Rapid Immunoassay Technique for Process Monitoring of Animal Cell Fermentations

Jervis, Eric; Kilburn, Douglas G.

doi:10.1021/bp00007a005

Cited by 13 publications

(1 citation statement)

References 17 publications

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“…Medium glucose concentration was monitored using a Glucose Analyzer 2 (Beckman, Fullerton, CAI. IgG antibody concentration was determined using a fluorescent concentration analyzer (Idexx Ltd., Westbrook, ME) by the method of Jervis and Kilburn (1991). Carboxylpolystyrene particles (0.8 pm, 0.25% (v/v), Idexx) coated with anti-mouse IgG (Sigma) were used to capture the antibody.…”

Section: Introductionmentioning

confidence: 99%

Batch and Semicontinuous Aggregation and Sedimentation of Hybridoma Cells by Acoustic Resonance Fields

Pui¹,

Trampler²,

Sonderhoff³

et al. 1995

Biotechnology Progress

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Ultrasound was used to enhance the sedimentation of hybridoma cells from medium in a 75 mL resonator chamber. Forces in the acoustic standing waves aggregated the cells, and the aggregates were then rapidly sedimented by gravity. Cell separation increased with acoustic treatment time and cell concentration. The separation efficiency was over 97% for cell concentrations between 10(6) and 10(7) cells/mL. During acoustic treatment at 180 W/L, the medium temperature increased at a rate of 1.3 degrees C/min. Ultrasonic exposures up to 220 W/L did not influence the viability or subsequent growth and antibody production of the cells. A decrease in cell viability was observed at a power level of 260 W/L. Batch separation efficiencies were as high as 98%. Acoustic separation was tested under semicontinuous operation, and above 90% separation efficiency was achieved at a flow rate of 0.7 L/h.

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Section: Introductionmentioning

confidence: 99%

Batch and Semicontinuous Aggregation and Sedimentation of Hybridoma Cells by Acoustic Resonance Fields

Pui¹,

Trampler²,

Sonderhoff³

et al. 1995

Biotechnology Progress

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Increasing GPI-anchored protein harvest concentrations from suspension and porous microcarrier CHO cell cultures

Sunderji¹,

Piret²,

Kennard³

1997

Biotechnol. Bioeng.

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Chinese hamster ovary (CHO) cells expressing the human melanoma tumour antigen, p97, were used to develop a controlled release process for the production of recombinant glycosyl‐phosphatidylinositol (GPI) anchored proteins. The cells were cultured either in suspension or immobilized on porous microcarriers and p97 was selectively cleaved from the cell surface by the bacterial enzyme, phosphatidylinositol‐phospholipase C (PI‐PLC). The kinetics of p97 cleavage from the cell surface by PI‐PLC was shown to be approximated by Michaelis‐Menten kinetics. The recovered p97 concentrations were increased by reusing the PI‐PLC enzyme solution to harvest multiple batches of cells. A convenient PI‐PLC assay was developed to monitor the harvesting process and to determine the stability of PI‐PLC under harvesting conditions. Although the Pl‐PLC was stable under harvesting conditions, it rapidly adsorbed to the cell surface and was depleted from the reused enzyme solution. In order to maintain PI‐PLC activity, it was necessary to add fresh PI‐PLC to the reused enzyme solution before harvesting a fresh batch of cells. The maximum p97 concentration that could be obtained from harvesting CHO cells cultured on porous microcarriers was limited by the dilution effects of sample removal, adding fresh PI‐PLC and liquid associated with settled microcarriers. A model was developed that adequately predicted the p97 concentration after each harvest and the maximum p97 concentration that could be achieved by this harvesting method. The dilution effects were minimized by harvesting from centrifuged suspension culture cells and the harvested p97 concentration was increased by over sixfold to 0.64 mg/mL. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 136–147, 1997.

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Controlled release process to recover heterologous glycosylphosphatidylinositol membrane anchored proteins from CHO cells

Kennard¹,

Food²,

Jefferies³

et al. 1993

Biotech & Bioengineering

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A semicontinuous process has been developed to recover heterologous proteins at increased concentrations and purities. Proteins attached to mammalian cell membranes by glycosylphosphatidylinositol (GPI) anchors can be selectively released into the supernatant by the enzyme phosphatidylinositol-phospholipase C (PI-PLC). Chinese hamster ovary (CHO) cells, genetically engineered to express the GPI anchored, human melanoma antigen (p97), were used as a model system. These cells were grown in protein containing growth medium. During a brief harvesting phase the medium was replaced by phosphate buffered saline (PBS) containing 10 mU/mL of PI-PLC and the GPI anchored protein was cleaved from the cell surface and recovered in soluble form at up to 30% purity. After harvesting, the cells were returned to growth medium where the protein was re-expressed within 40 h. The growth rate, viability, and protein production of cells, repeatedly harvested over a 44-day period, were not adversely affected. This continuous cyclic harvesting process allowed recovery of a heterologous protein at high purity and concentrations and could be applied to the recovery of other GPI anchored proteins and genetically engineered GPI anchored fusion proteins.

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Rapid Immunoassay Technique for Process Monitoring of Animal Cell Fermentations

Cited by 13 publications

References 17 publications

Batch and Semicontinuous Aggregation and Sedimentation of Hybridoma Cells by Acoustic Resonance Fields

Batch and Semicontinuous Aggregation and Sedimentation of Hybridoma Cells by Acoustic Resonance Fields

Increasing GPI-anchored protein harvest concentrations from suspension and porous microcarrier CHO cell cultures

Controlled release process to recover heterologous glycosylphosphatidylinositol membrane anchored proteins from CHO cells

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