An understanding of potential and limitations of alginate/PLL microcapsules as a cell retention system for perfusion cultures.

Microcapsules for high cell density culture of mammalian cells have found an increasing interest, however, the poor stability of the microcapsules and the lack of characterisation methods led to few quantitative results. Alginate-poly-L-lysine (PLL) microcapsules have been studied in detail in order to form a basis for comparison of capsules made from different polymers. Since the microcapsules can be easily retained in the bioreactor without the need for a cell separation device, high cell densities were achieved with a maximum of 4 × 10(7) cell/ml(microcapsules), corresponding to a colonisation of 5% of the internal capsule volume. Measurement of microcapsule integrity and mechanical resistance showed that alginate-PLL microcapsules are not suitable for perfusion cultures since they are very sensitive to media composition, mainly the presence of non-gelling ions that have a higher affinity for alginate than PLL and Ca(2+), leading to the leakage of PLL and Ca(2+), and to microcapsule rupture.

Quantitative host cell protein analysis using two dimensional data independent LC-MS(E).

Host cell proteins (HCPs) are bioprocess-related impurities arising from cell-death or secretion from nonhuman cells used for recombinant protein production. Clearance of HCPs through downstream purification (DSP) is required to produce safe and efficacious therapeutic proteins. While traditionally measured using anti-HCP ELISA, more in-depth approaches for HCP characterization may ensure that risks to patients from HCPs are adequately assessed. Mass spectrometry methods provide rationale for targeted removal strategies through the provision of both qualitative and quantitative HCP information. A high pH, low pH, reversed-phase data independent 2D-LC-MS(E) proteomic platform was applied to determine HCP repertoires in the Protein A purified monoclonal antibody (mAb) samples as a function of culture harvest time, elution buffer used for DSP and also following inclusion of additional DSP steps. Critical quality attributes (CQAs) were examined for mAbs purified with different Protein A elution buffers to ensure that the selected buffers not only minimized the HCP profile but also exhibited no adverse effect on product quality. Results indicated that an arginine based Protein A elution buffer minimized the levels of HCPs identified and quantified in a purified mAb sample and also demonstrated no impact on product CQAs. It was also observed that mAbs harvested at later stages of cell culture contained higher concentrations of HCPs but that these were successfully removed by the addition of DSP steps complementary to Protein A purification. Taken together, our results showed how mass spectrometry based methods for HCP determination in conjunction with careful consideration of processing parameters such as harvest time, Protein A elution buffers, and subsequent DSP steps can reduce the HCP repertoire of therapeutic mAbs.

In-vitro characterisation of a novel celecoxib microbead formulation for the treatment and prevention of colorectal cancer.

OBJECTIVES: Colorectal cancer (CRC) is a life-threatening disease that can develop as a consequence of a sustained chronic inflammatory pathology of the colon. Although not devoid of side effects, the anti-inflammatory drug celecoxib (CLX) has been shown to exert protective effects in CRC therapy. The purpose of this study was to develop and characterise a novel CLX microbead formulation suitable for use in the treatment and prevention of CRC, which has the potential to minimise the side effects associated with CLX. METHODS: The study involved the assessment of the effectiveness of CLX formulations in an in-vitro cell model (HT29 cells) and a comparison of these effects to that of the marketed CLX product, Celebrex. Liquid CLX formulations were developed as precursors to microbead formulations. The effect of liquid CLX formulations on HT29 cell viability (MTT and flow cytometry apoptotic assays) and motility (scratch wound assay) were assessed and compared with the effect of Celebrex. A correlation between the in-vitro dissolution performance of the formulations and the effect in the cell model was also explored. Liquid CLX formulations were translated into an optimised CLX microbead formulation, and a colonic targeted sustained release coat (Surelease) was applied to the beads with the aim of producing a formulation for a future in-vivo study to compare the effect of the coated CLX microbeads versus Celebrex in the attenuation of CRC tumours and inflammation in a CRC murine model. The production of CLX microbeads was scaled-up using vibrating-jet encapsulation technology to allow for the development of an optimised dissolution profile to enable colonic release. KEY FINDINGS: In-vitro cell viability and motility were shown to be significantly reduced after treatment with CLX liquid formulations relative to the control, whereas the results for treatment with Celebrex were comparable with the control. Dissolution experiments and correlation analysis demonstrated that the formulations that showed a greater extent of drug release had reduced cell viability and motility. The CLX liquid formulations were translated into colon-targeted CLX microbeads suitable for use in a future in-vivo mouse study. CONCLUSIONS: These results represent a significant step forward in the chemopreventative treatment of CRC using CLX, as the microbead formulation developed suggests the possibility of presenting CLX in a format that has the potential to minimise gastrointestinal and cardiovascular side effects.

