Production of recombinant human antithrombin III on 20-L bioreactor scale: Correlation of supernatant neuraminidase activity, desialylation, and decrease of biological activity of recombinant glycoprotein

Munzert, E.; Heidemann, Rüdiger; Lehmann, Jürgen; Müthing, Johannes

doi:10.1002/(sici)1097-0290(19971120)56:4<441::aid-bit9>3.0.co;2-o

Cited by 25 publications

(10 citation statements)

References 36 publications

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“…Our results indicate that the cellular machinery is not significantly affected by the progress of infection, in contrast to a previous report using Sf-21 cells (7). Moreover, the sialylated structures do not undergo significant extracellular degradation, in contrast to glycoproteins produced in CHO cells (18,19).…”

Section: Discussionmentioning

confidence: 99%

“…Similarly, Licari et al report an activity of 0.14 mU/µg total protein (i.e., 8.4 nmol/h‚µg total protein) in Tn5B1-4 cells (8). It appears that our Tn-4s variant distinguishes itself from regular Tn5B1-4 cells and Tn4h cells, as well as from CHO cells (15,18,19), by the absence or by very low levels of sialidase. This absence can be a considerable advantage for the production of sialylated glycoproteins.…”

Section: Discussionmentioning

confidence: 99%

“…This absence can be a considerable advantage for the production of sialylated glycoproteins. The ability of extracellular sialidase in CHO cultures to release terminal sialic acid from glycoproteins has been demonstrated (18,19), and it was suggested that those cultures may have to be harvested at suboptimal product concentrations in order to minimize product degradation by sialidase (19). The apparent lack of sialidase activity as well as the constant degree of sialylation at three harvest times in our study suggests that such degradation is not a concern with the Tn-4s cell line and that the product may be harvested at the time its concentration is maximum, without an adverse effect on its glycosylation.…”

Section: Discussionmentioning

confidence: 99%

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Production of a Sialylated N‐Linked Glycoprotein in Insect Cells: Role of Glycosidases and Effect of Harvest Time on Glycosylation

Joosten

Shuler

2003

Biotechnology Progress

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Using a nonengineered Trichoplusia ni insect cell line, Tn-4s, infected with an Autographa californica recombinant baculovirus, 20% sialylation of human secreted placental alkaline phosphatase (SEAP) was observed. In contrast to this level of sialylation, intermediate complex forms with terminal galactose or N-acetylglucosamine were found in low proportions (<3% and <1%, respectively). We tested whether time of harvest or degradation of intermediate complex forms is responsible for this distribution of glycoforms. Spinner-flask cultures were infected with the SEAP baculovirus expression vector, and the cultures were harvested 48, 72, and 96 h post-infection. Structural analysis revealed that the glycoform distribution of SEAP was very similar at the different times of harvest, indicating that the cellular machinery was not significantly affected by the progress of infection and that the glycoforms obtained were stable. High levels of beta-galactosidase and N-acetylglucosaminidase activity were detected throughout infection. In contrast, sialidase activity was below detection level both in cell extracts and in supernatants. These levels of glycosidases activities raise the possibility that intermediate complex glycoforms may be degraded while sialylated forms should not experience significant degradation in this cell line. However, culture in the presence of extracellular beta-galactosidase and N-acetylglucosaminidase inhibitors did not significantly improve glycosylation, suggesting that extracellular degradation processes are not taking place. Instead, results suggest that the intracellular machinery of the Tn-4s cells tends to either shunt the glycans to paucimannosidic forms or drive them completely to sialylation.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Production of a Sialylated N‐Linked Glycoprotein in Insect Cells: Role of Glycosidases and Effect of Harvest Time on Glycosylation

Joosten

Shuler

2003

Biotechnology Progress

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“…This enzymatic activity increased over the batch process to reach 430 nmol/h/L in the supernatant at the end of the culture. Though quantitative information concerning sialidase activity remains very sparse, levels higher than 3000 nmol/h/L have been reported in CHO cell culture supernatants (33,56,57). Thus, the sialidase activity measured in our serum-containing medium seems to be relatively low, which is in good agreement with the slow but continuous desialylation of murine EPO observed by HPCE over the batch process.…”

