High-producing Cell Research Articles

Minimally manipulated adipose derived mesenchymal stromal cells and osteoarthritis: A narrative review.

Human mesenchymal stromal cells (MSCs) have increasingly been used to treat osteoarthritis (OA) related pain and dysfunction, due to their capacity for regeneration and anti-inflammatory effects. Adipose-derived MSCs are characterized by their abundance, ease of access, easy isolation procedures, high lipoaspirate stromal cell production, quicker multiplication of cells, and less pain and morbidity during harvesting. These cells are typically enzymatically derived from adipose tissue but this technique has complicated regulatory problems. To address this problem, a new technique has been created to extract and process adipose tissue without expansion and the use of enzymes to produce autologous minimally manipulated adipose-derived MSCs. Recent studies have confirmed that this treatment is an effective and promising method for treating pain and improving joint function in patients affected by OA with a very low percentage of complications at short to mid-term follow-up. (www.actabiomedica.it)

Novel cell line development strategy for monoclonal antibody manufacturing using translational enhancing technology

Chinese hamster ovary (CHO) cells are widely used for constructing expression systems to produce therapeutic proteins. However, the establishment of high-producer clones remains a laborious and time-consuming process, despite various progresses having been made in cell line development. We previously developed a new strategy for screening high monoclonal antibody (mAb)-producing cells using flow cytometry (FCM). We also reported that p180 and SF3b4 play key roles in active translation on the endoplasmic reticulum, and that the productivity of secreted alkaline phosphatase was enhanced by the overexpression of p180 and SF3b4. Here, we attempted to apply the translational enhancing technology to high mAb-producing cells obtained after high-producer cell sorting. A high mAb-producing CHO clone, L003, which showed an mAb production level of >3g/L in fed-batch culture, was established from a high mAb-producing cell pool fractionated by FCM. Clones generated by the overexpression of p180 and SF3b4 in L003cells were evaluated by fed-batch culture. The specific productivity of clones overexpressing these two factors was ∼3.1-fold higher than that of parental L003cells in the early phase of the culture period. Furthermore, the final mAb concentration was increased to 9.5g/L during 17 days of fed-batch culture after optimizing the medium and culture process. These results indicate that the overexpression of p180 and SF3b4 would be promising for establishing high-producer cell lines applicable to industrial production.

Cheating the Cheater: Suppressing False-Positive Enrichment during Biosensor-Guided Biocatalyst Engineering.

Transcription factor (TF)-based biosensors are very desirable reagents for high-throughput enzyme and strain engineering campaigns. Despite their potential, they are often difficult to deploy effectively as the small molecules being detected can leak out of high-producer cells, into low-producer cells, and activate the biosensor therein. This crosstalk leads to the overrepresentation of false-positive/cheater cells in the enriched population. While the host cell can be engineered to minimize crosstalk (e.g., by deleting responsible transporters), this is not easily applicable to all molecules of interest, particularly those that can diffuse passively. One such biosensor recently reported for trans-cinnamic acid (tCA) suffers from crosstalk when used for phenylalanine ammonia-lyase (PAL) enzyme engineering by directed evolution. We report that desensitizing the biosensor (i.e., increasing the limit of detection) suppresses cheater population enrichment. Furthermore, we show that, if we couple the biosensor-based screen with an orthogonal prescreen that eliminates a large fraction of true negatives, we can successfully reduce the cheater population during the fluorescence-activated cell sorting. Using the approach developed here, we were successfully able to isolate PAL variants with ∼70% higher kcat after a single sort. These mutants have tremendous potential in phenylketonuria (PKU) treatment and flavonoid production.

Developing a secretory AcGFP1-based IRES expression system for efficient production of mammalian recombinant proteins