Microbeads: a novel multiparticulate drug delivery technology for increasing the solubility and dissolution of celecoxib.

The purpose of this study was to develop a novel multipaticulate drug delivery technology suitable for the delivery of pre-solubilized celecoxib to the gastrointestinal tract and more specifically to the colon. The solubility of celecoxib in a range of oils, surfactants and co-solvents was evaluated. Celecoxib was solubilized in mixtures of these vehicles to produce liquid formulations. The in vitro dissolution of these liquid formulations was assessed and the data obtained was used to design microbead formulations containing celecoxib dissolved within an emulsion/micellar solution core. Microbead formulations were optimized to increase drug loading, avoid precipitation and to achieve good in vitro dissolution performance. An optimized formulation with a celecoxib loading of 6% w/w was produced and yielded an in vitro dissolution result of 80% over 6 h. The structure of these microbead formulations was characterized using light microscopy to reveal a correlation between droplet size and dissolution performance.

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

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 associated with different phenotypes. Using systems biology to understand these important host cells at the cellular level will undoubtedly result in further progression in the development and expression of biopharmaceutical products in CHO cells.

The choice of suitable online analytical techniques and data processing for monitoring of bioprocesses.

With increasing pressure from regulatory authorities on industry to develop processes embracing process analytical technology (PAT) initiatives, there is a growing demand to establish reliable tools and systems capable of meeting this need. With regard to monitoring and control of bioprocesses, this need translates to a search for robust instrumentation capable of monitoring the critical process parameters in real time. The application of such technologies at all stages of the process, from the initial R&D phase to process optimisation and production, enhances process understanding and paves the way for the development of control platforms. An examination of the PAT concept and selected tools (NIR, MIR, Raman, dielectric spectroscopy and calorimetry) are presented here. A description of each tool is given, with particular emphasis on the nature of the signal produced and how these relate to measurements of biomass, metabolites and product. A description of the signal processing that is necessary to gain meaningful results from the different tools is also given, together with online data reconciliation techniques based on mass and energy balances. Many techniques such as those based on vibrational spectroscopy are of particular interest, since they are capable of monitoring several critical process parameters which are typically controlled in a bioprocess. A window of application for each of the techniques, when used in the area of bioprocessing, is suggested based on their uses and inherent limitations.

Real-time monitoring and control of microbial bioprocesses with focus on the specific growth rate: current state and perspectives.

Understanding the growth characteristics of microorganisms is an essential step in bioprocessing, not only because product formation may be growth-associated but also because they might influence cell physiology and thereby product quality. The specific growth rate, a key variable of many bioprocesses, cannot be measured directly and relies on the estimation through other measurable variables such as biomass, substrate, or product concentrations. Techniques for real-time estimation of the specific growth rate in microbial fed-batch cultures are discussed in the present paper. The advantages and limitations of different models and various monitoring techniques are discussed, highlighting the importance of the specific growth rate in the development of fast, reliable, and robust processes for the production of high-value products such as recombinant proteins.

Investigation of the potential of biocalorimetry as a process analytical technology (PAT) tool for monitoring and control of Crabtree-negative yeast cultures.

Biological reaction calorimetry, also known as biocalorimetry, has led to extensive applications in monitoring and control of different bioprocesses. A simple real-time estimator for biomass and growth rate was formulated, based on in-line measured metabolic heat flow values. The performance of the estimator was tested in a unique bench-scale calorimeter (BioRC1), improved to a sensitivity range of 8 mW l(-1) in order to facilitate the monitoring of even weakly exothermic biochemical reactions. A proportional-integral feedback control strategy based on these estimators was designed and implemented to control the growth rate of Candida utilis, Kluyveromyces marxianus and Pichia pastoris by regulating an exponential substrate feed. Maintaining a particular specific growth rate throughout a culture is essential for reproducible product quality in industrial bioprocesses and therefore a key sequence for the step from quality by analysis to quality by design. The potential of biocalorimetry as a reliable biomass monitoring tool and as a key part of a robust control strategy for aerobic fed-batch cultures of Crabtree-negative yeast cells in defined growth medium was investigated. Presenting controller errors of less than 4% in the best cases, the approach paves the way for the development of a generally applicable process analytical technology platform for monitoring and control of microbial fed-batch cultures.

