Antonio R. Moreira

Disposable bioprocessing: the future has arrived.

Increasing cost pressures are driving the rapid adoption of disposables in bioprocessing. While well ensconced in lab-scale operations, the lower operating/ validation costs at larger scale and relative ease of use are leading to these systems entering all stages and operations of a typical biopharmaceutical manufacturing process. Here, we focus on progress made in the incorporation of disposable equipment with sensor technology in bioprocessing throughout the development cycle. We note that sensor patch technology is mostly being adapted to disposable cell culture devices, but future adaptation to downstream steps is conceivable. Lastly, regulatory requirements are also briefly assessed in the context of disposables and the Process Analytical Technologies (PAT) and Quality by Design (QbD) initiatives.

Utilization of Enzymes for Environmental Applications

ABSTRACT Enzymes are powerful tools that help sustain a clean environment in several ways. They are utilized for environmental purposes in a number of industries including agro-food, oil, animal feed, detergent, pulp and paper, textile, leather, petroleum, and specialty chemical and biochemical industry. Enzymes also help to maintain an unpolluted environment through their use in waste management. Recombinant DNA technology, protein engineering, and rational enzyme design are the emerging areas of research pertaining to environmental applications of enzymes. The future will also see the employment of various technologies including gene shuffling, high throughput screening, and nanotechnology. This article presents an overview of the enzymatic applications in pollution control and the promising research avenues in this area.

Production of extracellular alkaline protease by immobilization of the marine bacterium Teredinobacter turnirae

Abstract Cells from a marine bacterium, Teredinobacter turnirae cells were immobilized in calcium alginate beads and used for alkaline protease production. The maximum protease activity was obtained at 3% (w/v) sodium alginate and 3% CaCl 2 concentrations with a 1/2 cell/alginate ratio, i.e. 2400 U/ml. There was no significant difference in the maximum protease activity between three bead sizes used. A drastic fall in protease production was observed when the beads were treated with glutaraldehyde. The beads were used for eight successive fermentation batches each lasting 72 h. It was also observed that there was a ∼3.5-fold increase in volumetric productivity of protease after the fourth cycle.

Advances in clone selection using high-throughput bioreactors†

Effective clone selection is a crucial step toward developing a robust mammalian cell culture production platform. Currently, clone selection is done by culturing cells in well plates and picking the highest producers. Ideally, clone selection should be done in a stirred tank bioreactor as this would best replicate the eventual production environment. The actual number of clones selected for future evaluation in bioreactors at bench‐scale is limited by the scale‐up and operational costs involved. This study describes the application of miniaturized stirred high‐throughput bioreactors (35 mL working volume; HTBRs) with noninvasive optical sensors for clone screening and selection. We investigated a method for testing several subclones simultaneously in a stirred environment using our high throughput bioreactors (up to 12 clones per HTBR run) and compared it with a traditional well plate selection approach. Importantly, it was found that selecting clones solely based on results from stationary well plate cultures could result in the chance of missing higher producing clones. Our approach suggests that choosing a clone after analyzing its performance in a stirred bioreactor environment is an improved method for clone selection.

Production of an endoglucanase by the shipworm bacterium, Teredinobacter turnirae

Abstract The nutritional behavior of a cellulolytic nitrogen-fixing shipworm bacterium, Teredinobacter turnirae, is described, with respect to various carbon and nitrogen sources, in terms of endoglucanase production. Also, the effects of various surfactants on enzyme production are reported. Among the carbon sources, sucrose results in the maximum enzyme production, followed by cellulose. Ammonium phosphate proves to be the best nitrogen source for endoglucanase production. Various surfactants enhance the enzyme titers, with Triton X-100 yielding the best results. A combination of the above-mentioned components improves the enzyme production by 3.6-fold.

Anaerobic Production of Chemicals

Anaerobic fermentations are among the oldest industrially practiced fermentation processes. The production of acetone and butanol, for example, by the well-known Weizmann process, dates as far back as 1916 [1]. In spite of such a long record, anaerobic processes are not, at the present time, among the most important fermentation-based industries. This has resulted from the tremendous development of the petrochemical industries starting in the 1950s and the consequent displacement of the fermentation routes by the more economically attractive petrochemical processes.

