Showing papers on "Bioprocess published in 2014"

More than meets the eye in bacterial cellulose: biosynthesis, bioprocessing, and applications in advanced fiber composites.

Abstract: Bacterial cellulose (BC) nanofibers are one of the stiffest organic materials produced by nature. It consists of pure cellulose without the impurities that are commonly found in plant-based cellulose. This review discusses the metabolic pathways of cellulose-producing bacteria and the genetic pathways of Acetobacter xylinum. The fermentative production of BC and the bioprocess parameters for the cultivation of bacteria are also discussed. The influence of the composition of the culture medium, pH, temperature, and oxygen content on the morphology and yield of BC are reviewed. In addition, the progress made to date on the genetic modification of bacteria to increase the yield of BC and the large-scale production of BC using various bioreactors, namely static and agitated cultures, stirred tank, airlift, aerosol, rotary, and membrane reactors, is reviewed. The challenges in commercial scale production of BC are thoroughly discussed and the efficiency of various bioreactors is compared. In terms of the application of BC, particular emphasis is placed on the utilization of BC in advanced fiber composites to manufacture the next generation truly green, sustainable and renewable hierarchical composites.

Allogeneic Cell Therapy Bioprocess Economics and Optimization: Single-Use Cell Expansion Technologies

Abstract: For allogeneic cell therapies to reach their therapeutic potential, challenges related to achieving scalable and robust manufacturing processes will need to be addressed. A particular challenge is producing lot-sizes capable of meeting commercial demands of up to 10 9 cells/ dose for large patient numbers due to the current limitations of expansion technologies. This article describes the application of a decisional tool to identify the most cost- effective expansion technologies for different scales of production as well as current gaps in the technology capabilities for allogeneic cell therapy manufacture. The tool integrates bioprocess economics with optimization to assess the economic competitiveness of planar and micro- carrier-based cell expansion technologies. Visualization methods were used to identify the production scales where planar technologies will cease to be cost-effective and where microcarrier-based bioreactors become the only option. The tool outputs also predict that for the industry to be sustainable for high demand scenarios, significant increases will likely be needed in the performance capabilities of microcarrier-based systems. These data are presented using a technology S-curve as well as windows of operation to identify the combination of cell productivities and scale of single-use bioreactors required to meet future lot sizes. The modeling insights can be used to identify where future RD111: 69-83. 2013 Wiley Periodicals, Inc.

Consolidated bioprocessing of lignocellulose by a microbial consortium

Abstract: Lignocellulosic biomass is uniquely suited as a sustainable feedstock for the biotechnological production of alternative fuels and chemicals. However, due to the biomass recalcitrance, the enzymatic conversion process is complex and needs to be simplified. To this end, we developed a process, which allows the consolidated bioprocessing of lignocellulose to ethanol in a single multi-species biofilm membrane reactor featuring both aerobic and anaerobic conditions necessary for the simultaneous fungal cellulolytic enzyme production and alcoholic yeast fermentation of the hydrolysis-derived sugars. The general feasibility of the concept was successfully demonstrated by producing ethanol with a 67% yield from undetoxified whole slurry dilute acid pretreated wheat straw by the combined action of Trichoderma reesei, Saccharomyces cerevisiae and Scheffersomyces stipitis. The results achieved underscore the potential of the process as a versatile cheap sugar platform for the production of fuels and chemicals based on lignocellulosic biomass by specifically compiled consortia of industrially proven robust microorganisms.

Open and continuous fermentation: products, conditions and bioprocess economy.

Abstract: Microbial fermentation is the key to industrial biotechnology. Most fermentation processes are sensitive to microbial contamination and require an energy intensive sterilization process. The majority of microbial fermentations can only be conducted over a short period of time in a batch or fed-batch culture, further increasing energy consumption and process complexity, and these factors contribute to the high costs of bio-products. In an effort to make bio-products more economically competitive, increased attention has been paid to developing open (unsterile) and continuous processes. If well conducted, continuous fermentation processes will lead to the reduced cost of industrial bio-products. To achieve cost-efficient open and continuous fermentations, the feeding of raw materials and the removal of products must be conducted in a continuous manner without the risk of contamination, even under 'open' conditions. Factors such as the stability of the biological system as a whole during long cultivations, as well as the yield and productivity of the process, are also important. Microorganisms that grow under extreme conditions such as high or low pH, high osmotic pressure, and high or low temperature, as well as under conditions of mixed culturing, cell immobilization, and solid state cultivation, are of interest for developing open and continuous fermentation processes.

