Ana Rita Costa

Technological progresses in monoclonal antibody production systems

Monoclonal antibodies (mAbs) have become vitally important to modern medicine and are currently one of the major biopharmaceutical products in development. However, the high clinical dose requirements of mAbs demand a greater biomanufacturing capacity, leading to the development of new technologies for their large‐scale production, with mammalian cell culture dominating the scenario. Although some companies have tried to meet these demands by creating bioreactors of increased capacity, the optimization of cell culture productivity in normal bioreactors appears as a better strategy. This review describes the main technological progresses made with this intent, presenting the advantages and limitations of each production system, as well as suggestions for improvements. New and upgraded bioreactors have emerged both for adherent and suspension cell culture, with disposable reactors attracting increased interest in the last years. Furthermore, the strategies and technologies used to control culture parameters are in constant evolution, aiming at the on‐line multiparameter monitoring and considering now parameters not seen as relevant for process optimization in the past. All progresses being made have as primary goal the development of highly productive and economic mAb manufacturing processes that will allow the rapid introduction of the product in the biopharmaceutical market at more accessible prices.

Critical review on biofilm methods

Abstract Biofilms are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions. They can be beneficial or have a negative impact particularly when formed in industrial settings or on medical devices. As such, research into the formation and elimination of biofilms is important for many disciplines. Several new methodologies have been recently developed for, or adapted to, biofilm studies that have contributed to deeper knowledge on biofilm physiology, structure and composition. In this review, traditional and cutting-edge methods to study biofilm biomass, viability, structure, composition and physiology are addressed. Moreover, as there is a lack of consensus among the diversity of techniques used to grow and study biofilms. This review intends to remedy this, by giving a critical perspective, highlighting the advantages and limitations of several methods. Accordingly, this review aims at helping scientists in finding the most appropriate and up-to-date methods to study their biofilms.

Glycosylation: impact, control and improvement during therapeutic protein production

Abstract The emergence of the biopharmaceutical industry represented a major revolution for modern medicine, through the development of recombinant therapeutic proteins that brought new hope for many patients with previously untreatable diseases. There is a ever-growing demand for these therapeutics that forces a constant technological evolution to increase product yields while simultaneously reducing costs. However, the process changes made for this purpose may also affect the quality of the product, a factor that was initially overlooked but which is now a major focus of concern. Of the many properties determining product quality, glycosylation is regarded as one of the most important, influencing, for example, the biological activity, serum half-life and immunogenicity of the protein. Consequently, monitoring and control of glycosylation is now critical in biopharmaceutical manufacturing and a requirement of regulatory agencies. A rapid evolution is being observed in this context, concerning the influence of glycosylation in the efficacy of different therapeutic proteins, the impact on glycosylation of a diversity of parameters/processes involved in therapeutic protein production, the analytical methodologies employed for glycosylation monitoring and control, as well as strategies that are being explored to use this property to improve therapeutic protein efficacy (glycoengineering). This work reviews the main findings on these subjects, providing an up-to-date source of information to support further studies.

The role of polysaccharide intercellular adhesin (PIA) in Staphylococcus epidermidis adhesion to host tissues and subsequent antibiotic tolerance

The aim of this study was to determine the role of polysaccharide intercellular adhesin (PIA) in Staphylococcus epidermidis adhesion to host tissues and subsequent antibiotic tolerance. The adherence of S. epidermidis 1457 and the mutant defective in PIA production (1457-M10) to urinary epithelium and endothelium was estimated by colony counting. Minimum bactericidal concentration and mean reduction of cellular activity (XTT) following antibiotic exposure was determined for planktonic and adhered bacteria. S. epidermidis 1457 adhered to a greater extent to both cells than the mutant strain. The adhered strains had a significantly higher antimicrobial tolerance than their planktonic counterparts. The mutant strain was, in general, the most susceptible to the antibiotics assayed. In conclusion, PIA may influence S. epidermidis adherence to host tissues and their antimicrobial susceptibility. Initial adhesion may be the main step for the acquisition of resistance in S. epidermidis.

Adaptation of the International Affective Picture System (IAPS) for European Portuguese

This study presents the results of the adaptation of the International Affective Picture System (IAPS) for European Portuguese (EP). Following the original procedure of Lang et al., 2000 native speakers of EP rated the 1,182 pictures of the last version of the IAPS set on the three affective dimensions of valence, arousal, and dominance, using the Self-Assessment Manikin (SAM). Results showed that the normative values of the IAPS for EP are properly distributed in the affective space of valence and arousal, showing the typical boomerang-shaped distribution observed in previous studies. Results also point to important differences in the way Portuguese females and males react to affective pictures that should be taken into consideration when planning and conducting research with Portuguese samples. Furthermore, the results from the cross-cultural comparisons between the EP ratings and the ratings from the American, Spanish, Brazilian, Belgian, Chilean, Indian, and Bosnian-Herzegovinian standardizations, showed that in spite of the fact that IAPS stimuli elicited affective responses that are similar across countries and cultures (at least in Western cultures), there are differences in the way Portuguese individuals react to IAPS pictures that strongly recommend the use of the normative values presented in this work. They can be downloaded as a supplemental archive at http://brm.psychonomic-journals.org/content/supplemental or at http://p-pal.di.uminho.pt/about/databases.