To generate stable cell lines that express high levels of recombinant genes often requires screening of a large number of transfected cells using ELISA. The most widely used alternative to ELISA screening is to use an intracellularly expressed GFP reporter construct which allows sorting of recombinant gene expression cells based on GFP fluorescence intensity. The disadvantage of cell sorting, however, is that the resulting population will be polyclonal with the danger of instability and overgrowth of low producers. In addition, GFP or its variants can be toxic to host cells at high concentrations, and thus may reduce growth and robustness of high producer cells or even cause them to become apoptotic. We have developed a new mammalian expression system in which a recombinant protein and a fluorescence protein, AcGFP1, are expressed on the same plasmid separated by an internal ribosome entry site (IRES). A signal peptide was incorporated upstream of AcGFP1 so that the fluorescent protein is secreted from cells, preventing cellular toxicity from intracellular accumulation and enabling convenient and accurate measurement of the protein. Expression tests of Ebola viral envelope GP1 and HIV gp120 proteins using this expression system in 293-H cells showed recombinant protein expression levels were closely correlated with AcGFP1 yield. Therefore, AcGFP1 can serve as an accurate reporter for recombinant protein expression and measuring AcGFP1 concentration provides a convenient, product independent and universal way for efficient clone screening.

Stable Sf9 cell pools as a system for rapid HIV‐1 virus‐like particle production

AbstractBACKGROUNDThe emergence of infectious diseases is accelerating the intensification of bioprocess strategies to support the increasing demand for the manufacture of higher quantities of vaccines in short timeframes. Here, the development of stable Sf9 cell pools producing human immunodeficiency virus serotype 1 (HIV‐1) Gag‐eGFP virus‐like particles (VLPs) is assessed.RESULTSFluorescence‐activated cell sorting (FACS) was employed to select high producing cells, achieving an 8.1‐fold increase in fluorescence intensity compared to unsorted cell pools after three rounds of cell sorting. The transferability of this system to bioreactor scale was also successfully achieved, attaining a 1.4‐fold increase in VLP production and maintaining a higher cell viability than shake flask controls. Analysis of the metabolism of stable cell pools and parental Sf9 cells did not show significant differences regarding metabolite consumption and production, even though a better performance and more efficient metabolism were observed in bioreactor compared with shake flask cultures, highlighting the flexibility of these cells to adapt to different culture conditions and heterologous recombinant protein production.CONCLUSIONSStable Sf9 cell pools represent a suitable system for shortening bioprocess development times and accelerating vaccine production. © 2021 Society of Chemical Industry (SCI).

Enhancement of anti-TNFα monoclonal antibody production in CHO cells through the use of UCOE and DHFR elements in vector construction and the optimization of cell culture media

Recently, there has been a high demand for anti-tumor necrosis factor-α monoclonal antibodies (mAbTNFα) in the treatment of rheumatoid arthritis and other autoimmune diseases. Thus, efficient strategies and stable high-producing cell lines need to be established to increase antibody production. In this study, we describe an efficient approach to establish a mAbTNFα high-producing clone through the optimization of expression vectors and cell culture media. The ubiquitous chromatin opening element (UCOE) and dihydrofolate reductase (DHFR)-based vectors encoding mAbTNFα were introduced into the CHO-DG44 cells using lipofection. Clones were obtained by selecting transfected cells with G418, amplifying them by treatment with methotrexate, and isolating them by limiting dilution. Different media formulated with commercial feeds and media were also screened to develop an improved medium. The antibody produced by the selected clone was purified, characterized, and compared to standard adalimumab. Using our established protocol, a cell clone obtained from stable mAbTNFα-expressing cell pools showed a 3.8-fold higher antibody titer compared to stable cell pools. Furthermore, the highest antibody yield of selected clones cultured in fed-batch mode using improved medium was 2450 ± 30 µg/mL, which was 13.2-fold higher than that of stable cell pool cultivated in batch mode using a basal medium. The purified antibody had primary chemical and biological characteristics similar to those of adalimumab. Therefore, the use of UCOE and DHFR vectors in combination with the optimization of cell culture media may help in establishing stable and high-producing CHO cell lines for therapeutic antibody production.

Screening and selection strategy for the establishment of biosimilar to trastuzumab-expressing CHO-K1 cell lines

