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In Situ Near-Infrared (NIR) Versus High-Throughput Mid- Infrared (MIR) Spectroscopy to Monitor Biopharmaceutical Production

Publication . Sales, Kevin C.; Rosa, Filipa; Sampaio, Pedro N.; Fonseca, LuÍs P.; B. Lopes, Marta; Calado, Cecília

The development of biopharmaceutical manufacturing processes presents critical constraints, with the major constraint being that living cells synthesize these molecules, presenting inherent behavior variability due to their high sensitivity to small fluctuations in the cultivation environment. To speed up the development process and to control this critical manufacturing step, it is relevant to develop high-throughput and in situ monitoring techniques, respectively. Here, high-throughput mid-infrared (MIR) spectral analysis of dehydrated cell pellets and in situ near-infrared (NIR) spectral analysis of the whole culture broth were compared to monitor plasmid production in recombinant Escherichia coil cultures. Good partial least squares (PLS) regression models were built, either based on MIR or NIR spectral data, yielding high coefficients of determination (R-2) and low predictive errors (root mean square error, or RMSE) to estimate host cell growth, plasmid production, carbon source consumption (glucose and glycerol), and by-product acetate production and consumption. The predictive errors for biomass, plasmid, glucose, glycerol, and acetate based on MIR data were 0.7 g/L, 9 mg/L, 0.3 g/L, 0.4 g/L, and 0.4 g/L, respectively, whereas for NIR data the predictive errors obtained were 0.4 g/L, 8 mg/L, 0.3 g/L, 0.2 g/L, and 0.4 g/L, respectively. The models obtained are robust as they are valid for cultivations conducted with different media compositions and with different cultivation strategies (batch and fed-batch). Besides being conducted in situ with a sterilized fiber optic probe, NIR spectroscopy allows building PLS models for estimating plasmid, glucose, and acetate that are as accurate as those obtained from the high-throughput MIR setup, and better models for estimating biomass and glycerol, yielding a decrease in 57 and 50% of the RMSE, respectively, compared to the MIR setup. However, MIR spectroscopy could be a valid alternative in the case of optimization protocols, due to possible space constraints or high costs associated with the use of multi-fiber optic probes for multi-bioreactors. In this case, MIR could be conducted in a high-throughput manner, analyzing hundreds of culture samples in a rapid and automatic mode.

2015-06Journal article

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In situ NIR spectroscopy monitoring of plasmid production processes effect of producing strain, medium composition and the cultivation strategy

Publication . Lopes, Marta B.; Gonçalves, Geisa A. L.; Felício-Silva, Daniel; Prather, Kristala L. J.; Monteiro, Gabriel; Prazeres, Duarte M. F.; Calado, Cecília

BACKGROUNDWhile the pharmaceutical industry keeps an eye on plasmid DNA production for new generation gene therapies, real-time monitoring techniques for plasmid bioproduction are as yet unavailable. This work shows the possibility of in situ monitoring of plasmid production in Escherichia coli cultures using a near infrared (NIR) fiber optic probe. RESULTSPartial least squares (PLS) regression models based on the NIR spectra were developed for predicting bioprocess critical variables such as the concentrations of biomass, plasmid, carbon sources (glucose and glycerol) and acetate. In order to achieve robust models able to predict the performance of plasmid production processes, independently of the composition of the cultivation medium, cultivation strategy (batch versus fed-batch) and E. coli strain used, three strategies were adopted, using: (i) E. coliDH5 cultures conducted under different media compositions and culture strategies (batch and fed-batch); (ii) engineered E. coli strains, MG1655endArecApgi and MG1655endArecA, grown on the same medium and culture strategy; (iii) diverse E. coli strains, over batch and fed-batch cultivations and using different media compositions. PLS models showed high accuracy for predicting all variables in the three groups of cultures. CONCLUSIONNIR spectroscopy combined with PLS modeling provides a fast, inexpensive and contamination-free technique to accurately monitoring plasmid bioprocesses in real time, independently of the medium composition, cultivation strategy and the E. coli strain used.

