Optimization and application of caprylic acid precipitation in the purification of monoclonal antibody / 生物工程学报

In response to the market demand for therapeutic antibodies, the upstream cell culture scale and expression titer of antibodies have been significantly improved, while the production efficiency of downstream purification process is relatively fall behind, and the downstream processing capacity has become a bottleneck limiting antibody production throughput. Using monoclonal antibody mab-X as experimental material, we optimized the caprylic acid (CA) precipitation process conditions of cell culture fluid and low pH virus inactivation pool, and studied two applications of using CA treatment to remove aggregates and to inactivate virus. Based on the lab scale study, we carried out a 500 L scale-up study, where CA was added to the low pH virus inactivation pool for precipitation, and the product quality and yield before and after precipitation were detected and compared. We found that CA precipitation significantly reduced HCP residuals and aggregates both before and after protein A affinity chromatography. In the aggregate spike study, CA precipitation removed about 15% of the aggregates. A virus reduction study showed complete clearance of a model retrovirus during CA precipitation of protein A purified antibody. In the scale-up study, the depth filtration harvesting, affinity chromatography, low pH virus inactivation, CA precipitation and depth filtration, and cation exchange chromatography successively carried out. The mixing time and stirring speed in the CA precipitation process significantly affected the CA precipitation effect. After CA precipitation, the HCP residue in the low pH virus inactivation solution decreased 895 times. After precipitation, the product purity and HCP residual meet the quality criteria of monoclonal antibodies. CA precipitation can reduce the chromatography step in the conventional purification process. In conclusion, CA precipitation in the downstream process can simplify the conventional purification process, fully meet the purification quality criterion of mab-X, and improve production efficiency and reduce production costs. The results of this study may promote the application of CA precipitation in the purification of monoclonal antibodies, and provide a reference for solving the bottleneck of the current purification process.

Factors influencing the sustainability of a community health volunteer programme — A scoping review

Background: Sustainability of any Community health worker programme is determined by several internal and external factors and is highly context and region specific. We aimed to identify factors that influence the sustainability of a community health volunteer programme across the globe.

Formulation Process Analysis of a Virus-based Biopesticide to Control the Tomato leafminer Tuta absoluta

Abstract: Virus-based biopesticides are effective biocontrol agents of crop insect pests. Development of suitable formulations and production processes are necessary to obtain high-quality products easily adopted by farmers. A detailed unit operation study was carried out for the production process of a Phthorimaea operculella granulovirus-based biopesticide to control the tomato leafminer, Tuta absoluta, one of the most important pests affecting this crop. Physicochemical, microbiological, and insecticidal parameters were implemented in the process and applied to the finished product, and a scaling strategy was developed. A Quantitative Polymerase Chain Reaction (Q-PCR) technique was implemented to quantify viral concentrations in the active ingredient (5.34 ± 1.44 x109 Occlusion Bodies mL-1) and in the finished product (>1.6x109 OB mL-1), without contaminant interferences. The Q-PCR methodology was also useful to select the appropriate solid mixing time following Lacey´s mixing index (8 min). Factors and similarity principles influencing the liquid mixing process were identified in the scaling evaluation. Furthermore, the drying kinetics analysis enabled identifying a drying temperature of 35 °C, with an efficacy under controlled conditions higher than 97%. Contaminant concentration was lower than 1%, indicating controlled and aseptic formulation process conditions. A simple statistical method was used to estimate the reproducibility and repeatability of the parameters assessed in the finished product. These results enable to establish and extrapolate important parameters in the standardization, scale-up, and quality control for the granulovirus-based biopesticide.

New opportunities and challenges for hybrid data and model driven bioprocess optimization and scale-up / 生物工程学报

Currently, biomanufacturing technology and industry are receiving worldwide attention. However, there are still great challenges on bioprocess optimization and scale-up, including: lacing the process detection methods, which makes it difficult to meet the requirement of monitoring of key indicators and parameters; poor understanding of cell metabolism, which arouses problems to rationally achieve process optimization and regulation; the reactor environment is very different across the scales, resulting in low efficiency of stepwise scale-up. Considering the above key issues that need to be resolved, here we summarize the key technological innovations of the whole chain of fermentation process, i.e., real-time detection-dynamic regulation-rational scale-up, through case analysis. In the future, bioprocess design will be guided by a full lifecycle in-silico model integrating cellular physiology (spatiotemporal multiscale metabolic models) and fluid dynamics (CFD models). This will promote computer-aided design and development, accelerate the realization of large-scale intelligent production and serve to open a new era of green biomanufacturing.