Microencapsulation using vibrating technology.

For over a half a century now, microencapsulation has played a very important role in many industries and in the recent decades, this versatile technology has been applied to numerous biotechnology and medical processes. However, successful application in these areas requires a methodology which has the capability to produce mono-dispersed, homogenous-shaped capsules, with a narrow size distribution, using a short production time. The manufacture of capsules using vibrating technology has gained significant interest mainly due to its simplistic approach to produce homogenous microcapsules with the desired characteristics for biotechnological and medical processes. However, certain limitations still exist for this methodology, which include the inability to manufacture microcapsules at large quantities and/or using highly viscous polymers. In this review, a detailed description of the theoretical and practical aspects behind the production of different types of alginate-based microcapsules, for application in biotechnological and medical processes, using vibrating technology, is given.

Capsular perstraction as a novel methodology for the recovery and purification of geldanamycin.

The molecular complex "Heat shock protein 90" has become a novel target for anticancer drugs in recent times on account of its ability to perform as a chaperone toward proteins involved in cancer progression. The geldanamycin binds to this complex with high affinity and prevents it from performing correctly, which results in tumor destruction. The aim of this study was to investigate the feasibility of applying liquid-core microcapsules as a novel technique (termed "capsular perstraction"), for the recovery and purification of geldanamycin from culture media. Results demonstrated how this procedure was capable of rapidly extracting >70% of geldanamycin from culture media using a liquid-core volume to medium ratio of only 1%. Optimum conditions for removal, including agitation speed, microcapsule size, and membrane thickness were examined, and it was shown how the stagnant aqueous film around the microcapsules was the main resistance to mass transfer. A volumetric mass transfer coefficient of 5.66×10(-6) m/s was obtained for the highest agitation speed (400 rpm), which was considerable greater compared to the value of 0.88×10(-6) m/s achieved for the lowest speed of 100 rpm. Removal of geldanamycin from microcapsules was also examined to fully investigate the potential of such particles for in situ product recovery, and it was demonstrated how the methodology can be used as a simple mechanism for purifying the compound (>99%) through solvent extraction and crystallization. The results of this work demonstrate the novel use of capsular perstraction as a methodology for the recovery and purification of geldanamycin from culture environments.

Simple control of specific growth rate in biotechnological fed-batch processes based on enhanced online measurements of biomass.

Reliable control of the specific growth rate (µ) in fed-batch fermentations depends on the availability of accurate online estimations of the controlled variable. Due to difficulties in measuring biomass, µ is typically estimated using reference models relating measurements of substrate consumption or oxygen uptake rate to biomass growth. However, as culture conditions vary, these models are adapted dynamically, resulting in complex algorithms that lack the necessary robustness for industrial applicability. A simpler approach is presented where biomass is monitored using dielectric spectroscopy. The measurements are subjected to online balances and reconciled in real time against metabolite concentrations and off-gas composition. The reconciled biomass values serve to estimate the growth rate and a simple control scheme is implemented to maintain the desired value of µ. The methodology is developed with the yeast Kluyveromyces marxianus, tested for disturbance rejection and validated with two other strains. It is applicable to other cellular systems with minor modifications.

Removal of pharmaceuticals from water: using liquid-core microcapsules as a novel approach.

In recent years ever-increasing amounts of pharmaceuticals are being detected in the aquatic environment and in some cases, they have even been discovered in drinking water. Their presence is attributed mainly to the inability of sewage treatment plants to adequately remove these compounds from the sewage influent. The aim of this study was to investigate the feasibility, kinetics and efficiency of using liquid-core microcapsules as a novel methodology, termed capsular perstraction, to remove seven pharmaceuticals commonly found in the environment, from water. The process involves the envelopment of pre-selected organic solvents within a porous hydrogel membrane to form liquid-core microcapsules, which can be used to extract a large range of compounds. Results indicate that this novel approach is capable of extracting the seven chosen compounds rapidly and with a variable efficiency. The simultaneous use of both dibutyl sebacate and oleic acid liquid-core microcapsules at a liquid volume ratio of only 4% (v/v) resulted in the following extractions within 50min of capsule addition to contaminated water: furosemide 15%; clofibric acid 19%; sulfamethoxazole 22%; carbamazepine 54%; warfarin 80%; metoprolol 90% and diclofenac 100%. The effects of different agitation rates, microcapsule size and membrane thickness on the rate of mass transfer of warfarin into the liquid-core (dibutyl sebacate) of microcapsules was also examined. Results showed that the main rate-limiting step to mass transfer was due to the stagnant organic film (microcapsule size) within the core of the microcapsules. A volumetric mass transfer coefficient of 2.28x10(-6)m/s was obtained for the smallest microcapsules, which was nearly 4-fold higher compared to the value (0.6x10(-6)m/s) obtained for the largest microcapsules used in this study. Even with this resistance liquid-core microcapsules are still capable of the rapid extraction of the tested compounds and may provide a platform for the safe disposal of the pharmaceuticals after removal.