The impact of cell adaptation to serum-free conditions on the glycosylation profile of a monoclonal antibody produced by Chinese hamster ovary cells

N-glycosylation is one of the most crucial parameters affecting the biological activity of therapeutic monoclonal antibodies (mAbs), and should therefore be closely monitored and controlled to guarantee a consistent and high-quality product in biopharmaceutical processes. In the present work, the effect of the time-consuming step of gradual cell adaptation to serum-free conditions on the glycosylation profile of a mAb produced by CHO-K1 cells was evaluated. High-performance liquid chromatography analysis revealed important changes in mAb glycosylation patterns in all steps of serum reduction. These changes could be grouped in two distinct phases of the process of adaptation: middle (2.5 to 0.15% serum) and final (0.075 and 0% serum). For intermediate levels of serum, a desirable increase of galactosylation and decrease of fucosylation, but an undesirable increase in sialylation were observed; while the inverse was obtained at the final stages of adaptation. These divergences may be related to the reduction of serum supplementation, to variations in the levels of cell density and viability achieved at these stages, and to the natural shift of the cell growth mode during adaptation from adherent to suspended. The divergent glycan profiles obtained in this study demonstrate a strong influence of the adaptation process on mAb glycosylation, suggesting that control and monitoring of product quality should be implemented at the early stages of process development.

ESCOLEX: A grade-level lexical database from European Portuguese elementary to middle school textbooks

In this article, we introduce ESCOLEX, the first European Portuguese children’s lexical database with grade-level-adjusted word frequency statistics. Computed from a 3.2-million-word corpus, ESCOLEX provides 48,381 word forms extracted from 171 elementary and middle school textbooks for 6- to 11-year-old children attending the first six grades in the Portuguese educational system. Like other children’s grade-level databases (e.g., Carroll, Davies, & Richman, 1971; Corral, Ferrero, & Goikoetxea, Behavior Research Methods, 41, 1009–1017, 2009; Lété, Sprenger-Charolles, & Colé, Behavior Research Methods, Instruments, & Computers, 36, 156–166, 2004; Zeno, Ivens, Millard, Duvvuri, 1995), ESCOLEX provides four frequency indices for each grade: overall word frequency (F), index of dispersion across the selected textbooks (D), estimated frequency per million words (U), and standard frequency index (SFI). It also provides a new measure, contextual diversity (CD). In addition, the number of letters in the word and its part(s) of speech, number of syllables, syllable structure, and adult frequencies taken from P-PAL (a European Portuguese corpus-based lexical database; Soares, Comesaña, Iriarte, Almeida, Simões, Costa, …, Machado, 2010; Soares, Iriarte, Almeida, Simões, Costa, França, …, Comesaña, in press) are provided. ESCOLEX will be a useful tool both for researchers interested in language processing and development and for professionals in need of verbal materials adjusted to children’s developmental stages. ESCOLEX can be downloaded along with this article or from http://p-pal.di.uminho.pt/about/databases.

Affective auditory stimuli: Adaptation of the International Affective Digitized Sounds (IADS-2) for European Portuguese

In this study, we present the normative values of the adaptation of the International Affective Digitized Sounds (IADS-2; Bradley & Lang, 2007a) for European Portuguese (EP). The IADS-2 is a standardized database of 167 naturally occurring sounds that is widely used in the study of emotions. The sounds were rated by 300 college students who were native speakers of EP, in the three affective dimensions of valence, arousal, and dominance, by using the Self-Assessment Manikin (SAM). The aims of this adaptation were threefold: (1) to provide researchers with standardized and normatively rated affective sounds to be used with an EP population; (2) to investigate sex and cultural differences in the ratings of affective dimensions of auditory stimuli between EP and the American (Bradley & Lang, 2007a) and Spanish (Fernández-Abascal et al., Psicothema 20:104–113 2008; Redondo, Fraga, Padrón, & Piñeiro, Behavior Research Methods 40:784–790 2008) standardizations; and (3) to promote research on auditory affective processing in Portugal. Our results indicated that the IADS-2 is a valid and useful database of digitized sounds for the study of emotions in a Portuguese context, allowing for comparisons of its results with those of other international studies that have used the same database for stimulus selection. The normative values of the EP adaptation of the IADS-2 database can be downloaded along with the online version of this article.

Comparison of commercial serum-free media for CHO-K1 cell growth and monoclonal antibody production.

The selection of a serum-free medium for a particular process of production using mammalian cells is a critical step for its success. In this study, seven commercially available serum-free media (EX-CELL, ISF-I, CD CHO, CDM4CHO, CHO-III-A, Octomed and HybridoMed) were evaluated and compared for cell growth and monoclonal antibody (mAb) production of a transfected CHO-K1 cell line. In the conditions assayed, EX-CELL and particularly CDM4CHO are the most recommended media for extended biopharmaceutical processes, on account of inducing superior levels of cell proliferation and mAb production, accentuated by a tendency to improve over time. Furthermore, the less positive results obtained with some media emphasize the importance and the impact of the correct medium selection.

Advances and Drawbacks of the Adaptation to Serum-Free Culture of CHO-K1 Cells for Monoclonal Antibody Production

Currently, mammalian cell technology has become the focus of biopharmaceutical production, with strict regulatory scrutiny of the techniques employed. Major concerns about the presence of animal-derived components in the culture media led to the development of serum-free (SF) culture processes. However, cell adaptation to SF conditions is still a major challenge and limiting step of process development. Thus, this study aims to assess the impact of SF adaptation on monoclonal antibody (mAb) production, identify the most critical steps of cell adaptation to the SF EX-CELL medium, and create basic process guidelines. The success of SF adaptation was dependent on critical steps that included accentuated cell sensitivity to common culture procedures (centrifugation, trypsinization), initial cell concentration, time given at each step of serum reduction, and, most importantly, medium supplements used to support adaptation. Indeed, only one of the five supplement combinations assessed (rhinsulin, ammonium metavanadate, nickel chloride, and stannous chloride) succeeded for the Chinese hamster ovary-K1 cell line used. This work also revealed that the chemically defined EX-CELL medium benefits mAb production in comparison with the general purpose Dulbecco’s Modified Eagle’s Medium, but the complete removal of serum attenuates these positive effects.