities. IFN-α2b produced in Pichia pastoris containing a significant product variant challenge was used as a case study. The product and product-related variants were screened on a set of 14 multimodal, ion exchange, and hydrophobic charge induction chromatography resins under various pH and salt linear gradient conditions. Data generated from reversed-phase chromatography of the fractions collected were used to generate a retention database for IFN-α2b and its variants. These data, in combination with a previously constructed process-related impurity database for P. pastoris, were input into an in silico process development tool that generated and ranked all possible integrated chromatographic sequences for their ability to remove both process and product-related impurities. Top-ranking outputs guided the experimental refinement of two successful three step purification processes, one comprising all bind-elute steps and the other having two bind-elute steps and a flowthrough operation. This approach suggests a new platform-like approach for rapidly designing purification processes for a range of proteins where separations of both process- and product-related impurities are needed.     

High‐throughput process development of purification alternatives for the protein avidin

With an increased number of applications in the field of the avidin‐biotin technology, the resulting demand for highly‐purified protein avidin has drawn our attention to the purification process of avidin that naturally occurs in chicken egg white. The high‐throughput process development (HTPD) methodology was exploited, in order to evaluate purification process alternatives to commonly used ion‐exchange chromatography. In a high‐throughput format, process parameters for aqueous two‐phase extraction, selective precipitation with salts and polyethylene glycol, and hydrophobic interaction and mixed‐mode column chromatography experiments were performed. The HTPD strategy was complemented by a high‐throughput tandem high‐performance liquid chromatography assay for protein quantification. Suitable conditions for the separation of avidin from the major impurities ovalbumin, ovomucoid, ovotransferrin, and lysozyme were identified in the screening experiments. By combination of polyethylene glycol precipitation with subsequent resolubilization and separation in a polyethylene glycol/sulfate/sodium chloride two‐phase system an avidin purity of 77% was obtained with a yield >90% while at the same time achieving a significant reduction of the process volume. The two‐phase extraction and precipitation results were largely confirmed in larger scale with scale‐up factors of 230 and 133, respectively. Seamless processing of the avidin enriched bottom phase was found feasible by using mixed‐mode chromatography. By gradient elution a final avidin purity of at least 97% and yield >90% was obtained in the elution pool. The presented identification of a new and beneficial alternative for the purification of the high value protein thus represents a successful implementation of HTPD for an industrially relevant purification task. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:957–973, 2015     

Integrated process development: The key to improve Fab production in E. coli

Bioprocess development and optimization is a challenging, costly, and time-consuming effort. In this multidisciplinary task, upstream processing (USP) and downstream processing (DSP) are conventionally considered distinct disciplines. This consideration fosters “one-way” optimization disregarding interdependencies between unit operations; thus, the full potential of the process chain cannot be achieved. Therefore, it is necessary to fully integrate USP and DSP process development to provide balanced biotechnological production processes. The aim of the present study was to investigate how different host/secretory signal/antigen binding fragment (Fab) combinations in E. coli expression systems influence USP, primary recovery performance and the final product quality. We ran identical fed-batch cultivations with 16 different expression clones to study growth and product formation kinetics, as well as centrifugation efficiency, viscosity, extracellular DNA, and endotoxin content, important parameters in DSP. We observed a severe influence on cell growth, product titer, extracellular product, and cell lysis, accompanied by a significant impact on the analyzed parameters of DSP performance. Our results provide the basis for future research on integrated process development considering interdependencies between USP and DSP; however, individual products need to be considered specifically. These interdependencies need to be understood for rational decision-making and efficient process development in research and industry.     

Viral clearance in end-to-end integrated continuous process for mAb purification: Total flow-through integrated polishing on two columns connected to virus filtration

There are few reports of the adoption of continuous processes in bioproduction, particularly the implementation of end-to-end continuous or integrated processes, due to difficulties such as feed adjustment and incorporating virus filtration. Here, we propose an end-to-end integrated continuous process for a monoclonal antibody (mAb) with three integrated process segments: upstream production processes with pool-less direct connection, pooled low pH virus inactivation with pH control and a total flow-through integrated polishing process in which two columns were directly connected with a virus filter. The pooled virus inactivation step defines the batch, and high impurities reduction and mAb recovery were achieved for batches conducted in succession. Viral clearance tests also confirmed robust virus reduction for the flow-through two-column chromatography and the virus filtration steps. Additionally, viral clearance tests with two different hollow fiber virus filters operated at flux ranging from 1.5 to 40 LMH (liters per effective surface area of filter in square meters per hour) confirmed robust virus reduction over these ranges. Complete clearance with virus logarithmic reduction value ≥4 was achieved even with a process pause at the lowest flux. The end-to-end integrated continuous process proposed in this study is amenable to production processes, and the investigated virus filters have excellent applicability to continuous processes conducted at constant flux.     