The high prices of biopharmaceuticals or biologics used in the treatment of many diseases limit the access of patients to these novel therapies. One example is the monoclonal antibody trastuzumab, successfully used for breast cancer treatment. An economic alternative is the generation of biosimilars to these expensive biopharmaceuticals. Since antibody therapies may require large doses over a long period of time, robust platforms and strategies for cell line development are essential for the generation of recombinant cell lines with higher levels of expression. Here, we obtained trastuzumab-expressing CHO-K1 cells through a screening and selection strategy that combined the use of host cells pre-adapted to protein-free media and suspension culture and lentiviral vectors. The results demonstrated that the early screening strategy obtained recombinant CHO-K1 cell populations with higher enrichment of IgG-expressing cells. Moreover, the measurement of intracellular heavy chain polypeptide by flow cytometry was a useful metric to characterize the homogeneity of cell population, and our results suggest this could be used to predict the expression levels of monoclonal antibodies in early stages of cell line development. Additionally, we propose an approach using 25 cm2 T-flasks in suspension and shaking culture conditions as a screening tool to identify high producing cell lines. Finally, trastuzumab-expressing CHO-K1 clones were generated and characterized by batch culture, and preliminary results related to HER2-recognition capacity were successful. Further optimization of elements such as gene optimization, vector selection, type of amplification/selection system, cell culture media composition, in combination with this strategy will allow obtaining high producing clones.

Targeted Knock-in of Transgenes into the CHO Cell Genome Using CRISPR-mediated Integration Systems

Biopharmaceutical proteins are usually produced by culturing recombinant Chinese hamster ovary (CHO) cells. High producer cell lines are screened from transfected cells with random integration of target genes. Since transgene expression is susceptible to the surrounding environment of the integrated genomic locus, producer cell lines should be selected from a large number of recombinant cells with heterogeneous transgene insertion. In contrast, targeted integration into a characterized genomic locus allows for predictable transgene expression and less clonal variability, and thus stable production of target proteins can be expected. Genome editing technology based on programmable nucleases has recently emerged as a versatile tool for precise editing of target locus in the cell genome. Here, we demonstrated targeted knock-in of transgenes into the hypoxanthine phosphoribosyltransferase (hprt) locus of CHO cells using CRISPR/Cas9 and CRISPR-mediated precise integration into target chromosome (PITCh) systems. We also generated knock-in CHO cells based on the homology-independent targeted integration (HITI) system. We evaluated the knock-in efficiency of transgenes into the hprt locus using these systems.

Accelerating HIV-1 VLP production using stable High Five insect cell pools.

Stable cell pools are receiving a renewed interest as a potential alternative system to clonal cell lines. The shorter development timelines and the capacity to achieve high product yields make them an interesting approach for recombinant protein production. In this study, stable High Five cell pools are assessed for the production of a simple protein, mCherry, and the more complex HIV-1 Gag-eGFP virus-like particles (VLPs). Random integration coupled to fluorescence-activated cell sorting (FACS) in suspension conditions is applied to accelerate the stable cell pool generation process and enrich it with high producer cells. This methodology is successfully transferred to a bioreactor for VLP production, resulting in a 2-fold increase in VLP yields with respect to shake flask cultures. In these conditions, maximum viable cell concentration improves by 1.5-fold, and by-product formation is significantly reduced. Remarkably, a global increase in the uptake of amino acids in the Gag-eGFP stable cell pool is observed when compared with parental High Five cells, reflecting the additional metabolic burden associated with VLP production. These results suggest that stable High Five cell pools are a robust and powerful approach to produce VLPs and other recombinant proteins, and put the basis for future studies aiming to scale up this system.

Understanding the Differences in the Growth and Toxin Production of Anatoxin-Producing Cuspidothrix issatschenkoi Cultured with Inorganic and Organic N Sources from a New Perspective: Carbon/Nitrogen Metabolic Balance.

Cyanotoxins are the underlying cause of the threat that globally pervasive Cyanobacteria Harmful algal blooms (CyanoHABs) pose to humans. Major attention has been focused on the cyanobacterial hepatotoxin microcystins (MCs); however, there is a dearth of studies on cyanobacterial neurotoxin anatoxins. In this study, we explored how an anatoxin-producing Cuspidothrix issatschenkoi strain responded to culture with inorganic and organic nitrogen sources in terms of growth and anatoxins production. The results of our study revealed that ʟ- alanine could greatly boost cell growth, and was associated with the highest cell productivity, while urea significantly stimulated anatoxin production with the maximum anatoxin yield reaching 25.86 μg/mg dry weight, which was 1.56-fold higher than that in the control group (BG11). To further understand whether the carbon/nitrogen balance in C. issatschenkoi would affect anatoxin production, we explored growth and toxin production in response to different carbon/nitrogen ratios (C/N). Anatoxin production was mildly promoted when the C/N ratio was within low range, and significantly inhibited when the C/N ratio was within high range, showing approximately a three-fold difference. Furthermore, the transcriptional profile revealed that anaC gene expression was significantly up-regulated over 2–24 h when the C/N ratio was increased, and was significantly down-regulated after 96 h. Overall, our results further enriched the evidence that urea can stimulate cyanotoxin production, and ʟ-alanine could boost C. issatschenkoi proliferation, thus providing information for better management of aquatic systems. Moreover, by focusing on the intracellular C/N metabolic balance, this study explained the anatoxin production dynamics in C. issatschenkoi in response to different N sources.