Preface for special issue on industrial microorganisms: innovation and breakthrough (2021) / 生物工程学报

Industrial microorganisms and their products are widely used in various fields such as industry, agriculture, and medicine, which play a pivotal role in economy. Efficient industrial strains are the key to improve production efficiency, and advanced fermentation technology as well as instrument platform is also important to develop microbial metabolic potential. In recent years, rapid development has been achieved in research of industrial microorganisms. Artificial intelligence, efficient genome-editing and synthetic biology technologies have been increasingly applied, and related industrial applications are being accomplished. In order to promote utilization of industrial microorganisms in biological manufacturing, we organized this special issue on innovation and breakthrough of industrial microorganisms. Progress including microbial strain diversity and metabolism, strain development technology, fermentation process optimization and scale-up, high-throughput droplet culture system, and applications of industrial microorganisms is summarized in this special issue, and prospects on future studies are proposed.

Development of biodegradation process for Poly(DL-lactic acid) degradation by crude enzyme produced by Actinomadura keratinilytica strain T16-1

Background: Plastic waste is a serious problem because it is difficult to degrade, thereby leading to global environment problems. Poly(lactic acid) (PLA) is a biodegradable aliphatic polyester derived from renewable resources, and it can be degraded by various enzymes produced by microorganisms. This study focused on the scale-up and evaluated the bioprocess of PLA degradation by a crude microbial enzyme produced by Actinomadura keratinilytica strain T16-1 in a 5 L stirred tank bioreactor. Results: PLA degradation after 72 h in a 5 L bioreactor by using the enzyme of the strain T16-1 under controlled pH conditions resulted in lactic acid titers (mg/L) of 16,651 mg/L and a conversion efficiency of 89% at a controlled pH of 8.0. However, the PLA degradation process inadvertently produced lactic acid as a potential inhibitor, as shown in our experiments at various concentrations of lactic acid. Therefore, the dialysis method was performed to reduce the concentration of lactic acid. The experiment with a dialysis bag achieved PLA degradation by weight loss of 99.93%, whereas the one without dialysis achieved a degradation of less than approximately 14.75%. Therefore, the dialysis method was applied to degrade a commercial PLA material (tray) with a conversion efficiency of 32%, which was 6-fold more than that without dialysis. Conclusions: This is the first report demonstrating the scale-up of PLA degradation in a 5 L bioreactor and evaluating a potential method for enhancing PLA degradation efficiency.

Scaling-up fermentation of Escherichia coli for production of recombinant P64k protein from Neisseria meningitidis

Background: P64k is a Neisseria meningitidis high molecular weight protein present in meningococcal vaccine preparations. The lpdA gene, which encodes for this protein, was cloned in Escherichia coli and the P64k recombinant protein was expressed in E. coli K12 GC366 cells under the control of a tryptophan promoter. P64k was expressed as an intracellular soluble protein about 28% of the total cellular protein. Several scale-up criteria of fermentation processes were studied to obtain the recombinant P64k protein at the pilot production scale. Results: The best operational conditions at a larger scale production of P64k recombinant protein were studied and compared using the four following criteria: Constant Reynold's number (Re constant), Constant impeller tip speed (n di constant), Constant power consumption per unit liquid volume (P/V constant) and Constant volumetric oxygen transfer coefficients (KLa/k constant). The highest production of the recombinant protein was achieved based on the constant KLa/k scale-up fermentation criterion, calculating the aeration rate (Q) and the impeller agitation speed (n) by iterative process, keeping constant the KLa/k value from bench scale. The P64k protein total production at the 50 l culture scale was 546 mg l -1 in comparison with the 284 mg l -1 obtained at 1.5 l bench scale. Conclusions: The methodology described herein, for the KLa/k scale-up fermentation criterion, allowed us to obtain the P64k protein at 50 l scale. A fermentation process for the production of P64k protein from N. meningitidis was established, a protein to be used in future vaccine formulations in humans.