Metagenomic scaffolds enable combinatorial lignin transformation

Abstract: Engineering the microbial transformation of lignocellulosic biomass is essential to developing modern biorefining processes that alleviate reliance on petroleum-derived energy and chemicals. Many current bioprocess streams depend on the genetic tractability of Escherichia coli with a primary emphasis on engineering cellulose/hemicellulose catabolism, small molecule production, and resistance to product inhibition. Conversely, bioprocess streams for lignin transformation remain embryonic, with relatively few environmental strains or enzymes implicated. Here we develop a biosensor responsive to monoaromatic lignin transformation products compatible with functional screening in E. coli. We use this biosensor to retrieve metagenomic scaffolds sourced from coal bed bacterial communities conferring an array of lignin transformation phenotypes that synergize in combination. Transposon mutagenesis and comparative sequence analysis of active clones identified genes encoding six functional classes mediating lignin transformation phenotypes that appear to be rearrayed in nature via horizontal gene transfer. Lignin transformation activity was then demonstrated for one of the predicted gene products encoding a multicopper oxidase to validate the screen. These results illuminate cellular and community-wide networks acting on aromatic polymers and expand the toolkit for engineering recombinant lignin transformation based on ecological design principles.

Lignin biodegradation and industrial implications

Abstract: Lignocellulose, which comprises the cell walls of plants, is the Earth’s most abundant renewable source of convertible biomass. However, in order to access the fermentable sugars of the cellulose and hemicellulose fraction, the extremely recalcitrant lignin heteropolymer must be hydrolyzed and removed—usually by harsh, costly thermochemical pretreatments. Biological processes for depolymerizing and metabolizing lignin present an opportunity to improve the overall economics of the lignocellulosic biorefinery by facilitating pretreatment, improving downstream cellulosic fermentations or even producing a valuable effluent stream of aromatic compounds for creating value-added products. In the following review we discuss background on lignin, the enzymology of lignin degradation, and characterized catabolic pathways for metabolizing the by-products of lignin degradation. To conclude we survey advances in approaches to identify novel lignin degrading phenotypes and applications of these phenotypes in the lignocellulosic bioprocess.

Stem cell engineering in bioreactors for large-scale bioprocessing

Abstract: Stem cells are promising cell sources for many biomedical applications including cell therapy, regenerative medicine, and drug discovery. However, the commonly used static tissue culture vessels can only generate a low number of cells. To provide an adequate number of stem cells for clinical applications, a scalable process based on bioreactors is needed. Stem cells can be either cultured as free cells/aggregates in suspension or as adherent cells on the solid substrates. Based on the cell property, different bioreactor configurations are developed to better expand stem cells while maintaining their differentiation capacity. In this review, several major types of bioreactor systems and their applications in stem cell engineering are discussed. Continued advancements in bioprocess and bioreactor research and development are important to engineer stem cells for their use in biomedical applications.

Application of multi-omics techniques for bioprocess design and optimization in chinese hamster ovary cells.

Abstract: Significant improvements in the productivity and quality of therapeutic proteins produced in Chinese hamster ovary (CHO) cells have been reported since their establishment as host cells for biopharmaceutical production. Initial advances in the field focused on engineering strategies to manipulate genes associated with proliferation, apoptosis, and various metabolic pathways. Process engineering efforts to optimize culture media, batch-feeding strategies and culture conditions, including temperature and osmolarity, were also reported. More recently, focus has shifted toward enhancing process consistency and product quality using systems biology quality by design-based approaches during process development. Integration of different data generated using omics technologies, such as genomics, transcriptomics, proteomics and metabolomics, has facilitated a greater understanding of CHO cell biology. These techniques have enabled the provision of global information on dynamic changes in cellular components associ...

Application of metabolic engineering for the biotechnological production of L-valine.