High-throughput methods for miniaturization and automation of monoclonal antibody purification processes

In the last decade, high-throughput downstream process development techniques have entered the biopharmaceutical industry. As chromatography is the standard downstream purification method, several high-throughput chromatographic methods have been developed and applied including miniaturized chromatographic columns for utilization on liquid handling stations. These columns were used to setup a complete downstream process on a liquid handling station for the first time. In this article, a monoclonal antibody process was established in lab-scale and miniaturized afterwards. The scale-down methodology is presented and discussed. Liquid handling in miniaturized single and multicolumn processes was improved and applicability was demonstrated by volume balances. The challenges of absorption measurement are discussed and strategies were shown to improve volume balances and mass balances in 96-well microtiter plates. The feasibility of miniaturizing a complete downstream process was shown. In the future, analytical bottlenecks should be addressed to gain the full benefit from miniaturized complete process development.     

Free-energy landscapes of the coupled conformational transition and inclusion processes of altro-cyclodextrins

Abstract

Mono-altro-cyclodextrin (altro-CD) may undergo a conformational change of its altropyranose unit when encapsulating guest molecules of different sizes. This conformational transition is found to be coupled to the inclusion processes. In the present contribution, the possible conformational transition pathways in the four (self-)inclusion processes of altro-α and -β-CDs with moieties of variant shapes are explored from the insights of free-energy calculations. The two-dimensional free-energy landscapes characterising the coupled (self-)inclusion and isomerisation processes are determined, and the lowest free-energy pathways (LFEP) connecting the minima of the landscapes are located. The conformational statistics of the altropyranose units along the LFEPs reveal different transition pathways in the four (self-)inclusion processes. It can be concluded that when accommodating a free bulky guest molecule, the altropyranose unit will adjust its conformation to match the guest. However, such induced fit effect in the self-complexation of altro-CD derivatives will be weakened. The conformation of the altropyranose unit changes accompanying the self-complexation, but always adopts the ⁴C₁ one in the self-inclusion complex, irrespective of the shape of the guest moieties. The present results help determine the transition states of the (self-)inclusion processes of CDs and further improve the understanding of the mechanical properties of CD-based molecular shuttles.     

Application of model discriminating experimental design for modeling and development of a fermentative fed-batch L-valine production process

A model discriminating experimental design approach for fed-batch processes has been developed and applied to the fermentative production of L-valine by a genetically modified Corynebacterium glutamicum strain possessing multiple auxotrophies as an example. Being faced with the typical situation of uncertain model information based on preliminary experiments, model discriminating design was successfully applied to improve discrimination between five competing models. Within the same modeling and experimental design framework, also the planning of an optimized production process with respect to the total volumetric productivity is shown. Simulation results were experimentally affirmed, yielding an increased total volumetric productivity of 6.2 mM L-valine per hour. However, also so far unknown metabolic mechanisms were observed in the optimized process, underlining the importance of process optimization during modeling to avoid problems of extreme extrapolation of model predictions during the final process optimization.     

Techno-economic evaluation of an inclusion body solubilization and recombinant protein refolding process