Double Fortified Salt in India: Coverage, Efficacy and Way Forward

Iron deficiency remains the world’s most widespread nutritional disorder and India is one of the countries very worst afflicted. India has successfully reduced the burden of iodine-deficiency disorders through mandatory iodization of salt for more than 20 years. This has resulted in a significant decrease in the prevalence of iodine deficiency diseases. Building on the success with iodization, double fortification of salt with iodine and iron is gaining ground and can be integrated with established iodization processes. DFS contemplates the creation and distribution of a powerful innovative product with demonstrated health effects, building on existing distribution platforms for salt through public distribution channels targeted to some of the most impoverished populations in the country at minimal expense and without requiring changes in cultural habits and compliance. Two formulations have been approved by Food Safety and Standard Authority of India (FSSAI) with iron either in the form of encapsulated ferrous fumarate or ferrous sulphate. A meta-analysis showed that DFS increased hemoglobin concentrations significantly. This intervention as part of a broader anemia strategy has the potential to effect large-scale anemia reduction across populations in India on a permanent and economically self-sustaining basis.

A review of methods to estimate the visibility factor for bias correction in network scale-up studies / 한국역학회지

Network scale-up is an indirect size estimation method, in which participants are questioned on sensitive behaviors of their social network members. Therefore, the visibility of the behavior affects the replies and estimates. Many attempts to estimate visibility have been made. The aims of this study were to review the main methods used to address visibility and to provide a summary of reported visibility factors (VFs) across populations. We systematically searched relevant databases and Google. In total, 15 studies and reports that calculated VFs were found. VF calculation studies have been applied in 9 countries, mostly in East Asia and Eastern Europe. The methods applied were expert opinion, comparison of NSU with another method, the game of contacts, social respect, and the coming-out rate. The VF has been calculated for heavy drug users, people who inject drugs (PWID), female sex workers (FSWs) and their clients, male who have sex with male (MSM), alcohol and methamphetamine users, and those who have experienced extra-/pre-marital sex and abortion. The VF varied from 1.4% in Japan to 52.0% in China for MSM; from 34.0% in Ukraine to 111.0% in China for FSWs; and from 12.0% among Iranian students to 57.0% in Ukraine for PWID. Our review revealed that VF estimates were heterogeneous, and were not available for most settings, in particular the Middle East and North Africa region, except Iran. More concrete methodologies to estimate the VF are required.

A review of methods to estimate the visibility factor for bias correction in network scale-up studies / 한국역학회지

Network scale-up is an indirect size estimation method, in which participants are questioned on sensitive behaviors of their social network members. Therefore, the visibility of the behavior affects the replies and estimates. Many attempts to estimate visibility have been made. The aims of this study were to review the main methods used to address visibility and to provide a summary of reported visibility factors (VFs) across populations. We systematically searched relevant databases and Google. In total, 15 studies and reports that calculated VFs were found. VF calculation studies have been applied in 9 countries, mostly in East Asia and Eastern Europe. The methods applied were expert opinion, comparison of NSU with another method, the game of contacts, social respect, and the coming-out rate. The VF has been calculated for heavy drug users, people who inject drugs (PWID), female sex workers (FSWs) and their clients, male who have sex with male (MSM), alcohol and methamphetamine users, and those who have experienced extra-/pre-marital sex and abortion. The VF varied from 1.4% in Japan to 52.0% in China for MSM; from 34.0% in Ukraine to 111.0% in China for FSWs; and from 12.0% among Iranian students to 57.0% in Ukraine for PWID. Our review revealed that VF estimates were heterogeneous, and were not available for most settings, in particular the Middle East and North Africa region, except Iran. More concrete methodologies to estimate the VF are required.

Scaling-up batch conditions for efficient sucrose hydrolysis catalyzed by an immobilized recombinant Pichia pastoris cells in a stirrer tank reactor

Background: Invert sugar is used greatly in food and pharmaceutical industries. This paper describes scaling-up batch conditions for sucrose inversion catalyzed by the recombinant Pichia pastoris BfrA4X whole cells expressing Thermotoga maritima invertase entrapped in calcium alginate beads. For the first time, we describe the application of a kinetic model to predict the fractional conversion expected during sucrose hydrolysis reaction in both, a model and a prototype bioreactor with 0.5- and 5-L working volume, respectively. Results: Different scaled-up criteria used to operate the 0.5-L bioreactor were analyzed to explore the invert sugar large scale production. After model inversion studies, a 5-L scaled-up reaction system was performed in a 7-L stirred reactor. Both scaled-up criteria, immobilized biocatalyst dosage and stirring speed, were analyzed in each type of bioreactors and the collected data were used to ensure an efficient scale-up of this biocatalyst. Conclusions: To date, there is not enough information to describe the large-scale production of invert sugar using different scaled-up criteria such as dose of immobilized biocatalyst and stirring speed effect on mass transfer. The present study results constitute a valuable tool to successfully carry out this type of high-scale operation for industrial purposes.