Abstract: The branched chain amino acid l-valine is an essential nutrient for higher organisms, such as animals and humans. Besides the pharmaceutical application in parenteral nutrition and as synthon for the chemical synthesis of e.g. herbicides or anti-viral drugs, l-valine is now emerging into the feed market, and significant increase of sales and world production is expected. In accordance, well-known microbial production bacteria, such as Escherichia coli and Corynebacterium glutamicum strains, have recently been metabolically engineered for efficient l-valine production under aerobic or anaerobic conditions, and the respective cultivation and production conditions have been optimized. This review summarizes the state of the art in l-valine biosynthesis and its regulation in E. coli and C. glutamicum with respect to optimal metabolic network for microbial l-valine production, genetic strain engineering and bioprocess development for l-valine production, and finally, it will shed light on emerging technologies that have the potential to accelerate strain and bioprocess engineering in the near future.

Biopharmaceutical Process Optimization with Simulation and Scheduling Tools

Abstract: Design and assessment activities associated with a biopharmaceutical process are performed at different levels of detail, based on the stage of development that the product is in. Preliminary "back-of-the envelope" assessments are performed early in the development lifecycle, whereas detailed design and evaluation are performed prior to the construction of a new facility. Both the preliminary and detailed design of integrated biopharmaceutical processes can be greatly assisted by the use of process simulators, discrete event simulators or finite capacity scheduling tools. This report describes the use of such tools for bioprocess development, design, and manufacturing. The report is divided into three sections. Section One provides introductory information and explains the purpose of bioprocess simulation. Section Two focuses on the detailed modeling of a single batch bioprocess that represents the manufacturing of a therapeutic monoclonal antibody (MAb). This type of analysis is typically performed by engineers engaged in the development and optimization of such processes. Section Three focuses on production planning and scheduling models for multiproduct plants.

Comprehensive clone screening and evaluation of fed-batch strategies in a microbioreactor and lab scale stirred tank bioreactor system: application on Pichia pastoris producing Rhizopus oryzae lipase

Abstract: In Pichia pastoris bioprocess engineering, classic approaches for clone selection and bioprocess optimization at small/micro scale using the promoter of the alcohol oxidase 1 gene (P AOX1 ), induced by methanol, present low reproducibility leading to high time and resource consumption An automated microfermentation platform (RoboLector) was successfully tested to overcome the chronic problems of clone selection and optimization of fed-batch strategies Different clones from Mut+ P pastoris phenotype strains expressing heterologous Rhizopus oryzae lipase (ROL), including a subset also overexpressing the transcription factor HAC1, were tested to select the most promising clones The RoboLector showed high performance for the selection and optimization of cultivation media with minimal cost and time Syn6 medium was better than conventional YNB medium in terms of production of heterologous protein The RoboLector microbioreactor was also tested for different fed-batch strategies with three clones producing different lipase levels Two mixed substrates fed-batch strategies were evaluated The first strategy was the enzymatic release of glucose from a soluble glucose polymer by a glucosidase, and methanol addition every 24 hours The second strategy used glycerol as co-substrate jointly with methanol at two different feeding rates The implementation of these simple fed-batch strategies increased the levels of lipolytic activity 80-fold compared to classical batch strategies used in clone selection Thus, these strategies minimize the risk of errors in the clone selection and increase the detection level of the desired product Finally, the performance of two fed-batch strategies was compared for lipase production between the RoboLector microbioreactor and 5 liter stirred tank bioreactor for three selected clones In both scales, the same clone ranking was achieved The RoboLector showed excellent performance in clone selection of P pastoris Mut+ phenotype The use of fed-batch strategies using mixed substrate feeds resulted in increased biomass and lipolytic activity The automated processing of fed-batch strategies by the RoboLector considerably facilitates the operation of fermentation processes, while reducing error-prone clone selection by increasing product titers The scale-up from microbioreactor to lab scale stirred tank bioreactor showed an excellent correlation, validating the use of microbioreactor as a powerful tool for evaluating fed-batch operational strategies

Exometabolome analysis reveals hypoxia at the up-scaling of a Saccharomyces cerevisiae high-cell density fed-batch biopharmaceutical process

