Perfusion Cell Culture Decreases Process and Product Heterogeneity in a Head‐to‐Head Comparison With Fed‐Batch

Walther, Johannes; Lü, Jiuyi; Hollenbach, Myles; Yu, Marcella; Hwang, Chris; McLarty, Jean; Brower, Kevin

doi:10.1002/biot.201700733

Cited by 63 publications

(49 citation statements)

References 48 publications

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“…In the literature, past studies produced contradictory conclusions on the IgG glycosylation when comparing the two cell culture modes, where some studies demonstrated similarities [9,10] and others showed discrepancies in the IgG glycan structures [13,14]. A recent report of a head-to-head comparison between perfusion and fed-batch cell cultures noted a strong similarity in the IgG glycosylation dynamics [45], an observation that aligns well with the result of our analysis.…”

Section: Discussionsupporting

confidence: 83%

Glycosylation Flux Analysis of Immunoglobulin G in Chinese Hamster Ovary Perfusion Cell Culture

et al. 2018

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The terminal sugar molecules of the N-linked glycan attached to the fragment crystalizable (Fc) region is a critical quality attribute of therapeutic monoclonal antibodies (mAbs) such as immunoglobulin G (IgG). There exists naturally-occurring heterogeneity in the N-linked glycan structure of mAbs, and such heterogeneity has a significant influence on the clinical safety and efficacy of mAb drugs. We previously proposed a constraint-based modeling method called glycosylation flux analysis (GFA) to characterize the rates (fluxes) of intracellular glycosylation reactions. One contribution of this work is a significant improvement in the computational efficiency of the GFA, which is beneficial for analyzing large datasets. Another contribution of our study is the analysis of IgG glycosylation in continuous perfusion Chinese Hamster Ovary (CHO) cell cultures. The GFA of the perfusion cell culture data indicated that the dynamical changes of IgG glycan heterogeneity are mostly attributed to alterations in the galactosylation flux activity. By using a random forest regression analysis of the IgG galactosylation flux activity, we were further able to link the dynamics of galactosylation with two process parameters: cell-specific productivity of IgG and extracellular ammonia concentration. The characteristics of IgG galactosylation dynamics agree well with what we previously reported for fed-batch cultivations of the same CHO cell strain.Processes 2018, 6, 176 2 of 15 and process analytical technology put further emphasis on implementing quantitative approaches for process improvements in biopharmaceutical manufacturing [4,5].Continuous manufacturing technology offers an effective and flexible way for the large scale and robust production of drug compounds [6]. In the biopharmaceutical industry, the application of continuous cell culture technology has thus far been limited to the production of unstable products that require constant recovery [7]. Nevertheless, continuous perfusion cell cultures have previously been demonstrated to be capable of producing antibodies at a volumetric rate that matches or exceeds that of fed-batch cultures [8]. In addition to the high productivity, the stable steady state operation mode in conjunction with the short residence time of perfusion cultures translates to a tight maintenance of product quality. Whether or not product qualities and their key controlling parameters can be directly translated from the traditional batch/fed-batch cell cultures to the perfusion cell cultivations is still unresolved. Past work on converting mAb production from batch/fed-batch to perfusion cell cultures gave conflicting reports on product qualities, where a few studies demonstrated consistent product qualities [9,10], and many others showed differences between the two production modes [11][12][13][14][15].Among the key critical quality attributes (CQAs) of therapeutic mAbs is the glycan structures of the Fc domain [16]. The N-linked glycosylation is a common post-translational modification of proteins, ...

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Section: Discussionsupporting

confidence: 83%

Glycosylation Flux Analysis of Immunoglobulin G in Chinese Hamster Ovary Perfusion Cell Culture

et al. 2018

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“…The biopharmaceutical industry is following other industries in moving from discrete batch operation to integrated continuous manufacturing, especially for high demand products, such as monoclonal antibodies (MAbs; Shukla, Wolfe, Mostafa, & Norman, 2017; Walsh, 2018). The drivers for continuous processing are many‐fold; process intensification and cost savings (Baur, Angarita, Müller‐Späth, Steinebach, & Morbidelli, 2016; Hummel et al, 2018; Pagkaliwangan, Hummel, Gjoka, Bisschops, & Schofield, 2018; Pollock, Coffman, Ho, & Farid, 2017) might emerge as the most obvious ones but steady‐state operation and thus better, more reproducible quality have also been associated with continuous biomanufacturing (Karst et al, 2017; Kaufman, Wasley, & Dorner, 1988; Walther et al, 2019). While upstream processing is ahead in this transition, where chemostat and perfusion reactors are commonly employed at the manufacturing scale (Arathoon & Birch, 1986; Shukla et al, 2017; Warnock & Al‐Rubeai, 2006), downstream operations have only in recent years started this transition.…”

