Assessment of the interactions between bioprocess conditions and protein glycosylation in antibody- producing mammalian cell cultures
Author(s)
Jimenez Del Val, Ioscani
Type
Thesis
Abstract
The pharmaceutical industry is going through a rather turbulent period. Many
blockbuster drugs have fallen off patent over the past two years and many more are
expected to do so in the near future. In response, pharmaceutical companies have
continued searching for products that will replace those that have lost patent
protection. However, drug development and approval is extremely time-consuming and
costly. So that this critical issue is addressed, industry experts and regulatory agencies
have jointly proposed the implementation of Quality by Design (QbD) principles in the
development and manufacture of all new drugs. Adoption of QbD is expected to reduce
drug development cost and approval time. It is also expected to encourage innovation
by developing drugs, and the processes used to manufacture them, around the
mechanisms that relate process inputs with end product quality. Within this context,
monoclonal antibodies (mAbs) are currently the highest-selling products of the
biopharmaceutical industry and are projected to account for nearly half of the world’s
top-selling drugs by 2018. All currently commercialized mAbs contain N-linked glycans
(complex carbohydrates) bound to their protein backbone. These carbohydrates, in
turn, have been widely reported to impact the safety and efficacy of mAbs. Furthermore,
it has widely been reported that bioprocess conditions heavily impact the composition
and distribution of these glycans. For these reasons, mAb glycosylation is considered a
critical quality attribute (CQA) of these therapeutic proteins under the QbD scope.
Based on QbD principles, the objective of this thesis was to generate a mathematical
model that mechanistically relates the effect of nutrient availability throughout cell
culture with the glycan profile of a mAb. The model was constructed from three
individual ones. The first model describes the N-linked glycosylation process which
occurs in the Golgi apparatus. The second model is unstructured and describes cell
culture dynamics. The third and final model describes the biosynthetic pathway for
nucleotide sugars. All three models were developed independently, but were adapted
with features so that they could be interconnected. The glycosylation model
approximates the Golgi apparatus to a single plug flow reactor where resident proteins
(glycosylation enzymes and transport proteins) are recycled from distal portions of the
Golgi space to proximal ones. Optimisation-based methods were developed to estimate
unknown parameters of the model. The cell culture dynamics model was developed to
represent cell growth, nutrient consumption and mAb synthesis. It was originally based
on Monod kinetics, but was adapted to include experimentally-encountered complexity.
The model for nucleotide metabolism was heuristically reduced from 35 constituting
reactions to 7. Additional mechanistic features were adapted or included to ensure
model fidelity. Experimentally, batch cultures were performed with hybridoma (CRL-1606 from
ATCC). Data for viable cell density, glucose, glutamine, lactate, ammonia and mAb titre
were collected. Intracellular samples were produced by perchloric acid extraction.
These samples were then analysed for nucleotide sugar content using a high
performance anion exchange chromatographic method which was optimized to quantify
eight nucleotide sugars and four nucleotides in 30min. mAb bound glycans were
analysed by MALDI mass spectrometry. The experimental data was used to estimate the
unknown parameters of the models.
The models – along with their associated parameters – were then combined to produce
a coupled model that mechanistically relates nutrient availability with mAb
glycosylation-associated quality. With further validation, such a model could be used for
bioprocess design, control and optimization.
blockbuster drugs have fallen off patent over the past two years and many more are
expected to do so in the near future. In response, pharmaceutical companies have
continued searching for products that will replace those that have lost patent
protection. However, drug development and approval is extremely time-consuming and
costly. So that this critical issue is addressed, industry experts and regulatory agencies
have jointly proposed the implementation of Quality by Design (QbD) principles in the
development and manufacture of all new drugs. Adoption of QbD is expected to reduce
drug development cost and approval time. It is also expected to encourage innovation
by developing drugs, and the processes used to manufacture them, around the
mechanisms that relate process inputs with end product quality. Within this context,
monoclonal antibodies (mAbs) are currently the highest-selling products of the
biopharmaceutical industry and are projected to account for nearly half of the world’s
top-selling drugs by 2018. All currently commercialized mAbs contain N-linked glycans
(complex carbohydrates) bound to their protein backbone. These carbohydrates, in
turn, have been widely reported to impact the safety and efficacy of mAbs. Furthermore,
it has widely been reported that bioprocess conditions heavily impact the composition
and distribution of these glycans. For these reasons, mAb glycosylation is considered a
critical quality attribute (CQA) of these therapeutic proteins under the QbD scope.
Based on QbD principles, the objective of this thesis was to generate a mathematical
model that mechanistically relates the effect of nutrient availability throughout cell
culture with the glycan profile of a mAb. The model was constructed from three
individual ones. The first model describes the N-linked glycosylation process which
occurs in the Golgi apparatus. The second model is unstructured and describes cell
culture dynamics. The third and final model describes the biosynthetic pathway for
nucleotide sugars. All three models were developed independently, but were adapted
with features so that they could be interconnected. The glycosylation model
approximates the Golgi apparatus to a single plug flow reactor where resident proteins
(glycosylation enzymes and transport proteins) are recycled from distal portions of the
Golgi space to proximal ones. Optimisation-based methods were developed to estimate
unknown parameters of the model. The cell culture dynamics model was developed to
represent cell growth, nutrient consumption and mAb synthesis. It was originally based
on Monod kinetics, but was adapted to include experimentally-encountered complexity.
The model for nucleotide metabolism was heuristically reduced from 35 constituting
reactions to 7. Additional mechanistic features were adapted or included to ensure
model fidelity. Experimentally, batch cultures were performed with hybridoma (CRL-1606 from
ATCC). Data for viable cell density, glucose, glutamine, lactate, ammonia and mAb titre
were collected. Intracellular samples were produced by perchloric acid extraction.
These samples were then analysed for nucleotide sugar content using a high
performance anion exchange chromatographic method which was optimized to quantify
eight nucleotide sugars and four nucleotides in 30min. mAb bound glycans were
analysed by MALDI mass spectrometry. The experimental data was used to estimate the
unknown parameters of the models.
The models – along with their associated parameters – were then combined to produce
a coupled model that mechanistically relates nutrient availability with mAb
glycosylation-associated quality. With further validation, such a model could be used for
bioprocess design, control and optimization.
Date Issued
2012-12
Date Awarded
2013-03
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
License URL
Advisor
Kontoravdi, Cleo
Nagy, Judit
Sponsor
Mexican National Council for Science and Technology ; Mario Molina Fund ; Secretaría de Educación Pública (Mexico) ; Biotechnology and Biological Sciences Research Council (Great Britain)
Publisher Department
Chemical Engineering
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)