Data reconciliation of concentration estimates from mid-infrared and dielectric spectral measurements for improved on-line monitoring of bioprocesses.

Real-time data reconciliation of concentration estimates of process analytes and biomass in microbial fermentations is investigated. A Fourier-transform mid-infrared spectrometer predicting the concentrations of process metabolites is used in parallel with a dielectric spectrometer predicting the biomass concentration during a batch fermentation of the yeast Saccharomyces cerevisiae. Calibration models developed off-line for both spectrometers suffer from poor predictive capability due to instrumental and process drifts unseen during calibration. To address this problem, the predicted metabolite and biomass concentrations, along with off-gas analysis and base addition measurements, are reconciled in real-time based on the closure of mass and elemental balances. A statistical test is used to confirm the integrity of the balances, and a non-negativity constraint is used to guide the data reconciliation algorithm toward positive concentrations. It is verified experimentally that the proposed approach reduces the standard error of prediction without the need for additional off-line analysis.

Cole-Cole, linear and multivariate modeling of capacitance data for on-line monitoring of biomass.

This work evaluates three techniques of calibrating capacitance (dielectric) spectrometers used for on-line monitoring of biomass: modeling of cell properties using the theoretical Cole-Cole equation, linear regression of dual-frequency capacitance measurements on biomass concentration, and multivariate (PLS) modeling of scanning dielectric spectra. The performance and robustness of each technique is assessed during a sequence of validation batches in two experimental settings of differing signal noise. In more noisy conditions, the Cole-Cole model had significantly higher biomass concentration prediction errors than the linear and multivariate models. The PLS model was the most robust in handling signal noise. In less noisy conditions, the three models performed similarly. Estimates of the mean cell size were done additionally using the Cole-Cole and PLS models, the latter technique giving more satisfactory results.

Enzymatic hydrolysis of organic-core microcapsules to produce aqueous-core microcapsules.

This paper describes the development of a new method to obtain aqueous-core microcapsules from organic-core capsules. The direct production of microcapsules, using tripropionin as organic material, followed by the hydrolysis of the core by a lipase was investigated. The enzymatic study showed that the enzyme obeyed a Michaelis-Menten mechanism and conditions for optimal activity were pH 7.5, 25-37 degrees C and 0% NaCl. Under these conditions, incubation of tripropionin-alginate microcapsules in a buffer containing the enzyme successfully produced aqueous-core capsules with reduced accumulation of alginate in the core in approximately 3 h.

On-line monitoring of nine different batch cultures of E. coli by mid-infrared spectroscopy, using a single spectra library for calibration.

A single spectra library was used to monitor on-line, by mid-infrared spectroscopy, nine different batch cultures of Escherichia coli performed with various medium compositions, including chemically complex formulations. Whereas the classic chemometrics approach would have required the preparation and measurement of hundreds of standards, only six spectra were included in the library. These included the molar absorbance of the four main metabolites (i.e. glucose, glycerol, ammonium and acetate), and the remaining two were drift spectra found by factor analysis. The accuracy of the prediction was not altered by a change of the carbon source, the ammonium concentration or even the addition of chemically undefined compounds, such as yeast extract and peptone. The standard errors of prediction averaged over the nine experiments were 8.0, 12.3, 5.9 and 5.6 mM for glucose, glycerol, ammonium and acetate, respectively. Inclusion of two drift spectra in the library provided an estimation of how noisy an experiment was. This also allowed detection of batch cultures that require further investigation, namely runs which were subject to large signal drift or during which an unexpected compound was produced, without having to carry out time-consuming off-line analyses.

Influence of specific growth rate on specific productivity and glycosylation of a recombinant avidin produced by a Pichia pastoris Mut+ strain.