Abstract: Scale-up to industrial production level of a fermentation process occurs after optimization at small scale, a critical transition for successful technology transfer and commercialization of a product of interest. At the large scale a number of important bioprocess engineering problems arise that should be taken into account to match the values obtained at the small scale and achieve the highest productivity and quality possible. However, the changes of the host strain’s physiological and metabolic behavior in response to the scale transition are still not clear. Heterogeneity in substrate and oxygen distribution is an inherent factor at industrial scale (10,000 L) which affects the success of process up-scaling. To counteract these detrimental effects, changes in dissolved oxygen and pressure set points and addition of diluents were applied to 10,000 L scale to enable a successful process scale-up. A comprehensive semi-quantitative and time-dependent analysis of the exometabolome was performed to understand the impact of the scale-up on the metabolic/physiological behavior of the host microorganism. Intermediates from central carbon catabolism and mevalonate/ergosterol synthesis pathways were found to accumulate in both the 10 L and 10,000 L scale cultures in a time-dependent manner. Moreover, excreted metabolites analysis revealed that hypoxic conditions prevailed at the 10,000 L scale. The specific product yield increased at the 10,000 L scale, in spite of metabolic stress and catabolic-anabolic uncoupling unveiled by the decrease in biomass yield on consumed oxygen. An optimized S. cerevisiae fermentation process was successfully scaled-up to an industrial scale bioreactor. The oxygen uptake rate (OUR) and overall growth profiles were matched between scales. The major remaining differences between scales were wet cell weight and culture apparent viscosity. The metabolic and physiological behavior of the host microorganism at the 10,000 L scale was investigated with exometabolomics, indicating that reduced oxygen availability affected oxidative phosphorylation cascading into down- and up-stream pathways producing overflow metabolism. Our study revealed striking metabolic and physiological changes in response to hypoxia exerted by industrial bioprocess up-scaling.

Robotic platform for parallelized cultivation and monitoring of microbial growth parameters in microwell plates

Abstract: The enormous variation possibilities of bioprocesses challenge process development to fix a commercial process with respect to costs and time. Although some cultivation systems and some devices for unit operations combine the latest technology on miniaturization, parallelization, and sensing, the degree of automation in upstream and downstream bioprocess development is still limited to single steps. We aim to face this challenge by an interdisciplinary approach to significantly shorten development times and costs. As a first step, we scaled down analytical assays to the microliter scale and created automated procedures for starting the cultivation and monitoring the optical density (OD), pH, concentrations of glucose and acetate in the culture medium, and product formation in fed-batch cultures in the 96-well format. Then, the separate measurements of pH, OD, and concentrations of acetate and glucose were combined to one method. This method enables automated process monitoring at dedicated intervals (e.g., also during the night). By this approach, we managed to increase the information content of cultivations in 96-microwell plates, thus turning them into a suitable tool for high-throughput bioprocess development. Here, we present the flowcharts as well as cultivation data of our automation approach.

Application of spectroscopic methods for monitoring of bioprocesses and the implications for the manufacture of biologics

Abstract: The ability to monitor and control bioreactor processes is an integral component to the implementation of Process Analytical Technology and Quality by Design principles. Desirable attributes of monitoring methods include the ability to monitor multiple analytes in real time with little to no sample processing. Spectroscopic methods fit these criteria and significant advancements in their application have been made. However, implementation of these systems has been hampered by their complexity. Here, we present an overview of near IR, mid-IR, Raman and fluorescence spectroscopy technologies, and the steps taken to enable their implementation as effective bioprocess monitoring tools. Specific applications for monitoring of microbial and mammalian cell bioreactors, and screening and classification of raw materials are discussed.

Rationally Engineered Synthetic Coculture for Improved Biomass and Product Formation

Abstract: In microbial ecosystems, bacteria are dependent on dynamic interspecific interactions related to carbon and energy flow. Substrates and end-metabolites are rapidly converted to other compounds, which protects the community from high concentrations of inhibitory molecules. In biotechnological applications, pure cultures are preferred because of the more straight-forward metabolic engineering and bioprocess control. However, the accumulation of unwanted side products can limit the cell growth and process efficiency. In this study, a rationally engineered coculture with a carbon channeling system was constructed using two well-characterized model strains Escherichia coli K12 and Acinetobacter baylyi ADP1. The directed carbon flow resulted in efficient acetate removal, and the coculture showed symbiotic nature in terms of substrate utilization and growth. Recombinant protein production was used as a proof-of-principle example to demonstrate the coculture utility and the effects on product formation. As a result, the biomass and recombinant protein titers of E. coli were enhanced in both minimal and rich medium simple batch cocultures. Finally, harnessing both the strains to the production resulted in enhanced recombinant protein titers. The study demonstrates the potential of rationally engineered cocultures for synthetic biology applications.