Selecting cells expressing high levels of recombinant proteins using the GPI-anchored protein with selenocysteine system

Most biopharmaceutical proteins are produced in mammalian cells because they have the advantageous capacity for protein folding, assembly, and posttranslational modifications. To satisfy the increasing demand for these proteins for clinical purposes and studies, traditional methods to improve protein productivity have included gene amplification, host cell engineering, medium optimization, and screening methods. However, screening and selection of high-producing cell lines remain complex and time consuming. In this study, we established a glycosylphosphatidylinositol (GPI)-anchored protein with a selenocysteine (GPS) system to select cells producing high levels of target secretory proteins. Recombinant lysosomal acid lipase (LIPA) and α-galactosidase A (GALA) were fused with a GPI attachment signal sequence and a selenocysteine insertion sequence after an in-frame UGA codon. Under these conditions, most of the recombinant proteins were secreted into the culture medium, but some were found to be GPI-anchored proteins on the cell surface. When sodium selenite was supplied into the culture medium, the amount of GPI-anchored LIPA and GALA was increased. High-expressing cells were selected by detecting surface GPI-anchored LIPA. The GPI-anchored protein was then eliminated by knocking out the GPI biosynthesis gene PIGK, in these cells, all LIPA was in secreted form. Our system provides a promising method of isolating cells that highly express recombinant proteins from large cell populations.

Improving Influenza HA-Vlps Production in Insect High Five Cells via Adaptive Laboratory Evolution.

The use of non-standard culture conditions has proven efficient to increase cell performance and recombinant protein production in different cell hosts. However, the establishment of high-producing cell populations through adaptive laboratory evolution (ALE) has been poorly explored, in particular for insect cells. In this study, insect High Five cells were successfully adapted to grow at a neutral culture pH (7.0) through ALE for an improved production of influenza hemagglutinin (HA)-displaying virus-like particles (VLPs). A stepwise approach was used for the adaptation process, in which the culture pH gradually increased from standard 6.2 to 7.0 (ΔPh = 0.2–0.3), and cells were maintained at each pH value for 2–3 weeks until a constant growth rate and a cell viability over 95% were observed. These adapted cells enabled an increase in cell-specific HA productivity up to three-fold and volumetric HA titer of up to four-fold as compared to non-adapted cells. Of note, the adaptation process is the element driving increased specific HA productivity as a pH shift alone was inefficient at improving productivities. The production of HA-VLPs in adapted cells was successfully demonstrated at the bioreactor scale. The produced HA-VLPs show the typical size and morphology of influenza VLPs, thus confirming the null impact of the adaptation process and neutral culture pH on the quality of HA-VLPs produced. This work strengthens the potential of ALE as a bioprocess engineering strategy to improve the production of influenza HA-VLPs in insect High Five cells.

Multicopy Targeted Integration for Accelerated Development of High-Producing Chinese Hamster Ovary Cells.

The ever-growing biopharmaceutical industry relies on the production of recombinant therapeutic proteins in Chinese hamster ovary (CHO) cells. The traditional timelines of CHO cell line development can be significantly shortened by the use of targeted gene integration (TI). However, broad use of TI has been limited due to the low specific productivity (qP) of TI-generated clones. Here, we show a 10-fold increase in the qP of therapeutic glycoproteins in CHO cells through the development and optimization of a multicopy TI method. We used a recombinase-mediated cassette exchange (RMCE) platform to investigate the effect of gene copy number, 5' and 3' gene regulatory elements, and landing pad features on qP. We evaluated the limitations of multicopy expression from a single genomic site as well as multiple genomic sites and found that a transcriptional bottleneck can appear with an increase in gene dosage. We created a dual-RMCE system for simultaneous multicopy TI in two genomic sites and generated isogenic high-producing clones with qP of 12-14 pg/cell/day and product titer close to 1 g/L in fed-batch. Our study provides an extensive characterization of the multicopy TI method and elucidates the relationship between gene copy number and protein expression in mammalian cells. Moreover, it demonstrates that TI-generated CHO cells are capable of producing therapeutic proteins at levels that can support their industrial manufacture.

