Application of Population Balance Model to the Loss of Hybridoma Antibody Productivity

Lee, Gyun Min; Varma, Amit; Palsson, Bernhard Ø.

doi:10.1021/bp00007a013

Cited by 37 publications

(21 citation statements)

References 17 publications

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“…Hybridoma cells producing MRSA-antiPBP2a mAb showed a too early decline in productivity. This is similar to what Lee et al (1991) reported upon studying other hybridoma cell lines, different from the MRSA-antiPBP2a mAb producing hybridomas, which had also lost production ability of around 10 -2 to 10 -5 per cell generation. HAb18 hybridoma cells reduced the antibody productivity at 15 % after 3 months of subculture (Li et al 2006).…”

Section: Monoclonal Antibody Productionsupporting

confidence: 87%

“…In addition to cell line stability, growth and metabolite characteristics affecting process robustness and scalability also need to be assessed (Li et al 2010). Despite the mAb tendency to lose productivity over a time, the limit in the number of passages that a given cell can be subjected to without impinging on its stability is singular to each cell line (Lee et al 1991).…”

Section: Introductionmentioning

confidence: 99%

“…Considering cost-effectiveness, it is important to have a cell line secreting proteins or antibodies of interest for development of diagnostics kits (Barnes et al 2003;Schmid et al 1990;Xin and Cutler 2006;Beckmann et al 2012;Kim et al 2011;Li et al 2010;Lee et al 1991). …”

Section: Introductionmentioning

confidence: 99%

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Effects of passage number on growth and productivity of hybridoma secreting MRSA anti-PBP2a monoclonal antibodies

Corrêa

Senna²,

Sousa³

2015

Cytotechnology

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Monoclonal antibodies (mAb) are high added value glycoproteins recommended for immunotherapy, diagnosis, and also for the treatment of bacterial infections resistant to multiple drugs such as Methicillin Resistant Staphylococcus aureus (MRSA). In addition to environmental conditions related to cell cultures, the intrinsic characteristics of hybridoma cells, like the secretion stability of monoclonal antibodies by the cells through successive subcultures, are relevant for the characterization of cell lines related to the productivity of mAb. The rate of mAb production differs significantly between different cell lines and different passage numbers, and it is an important variable in characterization of cell lines. In order to find a more robust, fastergrowing, and higher-productivity cell line of hybridoma, cultivations in 24-well plates were performed in different subculture periods, or cell passages (P), of hybridoma cells producing MRSA anti-PBP2a monoclonal antibodies ]. The objective of this study was to study the effects of cell growth and production of MRSA-antiPBP2a mAb secreted by murine hybridoma cells grown in different passages as well as determine the which passages the hybridomas can be cultivated without harming their growth and productivity. So, cell growth profiles of hybridomas secreting MRSA-antiPBP2a (mAb) and the production of MRSA-antiPBP2a mAb in different subculture periods or cell passages (P) were studied. Cell growth tests, monoclonal antibody productivity, and metabolite characteristics revealed substantial differences in those cells kept between P10 and P50. Similarities in the secretion of monoclonal antibody, growth, and metabolic profiles, were noted in the MRSA-antiPBP2a mAb producing hybridoma cells kept between P10 and P20. Also, glucose consumption (g/L) and lactate production (g/L) in the latter cell cultures were monitored daily through biochemical analyzer. As of P30, it was observed a 4.4 times reduction in productivity, a 13 % reduction in metabolic yield, and a significant change in cell growth. Secretion of MRSA-antiPBP2a mAb should be obtained through the culture of hybridomas up to P20 in order to keep its stability.

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Section: Monoclonal Antibody Productionsupporting

confidence: 87%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Effects of passage number on growth and productivity of hybridoma secreting MRSA anti-PBP2a monoclonal antibodies

Corrêa

Senna²,

Sousa³

2015

Cytotechnology

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“…26 In fact, in many other cases, it has been observed that P and NP are balanced in the culture.2'21 The population balance model also supports that P and NP can be balanced in the culture at particular conditions of mutation rate and relative growth rate of P and NP. 15 In conclusion, based on the data obtained here, the following production strategies are suggested. The H69K-1 transfectoma is better than the 6-31 transfectoma for chimeric antibody production.…”

Section: Discussionmentioning

confidence: 80%

Stability of transfectomas producing chimeric antibody against the pre‐S2 surface antigen of hepatitis B virus during a long‐term culture