Current research and future perspectives of phytase bioprocessing

Abstract: Phosphorus is one of nature's paradoxes as it is life's bottleneck for subsistence on earth but at same time is detrimental in surplus quantities in an aquatic environment. Phosphorus cannot be manufactured, though fortunately it can be recovered and reused. The only way to avert a supply crisis is to implement the “3R's” of sustainability, “Reduce, Reuse and Recycle.” Phytase is likely to play a critical role in the dephosphorylation of antinutritional and indigestible phytate, a phosphorus locking molecule, to digestible phosphorus, calcium and other mineral nutrients in the coming years. Hence, efforts are required to produce cost effective phytase with fast upstream and economic downstream processing because the current available process is expensive and time consuming. This review summarizes the present state of methods studied for phytase bioprocessing. Production, extraction and purification incur a large cost in product development. In addition, the process has several limitations such as dilute concentration of enzyme, extensive downstream procedures and treatment of generated effluents. However, these methods are currently employed due to lack of alternative methods. Thus, there is a clear need for an efficient, scalable and economical process for phytase production and bioseparation, and improvements are especially required with regard to yield, purity, and energy consumption. Perspectives for an improved bioprocess development for phytase are discussed based on our own experience and recent studies. It is argued that the optimization of production techniques, strain improvement and liquid–liquid extraction deserves more attention in the future.

Verification of a new biocompatible single-use film formulation with optimized additive content for multiple bioprocess applications

Abstract: Single-use bioprocessing bags and bioreactors gained significant importance in the industry as they offer a number of advantages over traditional stainless steel solutions. However, there is continued concern that the plastic materials might release potentially toxic substances negatively impacting cell growth and product titers, or even compromise drug safety when using single-use bags for intermediate or drug substance storage. In this study, we have focused on the in vitro detection of potentially cytotoxic leachables originating from the recently developed new polyethylene (PE) multilayer film called S80. This new film was developed to guarantee biocompatibility for multiple bioprocess applications, for example, storage of process fluids, mixing, and cell culture bioreactors. For this purpose, we examined a protein-free cell culture medium that had been used to extract leachables from freshly gamma-irradiated sample bags in a standardized cell culture assay. We investigated sample bags from films generated to establish the operating ranges of the film extrusion process. Further, we studied sample bags of different age after gamma-irradiation and finally, we performed extended media extraction trials at cold room conditions using sample bags. In contrast to a nonoptimized film formulation, our data demonstrate no cytotoxic effect of the S80 polymer film formulation under any of the investigated conditions. The S80 film formulation is based on an optimized PE polymer composition and additive package. Full traceability alongside specifications and controls of all critical raw materials, and process controls of the manufacturing process, that is, film extrusion and gamma-irradiation, have been established to ensure lot-to-lot consistency. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1171–1176, 2014

Advanced near-infrared monitor for stable real-time measurement and control of Pichia pastoris bioprocesses

Abstract: Near-infrared spectroscopy is considered to be one of the most promising spectroscopic techniques for upstream bioprocess monitoring and control. Traditionally the nature of near-infrared spectroscopy has demanded multivariate calibration models to relate spectral variance to analyte concentrations. The resulting analytical measurements have proven unreliable for the measurement of metabolic substrates for bioprocess batches performed outside the calibration process. This paper presents results of an innovative near-infrared spectroscopic monitor designed to follow the concentrations of glycerol and methanol, as well as biomass, in real time and continuously during the production of a monoclonal antibody by a Pichia pastoris high cell density process. A solid state instrumental design overcomes the ruggedness limitations of conventional interferometer-based spectrometers. Accurate monitoring of glycerol, methanol, and biomass is demonstrated over 274 days postcalibration. In addition, the first example of feedback control to maintain constant methanol concentrations, as low as 1 g/L, is presented. Postcalibration measurements over a 9-month period illustrate a level of reliability and robustness that promises its adoption for online bioprocess monitoring throughout product development, from early laboratory research and development to pilot and manufacturing scale operation. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:749–759, 2014

Monitoring cell growth, viability, and apoptosis.