Section: Introductionmentioning

confidence: 99%

Truly continuous low pH viral inactivation for biopharmaceutical process integration

Martins

Sencar

Hammerschmidt

et al. 2020

Biotech & Bioengineering

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Continuous virus inactivation (VI) has received little attention in the efforts to realize fully continuous biomanufacturing in the future. Implementation of continuous VI must assure a specific minimum incubation time, typically 60 min. To guarantee the minimum incubation time, we implemented a packed bed continuous viral inactivation reactor (CVIR) with narrow residence time distribution (RTD) for low pH incubation. We show that the RTD does not broaden significantly over a wide range of linear flow velocities—which highlights the flexibility and robustness of the design. Prolonged exposure to acidic pH has no impact on bed stability, assuring constant RTD throughout long term operation. The suitability of the packed bed CVIR for low pH inactivation is shown with two industry‐standard model viruses, that is xenotropic murine leukemia virus and pseudorabies virus. Controls at neutral pH showed no system‐induced VI. At low pH, significant VI is observed, even after only 15 min. Based on the low pH inactivation kinetics, the continuous process is equivalent to traditional batch operation. This study establishes a concept for continuous low pH inactivation and, together with previous reports, highlights the versatility of the packed bed reactor for continuous VI, regardless of the inactivation method.

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“…Perhaps because a significant fraction of cellular energy resources are devoted to continued biomass generation, cells in a continuous sustainable perfusion system may have lower per cell, or specific, productivity (usually measured in pg·cell −1 ·day −1 ) when compared with a less sustainable perfusion process (Gagnon, Nagre, Wang, & Hiller, ), or in the extreme with a fed‐batch process (Bi, Shuttleworth, & Al‐Rubeai, ; Suzuki & Ollis, ). Significant differences have also been noted in the product quality of proteins produced in a sustainable perfusion system compared with that produced from a more traditional fed‐batch operation (Meuwly et al, ; Walther et al, ). In continuous perfusion cultures that maintain a cell culture at minimal growth rates, a constant, low cell bleed rate may also serve to continuously remove accumulated biomass debris resulting mostly from cell death due to some process insult (e.g.…”

Section: Introductionmentioning

confidence: 99%

Novel, linked bioreactor system for continuous production of biologics

Gagnon

Nagre

Wang

et al. 2019

Biotech & Bioengineering

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A novel, alternative intensified cell culture process comprised of a linked bioreactor system is presented. An N-1 perfusion bioreactor maintained cells in a highly proliferative state and provided a continuous inoculum source to a second bioreactor operating as a continuous-flow stirred-tank reactor (CSTR). An initial study evaluated multiple system steady-states by varying N-1 steady-state viable cell densities, N-1 to CSTR working volume ratios, and CSTR dilution rates. After identifying near optimum system steady-state parameters yielding a relatively high volumetric productivity while efficiently consuming media, a subsequent lab-scale experiment demonstrated the startup and long-term operation of the envisioned manufacturing process for 83 days. Additionally, to compensate for the cell-specific productivity loss over time due to cell line instability, the N-1 culture was also replaced with younger generation cells, without disturbing the steady-state of the system. Using the model cell line, the system demonstrated a two-fold volumetric productivity increase over the commercial-ready, optimized fed-batch process.

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Perfusion Cell Culture Decreases Process and Product Heterogeneity in a Head‐to‐Head Comparison With Fed‐Batch

Cited by 63 publications

References 48 publications

Glycosylation Flux Analysis of Immunoglobulin G in Chinese Hamster Ovary Perfusion Cell Culture

Glycosylation Flux Analysis of Immunoglobulin G in Chinese Hamster Ovary Perfusion Cell Culture

Truly continuous low pH viral inactivation for biopharmaceutical process integration

Novel, linked bioreactor system for continuous production of biologics

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