Expression of recombinant proteins in Escherichia coli is normally accompanied by the formation of inclusion bodies (IBs). To obtain the protein product in an active (native) soluble form, the IBs must be first solubilized, and thereafter, the soluble, often denatured and reduced protein must be refolded. Several technically feasible alternatives to conduct IBs solubilization and on-column refolding have been proposed in recent years. However, rarely these on-column refolding alternatives have been evaluated from an economical point of view, questioning the feasibility of their implementation at a preparative scale. The presented study assesses the economic performance of four distinct process alternatives that include pH induced IBs solubilization and protein refolding (pH_IndSR); IBs solubilization using urea, dithiothreitol (DTT), and alkaline pH followed by batch size-exclusion protein refolding; inclusion bodies (IBs) solubilization using urea, DTT, and alkaline pH followed by simulated moving bed (SMB) size-exclusion protein refolding, and IBs solubilization using urea, DTT and alkaline pH followed by batch dilution protein refolding. The economic performance was judged on the basis of the direct fixed capital, and the production cost per unit of product (P(C)). This work shows that (1) pH_IndSR system is a relatively economical process, because of the low IBs solubilization cost; (2) substituting β-mercaptoethanol for dithiothreithol is an attractive alternative, as it significantly decreases the product cost contribution from the IBs solubilization; and (3) protein refolding by size-exclusion chromatography becomes economically attractive by changing the mode of operation of the chromatographic reactor from batch to continuous using SMB technology.     

Overexpression in Escherichia coli and functional reconstitution of anchovy trypsinogen from the bacterial inclusion body

We have synthesized and optimized a high-yielding Escherichia coli expression system to produce trypsinogen from anchovy Engraulis japonicus and have developed conditions for its successful refolding. Recombinant anchovy trypsinogen precipitated in E. coli Rosetta (DE3) placI strain as inclusion bodies was denatured by 6 M guanidine-HCl followed by refolding with drop wise addition to a large excess of a folding buffer containing 0.5 M non-detergent sulfobetaine (NDSB-251) and a redox potential of oxidized and reduced glutathiones. The folded trypsinogen was autocatalytically activated to its mature form, trypsin, and purified with a MonoQ ion-exchange column. NH₂-terminal amino acid sequencings revealed that E. coli efficiently processed NH₂-terminal methionine residue from the expressed trypsinogen and that trypsinogen was activated at the correct site to generate active trypsin. The recombinant enzyme showed kinetic properties comparable to those of the native enzyme and demonstrated a typical cleavage preference for arginine over lysine residue against a protein substrate. The optimized expression and folding procedures yielded 12 mg of purified, active trypsin from 1 L of bacterial culture or 45 g wet weight cells, which is quite enough for various analytical and semipreparative purposes.     

Conflicting processes in the evolution of body size and development time

Body size and development time of Manduca sexta are both determined by the same set of three developmental–physiological factors. These define a parameter space within which it is possible to analyse and explain how phenotypic change is associated with changes in the underlying factors. Body size and development time are determined by the identical set of underlying factors, so they are not independent, but because the mechanisms by which these factors produce each phenotype are different, the two phenotypes are only weakly correlated, and the correlation is context dependent. We use a mathematical model of this mechanism to explore the association between body size and development time and show that the correlation between these two life-history traits can be positive, zero or negative, depending entirely on where in parameter space a population is located, and on which of the underlying factors has a greater variation. The gradient within this parameter space predicts the unconstrained evolutionary trajectory under directional selection on each trait. Calculations of the gradients for body size and development time revealed that these are nearly orthogonal through much of the parameter space. Therefore, simultaneous directional selection on body size and development time can be neither synergistic nor antagonistic but leads to conflicting selection on the underlying developmental parameters.     

Baculoviral polyhedrin as a novel fusion partner for formation of inclusion body in Escherichia coli

Baculoviral polyhedrin, which originated from Autographa californica nuclear polyhedrosis virus (AcNPV), was employed for the first time as a novel fusion partner for expression of foreign proteins in an Escherichia coli system. We characterized the expression of recombinant polyhedrin protein fused to green fluorescent protein (GFP). The polyhedrin fusion protein ( approximately 58 kDa) was successfully expressed as an insoluble inclusion body comprising approximately 30% of the total cellular protein. The E. coli expressing polyhedrin-GFP fusion protein showed higher cell growth ( approximately 1.8-fold) and higher GFP yield ( approximately 3.5-fold) than the strain expressing soluble single GFP. Interestingly, the polyhedrin fusion portion showed almost the same characteristics as the native baculoviral polyhedrin; it was rapidly solubilized under alkaline conditions, similar to the conditions found in the insect midgut. In addition, the polyhedrin fusion portion was rapidly digested by alkaline proteases in insect Plutella xylostella midgut as well as by alpha-chymotrypsin, a protease that has similar properties to insect midgut polyhedra-associated alkaline proteases. These unique properties suggest that baculoviral polyhedrin might be an advantageous fusion partner for production of foreign proteins, especially harmful proteins, in E. coli expression systems.     