Pilot-scale purification of lipopeptide from marine-derived Bacillus marinus / 生物工程学报

This research was aimed at establishing the pilot-scale purification technology of lipopeptide from marine-derived Bacillus marinus. We studied lipopeptide surfactivity interferences on scale-up unit technologies including acid precipitation, methanol extraction, solvent precipitation, salting out, extraction, silica gel column chromatography and HZ806 macroporous absorption resin column chromatography. Then, the unit technologies were combined in a certain order, to remove the impurities gradually, and to gain purified lipopeptide finally, with high recovery rate throughout the whole process. The novel pilot-scale purification technology could effectively isolate and purify lipopeptide with 87.51% to 100% purity in hectograms from 1 ton of Bacillus marinus B-9987 fermentation broth with more than 81.73% recovery rate. The first practical hectogram production of highly purified lipopeptide derived from Bacillus marinus was achieved. With this new purification method, using complex media became possible in fermentation process to reduce the fermentation cost and scale-up the purification for lipopeptide production. For practicability and economy, foaming problem resulting from massive water evaporation was avoided in this technology.

Effectiveness of Scaling up the ‘Three Pillars’ Approach to Accelerating MDG 4 Progress in Ethiopia.

This paper describes the integrated approach taken by the Government of Ethiopia with support from the Essential Services for Health in Ethiopia (ESHE) Project and assesses its effect on the coverage of six child health practices associated with reducing child mortality. The ESHE Project was designed to contribute to reducing high child mortality rates at scale among 14.5 million people through the ‘three pillars’ approach. This approach aimed to (i) strengthen health systems, (ii) improve health workers’ performance, and (iii) engage the community. The intervention was designed with national and subnational stakeholders’ input. To measure the Project’s effect on the coverage of child health practices, we used a quasi-experimental design, with representative household survey data from the three most populous regions of Ethiopia, collected at the 2003-2004 baseline and 2008 endline surveys of the Project. A difference-in-differences analysis model detected an absolute effect of the ESHE intervention of 8.4% points for DTP3 coverage (p=0.007), 12.9% points for measles vaccination coverage (p<0.001), 12.6% points for latrines (p=0.002), and 9.8% points for vitamin A supplementation (p<0.001) across the ESHE-intervention districts (woredas) compared to all non-ESHE districts of the same three regions. Improvements in the use of modern family planning methods and exclusive breastfeeding were not significant. Important regional variations are discussed. ESHE was one of several partners of the Ministry of Health whose combined efforts led to accelerated progress in the coverage of child health practices

Scale up of production in a bioreactor of a halotolerant protease from moderately halophilic Bacillus sp. isolated from soil

Studies were conducted on the production of protease by moderately halophilic Bacillus sp. on agro-industrial waste materials. The bacterium could efficiently use many agro wastes as substrates but wheat bran supported maximum enzyme production. To ascertain the performance of the process in shake flasks and lab scale bioreactor, experiments were conducted to analyse protease activity utilizing wheat bran as cost effective substrate. The studies unveiled that pH 7.0, temperature 30°C and static conditions were optimal for enzyme production in flask level fermentation. In scale-up fermentation, at optimal pH and temperature, agitation rate of 50 rpm was best for protease production. The enzymatic nature was studied in 10% SDS gels with BSA (2.5 mg/mL) as substrate and banding pattern was compared with undigested BSA as control. The endoprotease nature and the kinetics of protease activity were confirmed. The enzyme retained 37% of its activity even at 5 M NaCl concentration. The proteolytic activity was also confirmed by casein zymogram analysis. The fermentation medium containing inexpensive substrates, physical conditions and ability of Bacillus sp. to exhibit protease activity on a large scale could collectively be useful for commercial production.

Preparation and scale up of extended-release tablets of bromopride

Reproducibility of the tablet manufacturing process and control of its pharmaceutics properties depends on the optimization of formulation aspects and process parameters. Computer simulation such as Design of Experiments (DOE) can be used to scale up the production of this formulation, in particular for obtaining sustained-release tablets. Bromopride formulations are marketed in the form of extended-release pellets, which makes the product more expensive and difficult to manufacture. The aim of this study was to formulate new bromopride sustained release formulations as tablets, and to develop mathematical models to standardize the scale up of this formulation, controlling weight and hardness of the tablets during manufacture according to the USP 34th edition. DOE studies were conducted using Minitab(tm) software. Different excipient combinations were evaluated in order to produce bromopride sustained-release matrix tablets. In the scale-up study, data were collected and variations in tableting machine parameters were measured. Data were processed by Minitab(tm) software, generating mathematical equations used for prediction of powder compaction behavior, according to the settings of the tableting machine suitable for scale-up purposes. Bromopride matrix tablets with appropriate characteristics for sustained release were developed. The scale-up of the formulation with the most suitable sustained release profile was established by using mathematical models, indicating that the formulation can be a substitute for the pellets currently marketed.