A rational approach to improving titer in Escherichia coli-based cell-free protein synthesis reactions.

Cell-free protein synthesis (CFPS) is an established method for rapid recombinant protein production. Advantages like short synthesis times and an open reaction environment make CFPS a desirable platform for new and difficult-to-express products. Most recently, interest has grown in using the technology to make larger amounts of material. This has been driven through a variety of reasons from making site specific antibody drug conjugates, to emergency response, to the safe manufacture of toxic biological products. We therefore need robust methods to determine the appropriate reaction conditions for product expression in CFPS. Here we propose a process development strategy for Escherichia coli lysate-based CFPS reactions that can be completed in as little as 48 hr. We observed the most dramatic increases in titer were due to the E. coli strain for the cell extract. Therefore, we recommend identifying a high-producing cell extract for the product of interest as a first step. Next, we manipulated the plasmid concentration, amount of extract, temperature, concentrated reaction mix pH levels, and length of reaction. The influence of these process parameters on titer was evaluated through multivariate data analysis. The process parameters with the highest impact on titer were subsequently included in a design of experiments to determine the conditions that increased titer the most in the design space. This proposed process development strategy resulted in superfolder green fluorescent protein titers of 0.686 g/L, a 38% improvement on the standard operating conditions, and hepatitis B core antigen titers of 0.386 g/L, a 190% improvement.

Small fluctuations in cell wall thickness in pine and spruce xylem: Signal from cambium?

In the conifer tree rings, each tracheid goes through three phases of differentiation before becoming an element of the stem water-conducting structure: division, extension, and cell wall thickening. These phases are long-lasting and separated temporally, especially cell wall thickening. Despite the numerous lines of evidence that external conditions affect the rate of growth processes and the final anatomical dimensions during the respective phases of tracheid differentiation, the influence of the environment on anatomical dimensions during the cell division phase (cambial activity) has not yet been experimentally confirmed. In this communication, we provide indirect evidence of such an effect through observations of the small fluctuations in the latewood cell wall thickness of rapidly growing tree rings, which exhibit a high cell production rate (more than 0.4 cells per day on average). Such small fluctuations in the cell wall thickness cannot be driven by variations in external factors during the secondary wall deposition phase, since this phase overlaps for several tens of latewood cells in the rings of fast-growing trees due to its long duration.

Identifying metabolic features and engineering targets for productivity improvement in CHO cells by integrated transcriptomics and genome-scale metabolic model

In this study, we presented an integrated systems biology approach to elucidate the key characteristics of cellular metabolism in Chinese hamster ovary (CHO) cells producing monoclonal antibodies (mAb). The cellular metabolism in high and low producers under batch conditions was interrogated dynamically both within and among cells. First, transcriptomics analysis was used to study the time-course change in the metabolic pathway within cells that was correlated with mAb productivity increase. Second, differentially regulated pathways between high and low producers were sought at each growth phase. Several up-regulated pathways were identified in the high producer at the late growth phase, including citrate cycle, oxidative phosphorylation, and pentose phosphate pathway. These activities were further analyzed by intracellular flux distributions estimated through a genome-scale CHO model. Our results revealed that these key pathways are identified to be characteristics of high mAb production, not only for the high-producing cell line but also a dynamic phenomenon in mAb-producing cell cultures. This study showed that the approach of integrating transcriptomics and flux analysis leads to a better understanding of cellular metabolism related to mAb productivity. In turn, this allows for the identification of metabolic bottlenecks and potential engineering targets for cell line development and process optimization.

Implementation of the aber biomass probe in Pall's iCELLis nano bioreactor provides a robust and reproducible method to assess cell density

Background & Aim In the past five years, Pall's iCELLis bioreactors have emerged as the leading bioreactor technology for clinical manufacturing of viral vectors and vaccines. Pall and InVitria have previously demonstrated the successful implementation of OptiPEAK HEK293T blood-free media formulation in the iCELLis bioreactor to produce viral vectors in adherent HEK293T, including rapid and uniform cell attachment to the iCELLis fixed-bed bioreactor and robust cell expansion kinetics found to be equivalent to those observed in serum-containing media. However, some questions remain regarding the impact of cell aggregation and fixed bed compaction on cell distribution, transfection efficiency, and subsequent cell productivity. Methods, Results & Conclusion In this poster, we look at the cell productivity as a function of aggregation in the cell seed and fixed bed compaction to better understand bioprocessing mechanisms that contribute to low and high cell productivity. We also demonstrate methods and techniques to enhance the cell production.