Machine learning-based model predictive controller design for cell culture processes

The biopharmaceutical industry continuously seeks to optimize the critical quality attributes to maintain the reliability and cost-effectiveness of its products. Such optimization demands a scalable and optimal control strategy to meet the process constraints and objectives. This work uses a model predictive controller (MPC) to compute an optimal feeding strategy leading to maximized cell growth and metabolite production in fed-batch cell culture processes. The lack of high-fidelity physics-based models and the high complexity of cell culture processes motivated us to use machine learning algorithms in the forecast model to aid our development. We took advantage of linear regression, the Gaussian process and neural network models in the MPC design to maximize the daily protein production for each batch. The control scheme of the cell culture process solves an optimization problem while maintaining all metabolites and cell culture process variables within the specification. The linear and nonlinear models are developed based on real cell culture process data, and the performance of the designed controllers is evaluated by running several real-time experiments.     

Optimization of biopharmaceutical downstream processes supported by mechanistic models and artificial neural networks

Downstream process development is a major area of importance within the field of bioengineering. During the design of such a downstream process, important decisions have to be made regarding the type of unit operations as well as their sequence and their operating conditions. Current computational approaches addressing these issues either show a high level of simplification or struggle with computational speed. Therefore, this article presents a new approach that combines detailed mechanistic models and speed‐enhancing artificial neural networks. This approach was able to simultaneously optimize a process with three different chromatographic columns toward yield with a minimum purity of 99.9%. The addition of artificial neural networks greatly accelerated this optimization. Due to high computational speed, the approach is easily extendable to include more unit operations. Therefore, it can be of great help in the acceleration of downstream process development. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:696–707, 2017     

A review of advanced small-scale parallel bioreactor technology for accelerated process development: current state and future need

The pharmaceutical and biotech industries face continued pressure to reduce development costs and accelerate process development. This challenge occurs alongside the need for increased upstream experimentation to support quality by design initiatives and the pursuit of predictive models from systems biology. A small scale system enabling multiple reactions in parallel (n ≥ 20), with automated sampling and integrated to purification, would provide significant improvement (four to fivefold) to development timelines. State of the art attempts to pursue high throughput process development include shake flasks, microfluidic reactors, microtiter plates and small-scale stirred reactors. The limitations of these systems are compared to desired criteria to mimic large scale commercial processes. The comparison shows that significant technological improvement is still required to provide automated solutions that can speed upstream process development.     

Intensified process for the purification of an enzyme from inclusion bodies using integrated expanded bed adsorption and refolding

This work describes the integration of expanded bed adsorption (EBA) and adsorptive protein refolding operations in an intensified process used to recover purified and biologically active proteins from inclusion bodies expressed in E. coli. Delta(5)-3-Ketosteroid isomerase with a C-terminal hexahistidine tag was expressed as inclusion bodies in the cytoplasm of E. coli. Chemical extraction was used to disrupt the host cells and simultaneously solubilize the inclusion bodies, after which EBA utilizing immobilized metal affinity interactions was used to purify the polyhistidine-tagged protein. Adsorptive refolding was then initiated in the column by changing the denaturant concentration in the feed stream from 8 to 0 M urea. Three strategies were tested for performing the refolding step in the EBA column: (i) the denaturant was removed using a step change in feed-buffer composition, (ii) the denaturant was gradually removed using a gradient change in feed-buffer composition, and (iii) the liquid flow direction through the column was reversed and adsorptive refolding performed in the packed bed. Buoyancy-induced mixing disrupted the operation of the expanded bed when adsorptive refolding was performed using either a step change or a rapid gradient change in feed-buffer composition. A shallow gradient reduction in denaturant concentration of the feed stream over 30 min maintained the stability of the expanded bed during adsorptive refolding. In a separate experiment, buoyancy-induced mixing was completely avoided by performing refolding in a settled bed, which achieved comparable yields to refolding in an expanded bed but required a slightly more complex process. A total of 10% of the available KSI-(His(6)) was recovered as biologically active and purified protein using the described purification and refolding process, and the yield was further increased to 19% by performing a second iteration of the on-column refolding operation. This process should be applicable for other polyhistidine tagged proteins and is likely to have the greatest benefit for proteins that tend to aggregate when refolded by dilution.