A reprodutibilidade do processo de fabricação de comprimidos e o controle das suas propriedades farmacotécnicas depende da otimização dos aspectos de formulação e dos parâmetros de processo. O planejamento de experimentos como o Desenho de Experimentos (DOE) pode ser utilizado para acelerar a produção desta formulação, em particular, para a obtenção de comprimidos de liberação prolongada. Formulações de bromoprida são comercializadas sob a forma de péletes de liberação prolongada, o que torna o produto caro e de difícil fabricação. O objetivo deste estudo foi preparar novas formulações de bromoprida de liberação prolongada na forma de comprimidos e desenvolver modelos matemáticos visando ao escalonamento destas formulações, controlando o peso e a dureza dos comprimidos durante a fabricação, de acordo com a 34ª Edição da USP. Estudos de DOE foram realizados utilizando o software Minitab(tm). Diferentes combinações de excipientes foram avaliadas visando à obtenção dos comprimidos de liberação prolongada de bromoprida. No estudo de scale-up, coletaram-se e mediu-se a influência das variações nos parâmetros da máquina de compressão. Processaram-se os dados obtidos pelo software Minitab (tm), gerando equações matemáticas aptas para a previsão do comportamento de compactação do pó em escala industrial. Os comprimidos obtidos apresentavam características adequadas em termos de liberação sustentada, sendo a cinética de liberação estabelecida utilizando modelos matemáticos, indicando que esta formulação pode ser uma substituta aos péletes de bromoprida atualmente comercializados.

Applications of recombinant Pichia pastoris in the healthcare industry

Since the 1970s, the establishment and development of the biotech industry has improved exponentially, allowing the commercial production of biopharmaceutical proteins. Nowadays, new recombinant protein production is considered a multibillion-dollar market, in which about 25% of commercial pharmaceuticals are biopharmaceuticals. But to achieve a competitive production process is not an easy task. Any production process has to be highly productive, efficient and economic. Despite that the perfect host is still not discovered, several research groups have chosen Pichia pastoris as expression system for the production of their protein because of its many features. The attempt of this review is to embrace several research lines that have adopted Pichia pastoris as their expression system to produce a protein on an industrial scale in the health care industry.

Scale-up of tuberculosis laboratory services in Gujarat, India.

M. tuberculosis has been classified as risk group 3 pathogen and has to be handled at appropriate containment level. The IRL, Ahmedabad got WHO accreditation in March 08 for Lowenstein-Jensen solid culture and first line anti-tuberculosis drug sensitivity. Immediately after WHO endorsement of line probe assay (LPA) for rapid MDR-TB testing in 2008, Gujarat state leaped forwarded and started research on “Genotype MTB DR Plus assay” in collaboration with WHO. In 2009 this effort gained momentum when the IRL, Ahmedabad was radically upgraded to BSL 3 facility. BSL 3 facility like this can balance risk of air-borne infection. As a minimum, countries embarking on drug-resistant tuberculosis programmes should establish laboratory capacity to diagnose MDR-TB and monitor culture conversion of patients on Category IV treatment. Gujarat has set the example of a truly dynamic tuberculosis management, demonstrating that rapid scale-up of laboratory services for MDR-TB diagnosis is feasible even at regional level, in resource-constrained settings.

Estimation on the size of men who have sex with men among college students in Beijing through the Network Scale-Up Method (NSUM) / 中华流行病学杂志

Objective Network Scale-Up Method (NSUM) was used to estimate the Personal Network Size (C) of college students in Beijing and the size of men who have sex with men (MSM).Methods A survey was conducted on Beijing college students to estimate the personal network size (C) through NSUM and then using the back-estimating and attitude adjustment to correct C value and the size of the target population.Results The mean of C among Beijing college students was 97 and the differences of C between different majors and different grades were both statistically significant.Among Beijing College students,3.96％ of them identified themselves as MSM,with the number as 16 260.Conclusion Using the NSUM to estimate C of Beijing college students and size of MSM seemed to be credible which worth popularized.