Toxicity of Bacillus thuringiensis Berl. KJ3P1 and DLM isolates towards pest of oil palm Oryctes rhinoceros [Coleoptera: Scarabaeidae

Oil palm plants are industrial plants that generate foreign exchange for Indonesia. Some obstacles are leading to production decrease. One of the important pests is Oryctes rhinoceros [Coleoptera: Scarabaeidae]. Both larval and imago stadia attack oil-palm in immature plants [IP] and mature plants [MP]. The method of controlling Oryctes often use chemical insecticides, in which continuously applications will cause residues in the environment and mortality of non-target insects. Therefore it is necessary to find an environmentally friendly control method that is easy, inexpensive and safe for the environment. The use of entomopathogenic bacteria Bacillus thuringiensis is an alternative. The study was aimed to study the toxicity of two B. thuringiensis isolates from the collection of Plant Protection Department, Faculty of Agriculture, Sriwijaya University [KJ3P1 and DLM isolate codes] on mortality of 3rd instar Oryctes larvae. Experiments were conducted at the Entomological Laboratory, Department of Plant Protection, Faculty of Agriculture, Sriwijaya University from March until May 2019. Bioinsecticide-based B. thuringiensis was made with 2 types of growth media, namely: 1] cow’s bio-urine enriched with 5% molasses, and 2]. Rice washing water enriched with 5% molasses. Treatments were included: 1] DLM5 isolate, 2] KJ3P1 isolate, 3] commercial B. thuringiensis and 4]. without treatment [as control]. The test insects used were 25 individuals of 1st instar Oryctes larvae in each treatment. The results showed KJ3PI isolate that propagated in bio-urine media showed the highest production of B. thuringiensis [5.69 x 106 spores/ml], the highest mortality [76% ] and the lowest LT50 value [5.28 days] as well. The use of agricultural and livestock waste as a medium to multiply B. thuringiensis bacteria may produce high cell and spore production and can be used as bioinsecticides to control O. rhinoceros.

Technological mapping and trends in photobioreactors for the production of microalgae.

Photobioreactors (PBRs) are equipment of central importance for the massive cultivation of microalgae, providing controlled conditions for high cell productivity. There are a few popular PBR designs, with contrasting advantages and limitations, such as poor light distribution, mass transfer, or hydrodynamic behavior. Due to the environmental concerns in recent decades and the discovery of new, useful microalgal metabolites, the interest in finding alternatives to solve technological bottlenecks of PBRs has intensified. In this process, new geometries, materials, and modes of light supply were developed, generating a significant scientific and technological output, reported in papers and patents. We present a technological landscape analysis of photobioreactor design, focusing on improvements of the classical geometries and trends in industrial photobioreactors. The analysis of 412 patent documents showed a surge in innovation filing since 2005 and a reduction in the number of new documents along the last decade. The recent efforts in design improvement, the leading countries, institutes and companies that innovate, and the trends in PBR technology are presented and discussed.

Создание стабильной высокопродуктивной платформы на основе клеток CHO, адаптированных к химическому и механическому стрессам

Возрастающий спрос на получение рекомбинантных молекул требует создания не только стабильных, но и высокопроизводительных клеточных линий, пригодных для масштабного производства. В настоящей работе предложен дизайн вектора для эффективной и стабильной экспрессии рекомбинатных моноклональных антител в клетках CHO и схема адаптации линии CHO-S (реципиента) к высокой осмоляльности среды в условиях повышенного содержания хлорида натрия (до 100 мМ), лактата натрия (до 170 мМ) и скорости перемешивания в биореакторе до 240 об/мин. Показана эффективность полученной клеточной платформы на примере экспрессии моноклонального антитела IgG2/4k, позволяющей получать до 2,5 г/л продукта без амплификации гена интереса и оптимизации условий культивирования в режиме fed-batch.