Abstract: The accurate determination of cell growth and viability is pivotal to monitoring a bioprocess Direct methods to determine the cell growth and/or viability in a bioprocess include microscopic counting, electronic particle counting, image analysis, in situ biomass monitoring, and dieletrophoretic cytometry These methods work most simply when a fixed volume sample can be taken from a suspension culture Manual microscopic counting is laborious but affords the advantage of allowing cell viability to be determined if a suitable dye is included Electronic particle counting is a rapid total cell count method for replicate samples, but some data distortion may occur if the sample has significant cell debris or cell aggregates Image analysis based on the use of digital camera images acquired through a microscope has advanced rapidly with the availability of several commercially available software packages replacing manual microscopic counting and viability determination Biomass probes detect cells by their dielectric properties or their internal concentration of NADH and can be used as a continuous monitor of the progress of a culture While the monitoring of cell growth and viability is an integral part of a bioprocess, the monitoring of apoptosis induction is also becoming more and more important in bioprocess control to increase volumetric productivity by extending bioprocess duration Different fluorescent assays allow for the detection of apoptotic characteristics in a cell sampleIndirect methods of cell determination involve the chemical analysis of a culture component or a measure of metabolic activity These methods are most useful when it is difficult to obtain intact cell samples However, the relationship between these parameters and the cell number may not be linear through the phases of a cell culture The determination of nucleic acid (DNA) or total protein can be used as an estimate of biomass, while the depletion of glucose from the media can be used as an estimate of cellular activity The state of cellular viability may be measured by the release of an enzyme such as lactate dehydrogenase or more directly from the intracellular adenylate energy charge from cell lysates Alternatively, radioactive techniques may be used for an accurate determination of cellular protein synthesis

Physiological description of multivariate interdependencies between process parameters, morphology and physiology during fed-batch penicillin production

Abstract: Optimization of productivity and economics of industrial bioprocesses requires characterization of interdependencies between process parameters and process performance. In the case of penicillin production, as in other processes, process performance is often closely interlinked with the physiology and morphology of the organism used for production. This study presents a systematic approach to efficiently characterize the physiological effects of multivariate interdependencies between bioprocess design parameters (spore inoculum concentration, pO2 control level and substrate feed rate), morphology, and physiology. Method development and application was performed using the industrial model process of penicillin production. Applying traditional, statistical bioprocess analysis, multivariate correlations of raw bioprocess design parameters (high spore inoculum concentration, low pO2 control as well as reduced glucose feeding) and pellet morphology were identified. A major drawback of raw design parameter correlation models; however, is the lack of transferability across different process scales and regimes. In this context, morphological and physiological bioprocess modeling based on scalable physiological parameters is introduced. In this study, raw parameter effects on pellet morphology were efficiently summarized by the physiological parameter of the biomass yield per substrate. Finally, for the first time to our knowledge, the specific growth rate per spore was described as time-independent determinant for switching from pellet to disperse growth during penicillin production and thus introduced as a novel, scalable key process parameter for pellet morphology and process performance. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:689–699, 2014

Fruit wastes fermentation for phenolic antioxidants production and their application in manufacture of edible coatings and films.

Abstract: Agro-industrial by-products are important sources of potent bioactive phenolic compounds. These compounds are of extreme relevance for food and pharmacological industries due to their great variety of biological activities. Fermentation represents an environmentally clean technology for production and extraction of these bioactive compounds, providing high quality and high activity extracts, which can be incorporated in foods using coatings/films wax-based in order to avoid alterations in their quality. In this document is presented an overview about importance and benefits of solid-state fermentation, pointing out this bioprocess as an alternative technology for use agro-industrial by-products as substrates to produce valuable secondary metabolites and their applications as food quality conservatives.

Model-based process development for the purification of a modified human growth hormone using multimodal chromatography.

Abstract: This study demonstrates how the multimodal Capto adhere resin can be used in concert with calcium chloride or arginine hydrochloride as mobile phase modifiers to create a highly selective purification process for a modified human growth hormone Importantly, these processes are shown to result in significant clearance of product related aggregates and host cell proteins Furthermore, the steric mass action model is shown to be capable of accurately describing the chromatographic process and the aggregate removal Finally, justification of the selected operating ranges is evaluated using the model together with Latin hypercube sampling The results in this article establish the utility of multimodal chromatography when used with appropriate mobile phase modifiers for the downstream bioprocessing of a modified human growth hormone and offer new approaches for bioprocess verification