Optimization of the culture temperature of recombinant CHO-DG44 cells expressing tumor necrosis factor receptor-Fc / 中国药学杂志

OBJECTIVE: To optimize the culture temperature of recombinant CHO-DG44 cells expressing tumor necrosis factor receptor-Fc fusion protein and to determine the bioactivity of expressed protein at the best culture temperature. METHODS: Recombinant CHO cells were batchly cultured at three different temperatures (37°C, 37°C shifting to 31°C and 31°C). Samples were daily tested for cell densities, viabilities, glucose concentration, lactic acid concentration and TNFR-Fc fusion protein concentration, then the best culture temperature was chosen to scale up the fermentation volume (10, 50, 200 mL, and 2 L). TNFR-Fc fusion protein was purified with Protein A affinity chromatography, determined for relative molecular mass, purity and neutralizing activity by SDS-PAGE, SE-HPLC and WST-8 separately. RESULTS: The culture condition of 37°C shifting to 31°C resulted in the maximum viable cell density, high viability and long culture time of the cells as well as high TNFR-Fc protein productivity, and this culture procedure could be successfully applied to the scale-up of recombinant CHO cells. The purified fusion protein, with a relative molecular mass about 150 000, reached the purity of more than 93% and neutralized the cytotoxic effect of TNFα. CONCLUSION: Compared to the traditional culture temperature of 37°C, the culture temperature of 37°C shifting to 31°C can obviously improve the TNFR-Fc fusion protein output by recombinant CHO-DG44 cells. And this technique can be applied to scale-up, which will lay a foundation of large scale industrial production.

Do método para estimar o tamanho médio das redes pessoais e o tamanho de populações difíceis de contar / Of a method for estimating the average population size of personal networks and the size of hard-to-count populations / /Sobre el método para estimar el tamaño medio de las redes personales y el tamaño de poblaciones difíciles de calcular

O método conhecido como Network Scale-Up (NSU) - método de ampliação das redes sociais -, utilizado para estimar "populações difíceis de contar" (hard-to-count populations), baseia-se na ideia de que as populações humanas se organizam numa rede complexa de interações sociais, na qual todos os indivíduos, independentemente de atributos pessoais distintos, estão conectados. Conhecendo-se, então, o padrão das redes pessoais associado a determinados atributos individuais, é possível estimar "parcelas" da população que possuem esses mesmos atributos. Os emigrantes internacionais, em especial os que se encontram em situação irregular, enquadram-se nesse tipo de subpopulação, cujo tamanho é desconhecido dada a dificuldade ou até mesmo a impossibilidade de mensurá-la diretamente. A descrição do método da ampliação das redes sociais e dos procedimentos metodológicos para obtenção dos dados necessários à aplicação desse método para estimar o número de emigrantes e de retornados internacionais de uma cidade brasileira de porte médio hipotética constitui o objetivo deste texto.

The method known as Network Scale-Up (NSU) - used for expanding social networks - is used to estimate hard-to-count populations, based on the idea that human populations are organized in a complex web of social interactions, to which all individuals, regardless of specific personal attributes, are connected. If we know the pattern of personal networks associated with certain individual attributes, we can estimate "parcels" of the population that have these same attributes. International migrants, especially those who are undocumented, fit into this type of subpopulation whose size is unknown because of the difficulty or even impossibility of measuring it directly. This article aims at describing the Network Scale-Up method and the methodological procedures necessary to estimate the number of international and returned migrants in a hypothetical, medium-sized, Brazilian city.

El método conocido como Network Scale-Up (NSU) -método de ampliación de las redes sociales-, utilizado para estimar "poblaciones difíciles de calcular" (hard-to-count populations), se basa en la idea de que las poblaciones humanas se organizan en una red compleja de interacciones sociales, en la que todos los individuos, independientemente de sus atributos personales diferenciados, están conectados. Conociéndose entonces el patrón de las redes personales, asociado a determinados atributos individuales, es posible estimar "porciones" de la población que posean estos mismos atributos. Los emigrantes internacionales, en especial los que se encuentran en situación irregular, se encuadran en este tipo de subpoblación, cuyo tamaño es desconocido, dada la dificultad o hasta incluso la imposibilidad de calcularlo directamente. La descripción del método de la ampliación de las redes sociales, y de los procedimientos metodológicos con el objeto de obtener los datos necesarios para la aplicación de este método en la estimación del número de emigrantes y de retornados internacionales de una hipotética ciudad brasileña de porte medio, constituye el objetivo de este trabajo.