Dissolved oxygen and pH profile evolution after cryovial thaw and repeated cell passaging in a T‐75 flask

Routine cell culture is done in small‐scale disposable vessels (typically 0.1–100 mL volumes) in academia and industry. Despite their wide use in bioprocess development (i.e., process optimization and process validation), miniature process scouting devices (PSDs) are considered “black boxes” because they are generally not equipped with sensors. In this study, we show that on‐line monitoring of dissolved oxygen (DO) and pH in a T‐75 flask‐based PSD can be achieved during cell passaging and that this information can be linked to different cellular metabolic states. In this case, on‐line monitoring of DO and pH show three distinctive metabolic regions in passages 1–18, 19–28, 29–54 and in particular, the shift in the pH curve, the specific oxygen uptake rate (qO2), and the lactate production rate to the oxygen consumption rate yield (YLac/ox) confirm the existence of these distinctive metabolic regions. These findings are particularly useful because they show that sensor equipped PSDs can help to monitor cell culture behavior after thaw, in pre‐ and seed culture prior to scale‐up and in development/optimization studies. Such routine monitoring will help to develop more consistent cell culture techniques. Biotechnol. Bioeng. 2010;105: 1040–1047.

A case study in converting disposable process scouting devices into disposable bioreactors as a future bioprocessing tool

In this study, we perform mass transfer characterization (kLa) on a novel mechanically driven/stirred Process Scouting Device, PSD, (SuperSpinner D 1000®, SSD) and demonstrate that this novel device can be viewed as disposable bioreactor. Using patch‐based optical sensors, we were able to monitor critical cell culture environmental conditions such as dissolved oxygen (DO) and pH in SSD for comparison to a 1 L standard spinner (SS) flask. We also coupled these mass transfer studies with mixing time studies where we observed relative high mixing times (5.2 min) that are typically observed in production scale bioreactors. Decreasing the mixing time 3.5‐fold resulted in 30% increase in kLa (from 2.3 to 3.0 h−1) and minimum DO level increased from 0% to 20% for our model hybridoma cell line. Finally, maximum viable cell density and protein titer stayed within ±20% of historical data, from our standard 5 L stirred bioreactor (Biostat®) operated under active DO control. Biotechnol. Bioeng. 2012; 109: 2790–2797.

Repeated batch production of alkaline protease using porous sintered glass as carriers

Abstract Whole cells of Teredinobacter turnirae were immobilized onto different sizes of porous sintered glass SIRAN. Production of alkaline protease by the immobilized whole cells was attempted in a batch system. Alkaline protease activity produced by immobilized whole cells was about 2.3 times higher than that produced by freely suspended cells under the same cultivation conditions. The reusability of the immobilized and freely suspended cells was examined. The activity of alkaline protease produced by freely suspended cells decreased with increasing use cycles while the activity of immobilized cells increased gradually and reached a steady state after 4 cycles. T. turnirae cells immobilized on different matrices were observed by scanning electron microscopy and the distribution of the cells on each matrix used either in and/or on the internal pores observed.

Genomic analysis of a hybridoma batch cell culture metabolic status in a standard laboratory 5 L bioreactor.

Currently, there is a gap in the knowledge of the culture responses to controlled bioreactor environment during the course of batch cell culture from early exponential phase to stationary‐phase. If available, such information could be used to designate gene transcripts for predicting cell status and as a quality predictor for a controlled bioreactor. In this study, we used oligonucleotide microarrays to obtain baseline gene expression profiles during the time‐course of a hybridoma batch cell culture in a 5 L bench‐scale bioreactor. Gene expression changes that were up or down modulated from early‐to‐late in batch culture, as well as invariant gene profiles with significant expression were identified using microarray. Typical cellular functions that seemed to be correlated with transcriptomics were oxidative stress response, DNA damage response, apoptosis, and cellular metabolism. As confirmatory evidence, microarray findings were verified with a more rigorous semiquantitative gene‐specific Reverse transcriptase‐polymerase chain reaction (RT‐PCR). The results of this study suggest that under predefined bioreactor culture conditions, significant gene changes from lag to log to stationary phase could be identified, which could then be used to track the culture state.