2015-02Journal article

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Monitoring the ex-vivo expansion of human mesenchymal stem/stromal cells in xeno-free microcarrier-based reactor systems by MIR spectroscopy

Publication . Rosa, Filipa; Sales, Kevin C.; Carmelo, Joana G.; Fernandes-Platzgummer, Ana; Silva, Cláudia L. da; Lopes, Marta B.; Calado, Cecília

Human mesenchymal stem/stromal cells (MSCs) have received considerable attention in the field of cell-based therapies due to their high differentiation potential and ability to modulate immune responses. However, since these cells can only be isolated in very low quantities, successful realization of these therapies requires MSCs ex-vivo expansion to achieve relevant cell doses. The metabolic activity is one of the parameters often monitored during MSCs cultivation by using expensive multi-analytical methods, some of them time-consuming. The present work evaluates the use of mid-infrared (MIR) spectroscopy, through rapid and economic high-throughput analyses associated to multivariate data analysis, to monitor three different MSCs cultivation runs conducted in spinner flasks, under xeno-free culture conditions, which differ in the type of microcarriers used and the culture feeding strategy applied. After evaluating diverse spectral preprocessing techniques, the optimized partial least square (PLS) regression models based on the MIR spectra to estimate the glucose, lactate and ammonia concentrations yielded high coefficients of determination (R2 ≥ 0.98, ≥0.98, and ≥0.94, respectively) and low prediction errors (RMSECV ≤ 4.7%, ≤4.4% and ≤5.7%, respectively). Besides PLS models valid for specific expansion protocols, a robust model simultaneously valid for the three processes was also built for predicting glucose, lactate and ammonia, yielding a R2 of 0.95, 0.97 and 0.86, and a RMSECV of 0.33, 0.57, and 0.09 mM, respectively. Therefore, MIR spectroscopy combined with multivariate data analysis represents a promising tool for both optimization and control of MSCs expansion processes.

2016-02-12Journal article

Open access

Metabolic proﬁling of recombinant Escherichia coli cultivations based on high-throughput FT-MIR spectroscopic analysis

Publication . Sales, Kevin C.; Rosa, Filipa; Ribeiro Da Cunha, Bernardo; Sampaio, Pedro; B. Lopes, Marta; Calado, Cecília

Escherichia coli is one of the most used host microorganism for the production of recombinant products, such as heterologous proteins and plasmids. However, genetic, physiological and environmental factors inﬂuence the plasmid replication and cloned gene expression in a highly complex way. To control and optimize the recombinant expression system performance, it is very important to understand this complexity. Therefore, the development of rap-id, highly sensitive and economic analytical methodologies, which enable the simultaneous characterization of the heterologous product synthesis and physiologic cell behavior under a variety of culture conditions, is highly desirable. For that, the metabolic proﬁle of recombinant E. coli cultures producing the pVAX-lacZ plasmid model was analyzed by rapid, economic and high-throughput Fourier Transform Mid-Infrared (FT-MIR) spectroscopy. The main goal of the present work is to show as the simultaneous multivariate data analysis by principal component analysis (PCA) and direct spectral analysis could represent a very interesting tool to monitor E. coli culture processes and acquire relevant information according to current quality regulatory guidelines. While PCA allowed capturing the energetic metabolic state of the cell, e.g. by identifying different C-sources consumption phases, direct FT-MIR spectral analysis allowed obtaining valuable biochemical and metabolic information along the cell culture, e.g. lipids, RNA, protein synthesis and turnover metabolism. The information achieved by spectral multivariate data and direct spectral analyses complement each other and may contribute to understand the complex interrelationships between the recombinant cell metabolism and the bioprocess environment towards more economic and robust processes design according to Quality by Design framework.

2017Journal article

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Optimization of fibers orientation in a composite specimen

Publication . Monte, Sara M. C.; Infante, Virginia; Madeira, JFA; Moleiro, Filipa

This article is devoted to the study of the optimal design of fibers orientation in a composite specimen with the objective to minimize the displacement. The composite specimen considered is within the scope of aerospace and mechanical applications. The objective function associated with the composite design is computed based on a static analysis of a finite element solid model, which allows one to define (or control) the fibers orientation. The recent global and local optimization using direct search methods (GLODS) is used for the optimization process. To validate and compare the numerical and optimized results, the specimens were manufactured and tested experimentally. The orientation of the layers that minimize the maximum displacement is achieved through the computational interaction of the optimization program, GLODS, in loop with the finite element program, ANSYS. It is shown that the optimized lamination schemes found by GLODS minimized about 60% of the displacement compared to the nonoptimized specimens.

2017-03Journal article

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