Bae

Hong

Lee

1995

Biotech & Bioengineering

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To design the scheme of large-scale production of chimeric antibody for the postexposure prophylaxis of hepatitis B virus (HBV) infection, the stability of transfectomas (H69K-1 and 6-31) in regard to antibody production was examined during a long-term, repeated fed-batch culture without selection pressure using antibiotics. Although the H69K-1 transfectoma was more stable than the 6-31 transfectoma, both displayed gradual decreases in specific antibody productivity (q(Ab)) for the first several weeks of cultivation. During this period, q(Ab) was de-creased by 40% to 50%. This loss of q(Ab) was due mainly to the appearance of a nonproducing population (NP) of transfectoma, which was monitored throughout the culture by flow cytometry and the limiting dilution method. However, an NP did not overtake the culture and was balanced with a producing population (P) of transfectoma, resulting in stable antibody production. The subclones of NP obtained at the end of long-term culture were further characterized by reverse transcription-polymerase chain reaction assay of the heavy and light chain mRNA. All the subclones of NP derived from H69K-1 transfectoma had only light chain mRNA. On the other hand, an NP in the 6-31 transfectoma culture was heterogeneous. Some subclones of NP derived from 6-31 transfectoma had only heavy chain mRNA and other subclones had only light chain mRNA. Taken together, the results obtained here suggest that selection pressure is necessary for a long-term, continuous culture, because stable antibody production in a long-term culture was achieved only after a significant loss of antibody productivity. Accordingly, a batch culture appears to be more appropriate for large-scale chimeric antibody production without selection pressure. (c) 1995 John Wiley & Sons, Inc.

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“…Although a manufacturing cell line should have a fast rate of biomass accumulation with a high maximum viable cell concentration (among other characteristics), a general correlation between increasing cell-specific growth rate and a decrease in productivity has been widely observed, presumably due to the higher metabolic burden associated with high-level recombinant protein production (Beckmann et al 2012;Kaneko et al 2010;Jiang et al 2006). Indeed, as a recombinant cell line or hybridoma loses productivity over extended culture due to de novo gene mutation, chromosome loss, or silencing, non-producer cells can rapidly overgrow the original population, particularly when non-producer status comes with a growth advantage (Kromenaker and Srienc 1994;Lee et al 1991). Other metabolic changes with extended cultivation may also be observed such as increasing resistance to lactate, higher expression of glycolytic enzymes, increased rate of glucose uptake and an increase in the expression of anti-stress proteins (Beckmann et al 2012;Hughes et al 2007).…”

Section: Impact Of Long-term Sub-culture On Cell Populationsmentioning

confidence: 99%

Building a Cell Culture Process with Stable Foundations: Searching for Certainty in an Uncertain World

O'Callaghan¹,

Racher

2014

Cell Engineering

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Considerable effort is expended during the mammalian cell line construction process to find a stably-transfected clone capable of supporting the largescale manufacture of a recombinant therapeutic protein. Such a clone must synthesise a sufficient volumetric concentration of the product with the correct biochemical characteristics (glycosylation, structural integrity, etc.). Furthermore, this performance must be maintained over the extended time period required to support a manufacturing campaign in 20,000 L bioreactors. However, a significant proportion of recombinant clonal cell lines show production instability over longterm sub-culture, where volumetric product yield and/or product quality are not maintained. This instability can potentially extend product development timelines and can affect the ability of a manufacturing process to meet market demand. In the worse-case scenario it can also jeopardise patient safety if product quality is impaired. In order to prevent this, industrial cell line construction processes include long-term stability studies where several candidate lead clones are serially sub-cultured and monitored for signs of instability before selecting the final production cell line. The roots of production instability are varied, but the epigenetic silencing of transgenes at sites of host cell chromosome integration, and the direct mutation or loss of transgenes are prominent molecular causes. Considerable research has been conducted by both industry and academic groups into the molecular mechanisms underpinning instability, with an emphasis on uncovering early predictive markers of incipient instability as well as preventing its occurrence. In this article we present a detailed overview of the industrial experience of production instability and its impact on the manufacturing of recombinant therapeutics. We also discuss our current understanding of the molecular causes of cell line instability and how this has been used to mitigate the impact of this phenomenon through novel vector redesigns and cell line screening.

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Application of Population Balance Model to the Loss of Hybridoma Antibody Productivity

Cited by 37 publications

References 17 publications

Effects of passage number on growth and productivity of hybridoma secreting MRSA anti-PBP2a monoclonal antibodies

Effects of passage number on growth and productivity of hybridoma secreting MRSA anti-PBP2a monoclonal antibodies

Stability of transfectomas producing chimeric antibody against the pre‐S2 surface antigen of hepatitis B virus during a long‐term culture

Building a Cell Culture Process with Stable Foundations: Searching for Certainty in an Uncertain World

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