Section: Profile Consistency Of the Desialylated N-linked Glycans Of mentioning

confidence: 99%

HPCE Monitoring of the N-Glycosylation Pattern and Sialylation of Murine Erythropoietin Produced by CHO Cells in Batch Processes

et al. 2004

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Using capillary electrophoresis coupled to laser-induced fluorescence (HPCE-LIF), it was possible to profile N-linked oligosaccharides from EPO, including species containing sialic acid, during the course of batch cultures performed either in serum-free or serum-containing medium. Although an unusual high heterogeneity of the N-linked oligosaccharides was observed by both SDS-PAGE and HPCE analysis, the patterns of mEPO glycans after desialylation by mild acid hydrolysis were found to be quite constant over the course of the cultures either with or without serum supplementation. In contrast, when the protein was analyzed by HPCE without acidic desialylation, fingerprints of N-linked oligosaccharides changed with time in serum-containing conditions. This phenomenon appeared to be mainly due to the desialylation of mEPO as a result of a sialidase activity released upon cell lysis. These results demonstrate that though a higher EPO titer was obtained in serum supplemented conditions, sialylation of EPO was severely affected by the presence of serum in the culture medium.

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“…Nevertheless, AT is available from human plasma (Büntemeyer et al 1994; Munzert et al 1997), from the view of product safety and possible virus infection (retrovirus and hepatotropic viruses), AT production must be only way through recombinant DNA (rDNA) technology (Zettlmeissl et al 1987). AT has been produced using different expression systems like E. coli , Chinese hamster ovary cells and Pichia pastoris , COS 1 monkey kidney cells, BHK (baby hamster kidney) cells, Spodoptera frugiperda insect cells, and transgenic goat milk (Bock et al 1982; Wasley et al 1987; Zettlmeissl et al 1988, 1989; Gillespie et al 1991; Edmunds et al 1998), but yeast-based recombinant human antithrombin (rhAT) has been demonstrated only by Broker et al (1987).…”

Section: Introductionmentioning

confidence: 99%

Efficient single step chromatographic purification of recombinant human antithrombin (rhAT) from Saccharomyces cerevisiae

2016

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Antithrombin (AT) is a glycoprotein that inactivates the several physiological target enzymes of coagulation system. The effect of purification strategies plays a crucial role in getting maximum recovery of yield, purity and biological activity of recombinant human antithrombin (rhAT). In the present work, the task of purifying rhAT from Saccharomyces cerevisiae BY4741 has been carried out using two different approaches such as cross flow filtration (CFF) system and chromatography methods. In the first approach, the protein was concentrated and partially purified through CFF to achieve maximum recovery yield and purity of 87 and 94 %, respectively. In the second approach, purification involved a single step chromatography with various types of ion exchange and size exclusion resins to analyze the maximum rhAT recovery yield and purity. From the experimental results, it has been observed that the size exclusion chromatography (SEC) technique with Superose 12 matrix was suitable for the purification of rhAT and achieved the maximum recovery yield and purity of 51 and 97 %, respectively. Further, to acquire a high recovery yield and purity of rhAT, the effect of various chromatographic conditions such as mobile phase, mobile phase pH, flow rate, sample volume and sample concentration were also investigated. Under the optimal chromatographic conditions, rhAT was significantly recovered and purified in a single step with maximum recovery yield, purity and biological activity of 67, 99 % and 410 IU/L, respectively. Based on these investigations, it was concluded that SEC with Superose 12 matrix was a more suitable and a potential method for the purification of rhAT.

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Production of recombinant human antithrombin III on 20-L bioreactor scale: Correlation of supernatant neuraminidase activity, desialylation, and decrease of biological activity of recombinant glycoprotein

Cited by 25 publications

References 36 publications

Production of a Sialylated N‐Linked Glycoprotein in Insect Cells: Role of Glycosidases and Effect of Harvest Time on Glycosylation

Production of a Sialylated N‐Linked Glycoprotein in Insect Cells: Role of Glycosidases and Effect of Harvest Time on Glycosylation

HPCE Monitoring of the N-Glycosylation Pattern and Sialylation of Murine Erythropoietin Produced by CHO Cells in Batch Processes

Efficient single step chromatographic purification of recombinant human antithrombin (rhAT) from Saccharomyces cerevisiae

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