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Determination of Bioprocessing Variables that Influence Particle and Aggregation Formation of Therapeutic IgGs

Davies, Stephanie Ann (2014) Determination of Bioprocessing Variables that Influence Particle and Aggregation Formation of Therapeutic IgGs. Doctor of Philosophy (PhD) thesis, University of Kent,. (Access to this publication is currently restricted. You may be able to access a copy if URLs are provided) (KAR id:48573)

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Abstract

One of the dominant classes of protein based drugs, which continues to contribute to the growth and success of biologics, are recombinant monoclonal antibody (mAb) therapies. In preparing recombinant antibodies for biotherapeutic applications, the stability and maintenance of this is of utmost importance; not just for the activity of the molecule but also in terms of the safety of the drug and this must be demonstrated to regulatory authorities before a biotherapeutic receives approval for use. One such measure of protein integrity, quality or stability is the assessment of soluble aggregates and sub-visible particles present in formulations after stressed stability studies. The presence of such particles and aggregates in recombinant biotheraputics is potentially of concern in the manufacture of such biologics due to the reported immunogenic responses observed in patients when these biologics are administered; which is believed to be associated with the presence of aggregated antibody species. In addition to this, the in vivo efficacy of the biologic is inevitably compromised upon formation of such species. As such the regulatory bodies require that aggregate and particle levels are monitored and reported in the specification of a biologic.

The results from these studies confirmed that washing of the protein A column during mAb purification can influence subsequent particle formation. The inclusion of a specific wash step during protein A purification was effective at lowering particle levels in formulations of a model mAb compared to those samples which were purified without. The wash step was shown to remove mAb fragments and molecular chaperones and other host cell proteins from the Chinese hamster ovary cell host. The re-introduction of these species back into mAb solutions resulted in a subsequent increase in particle formation suggesting that species present in the material removed by the wash fraction influenced the particle numbers observed. With regard to culture conditions and feeding of the cells, batch cultures produced a less stable molecule compared to the fed-batch cultures as determined by the propensity to form particles and this could be related to the intracellular stress being experienced by cells in during culture. Cell lysis is influenced by the intracellular stress levels of the cultures, which is in-turn influenced by the feeding regime and harvest day. The levels of mAb fragments and sub-visible particles increased in fed-batch and batch Mab-184 samples, as culture viability decreases; which was mirrored by changes in the expression of certain stress inducible genes. Purification of the samples with the wash step reduces the particle levels to similar levels across all harvest days suggesting that the wash step is not only removing misfolded protein released into the culture supernatant upon cell lysis, but also removes species that are present in the cell culture supernatant which contribute to antibody particle formation. These results highlight the need for better monitoring of intracellular stress levels during CHO culture for informing harvest decisions as the intracellular state of the cells influence the quality of the final therapeutic product. Finally, using AFM particle formation of a model mAb is characteristic of a coagulation mechanism after one hour of temperature stress. Collectively together these studies demonstrate the importance of both upstream and downstream processing decisions on the subsequent propensity of mAbs to form particles and hence the need to consider the whole bioprocess to obtain the best possible product with regard to aggregate and particle amounts.

Item Type: Thesis (Doctor of Philosophy (PhD))
Thesis advisor: Smales, Christopher Mark
Thesis advisor: Uddin, Shahid
Uncontrolled keywords: Bioprocess, Upstream/downstream processing, Monoclonal antibody, Aggregation formation, Particle formation, Atomic force microscopy
Subjects: Q Science > QP Physiology (Living systems) > QP506 Molecular biology
Q Science > QP Physiology (Living systems) > QP517 Biochemistry
Divisions: Divisions > Division of Natural Sciences > School of Biosciences
Depositing User: Users 1 not found.
Date Deposited: 19 May 2015 15:00 UTC
Last Modified: 20 May 2021 13:33 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/48573 (The current URI for this page, for reference purposes)
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