A recombinant avidin-producing Mut+ Pichia pastoris strain was used as a model organism to study the influence of the methanol feeding strategy on the specific product productivity (q(p)) and protein glycosylation. Fed-batch cultivations performed at various specific growth rates (micro) and residual methanol concentrations showed that the specific avidin productivity is growth-dependent. The specific productivity increases strongly with the specific growth rate for micro ranging from 0 to 0.02 h(-1), and increases only slightly with the specific growth rate above this limit. N-terminal glycosylation was also found to be influenced by the specific growth rate, since 9-mannose glycans were the most abundant form at low growth rates, whereas 10-mannose carbohydrate chains were favored at higher micro. These results show that culture parameters, such as the specific growth rate, may significantly affect the activity of glycoproteins produced in Pichia pastoris. In terms of process optimization, this suggests that a compromise on the specific growth rate may have to be found, in certain cases, to work with an acceptable productivity while avoiding the addition of many mannoses.

pH prediction and control in bioprocesses using mid-infrared spectroscopy.

An on-line pH monitoring method based on mid-infrared spectroscopy relevant to bioprocesses is presented. This approach is non-invasive and does not require the addition of indicators or dyes, since it relies on the analysis of species of common buffers used in culture media, such as phosphate buffer. Starting with titrations of phosphoric and acetic acid solutions over almost the entire pH range (2-12), it was shown that the infrared spectra of all samples can be expressed as a linear combination of the molar absorbance of the acids and their deprotonated forms. In other words, pH had no direct influence on the molar infrared spectra themselves, but only on deprotonation equilibria. Accurate prediction (standard error of prediction for pH < 0.15 pH units) was achieved by taking into account the non-ideal behavior of the solutions, using the Debye-Hückel theory to estimate the activity coefficients. Batch cultures of E. coli were chosen as a case study to show how this approach can be applied to bioprocess monitoring. The discrepancy between the spectroscopic prediction and the conventional electrochemical probe never exceeded 0.12 pH units, and the technique was fast enough to implement a feedback controller to maintain the pH constant during cultivation.

Characterization of alginate lyase activity on liquid, gelled, and complexed states of alginate.

A study of alginate lyase was carried out to determine if this enzyme could be used to remove alginate present in the core of alginate/poly-L-lysine (AG/PLL) microcapsules in order to maximize cell growth and colonization. A complete kinetic study was undertaken, which indicated an optimal activity of the enzyme at pH 7-8, 50 degrees C, in the presence of Ca2+. The buffer, not the ionic strength, influenced the alginate degradation rate. Alginate lyase was also shown to be active on gelled forms of alginate, as well as on the AG/PLL complex constituting the membrane of microcapsules. Batch cultures of CHO cells in the presence of alginate showed a decrease of the growth rate by a factor of 2, although the main metabolic flux rates were not modified. The addition of alginate lyase to cell culture medium increased the doubling time 5-7-fold and decreased the protein production rate, although cell viability was not affected. The addition of enzyme to medium containing alginate did not improve growth conditions. This suggests that alginate lyase is probably not suitable for hydrolysis of microcapsules in the presence of cells, in order to achieve high cell density and high productivity. However, the high activity may be useful for releasing cells from alginate beads or AG/PLL microcapsules.

A quantitative analysis of the benefits of mixed feeds of sorbitol and methanol for the production of recombinant avidin with Pichia pastoris.

The advantages of mixed feeds of sorbitol and methanol for the production of recombinant proteins with Pichia pastoris were analyzed quantitatively. The influence of the methanol-sorbitol ratio in the feed medium was investigated on growth stoichiometry and recombinant protein productivity with a P. pastoris Mut(+) strain secreting avidin by performing a transient nutrient gradient in continuous cultivation at a dilution rate of 0.03h(-1). Results showed that mixed feeds of sorbitol and methanol instead of methanol as sole carbon source can improve the productivity in recombinant avidin due to increased biomass yields during mixed substrate growth. The highest volumetric avidin productivity was achieved at a methanol fraction of 43%C-molC-mol(-1) in the feed medium: the volumetric avidin productivity was 1.3-fold higher than during chemostat culture on methanol. The heat production and the oxygen consumption rates were reduced by about 38% for a given dry cell weight concentration at this methanol fraction, features which are very useful for high cell density cultures. Results were in good agreement with a high cell density fed-batch culture performed with a mixed feed of 43% methanol and 57% sorbitol C-molC-mol(-1) at a specific growth rate of 0.03h(-1) during the induction phase. Moreover, it was confirmed that sorbitol accumulation in the culture medium does not affect recombinant avidin productivity, which can especially be advantageous during large-scale cultures where transient substrate accumulation can result from imperfect mixing.