Improving biopharmaceutical production of Chinese hamster ovary cells using targeted genome engineering Tools

Chinese Hamster Ovary (CHO) cells are the prominent cell line used in biopharmaceutical production. Over 70% of all therapeutically used recombinant proteins are produced by CHO cells with a market size predicted to exceed €250bn by 2021. Their favourable attributes over other cell lines are properties like resistance to viral infections, growth in chemical defined media, human like glycosylation patterns, good productivity and ease of genetic engineering. Due to the complexity of mammalian expression systems yields achieved are not phenomenal by any means. However, compared to 1986 with product concentrations of 50 mg/L, titres up to 10 g/L have been reported recently. Improvement of bioprocesses and media development contributed their part in facilitating higher titres but genetic engineering to improve host cells came to the foreground. MicroRNAs (miRNAs) have hereby been highlighted as attractive targets due to their involvement in processes like viability, secretion, productivity, product quality to mention only a few. MiRNAs are small non-coding RNAs which are about 22 nucleotides in length and were first discovered in the early 90’s. A single miRNA can target 100-200 mRNAs which highlights them as key regulators for translational control. The miRNA-23 cluster was first identified as upregulated during induced hypothermic conditions. Hypothermia is a commonly used process to reduce growth and thrive CHO cells to improved productivities. Therefore, we hypothesised involvement of the miR-23 cluster or individual miRNA members in viability and productivity phenotypes. In this work we investigated the depletion of the miR-23 cluster as well as miR-23, miR-24 and miR-27 in a panel of industrially relevant cell lines expressing various recombinant products. In fact, miR-24 was identified as thriver of productivity and growth by upregulating ribosomal biogenesis, assembly of ribosomal subunits, translation as well as unfolded protein response (UPR). This was demonstrated in several cell lines and was not product specific. Furthermore, the depletion of the whole miR-23 cluster as well as miR-27 has been shown to improve productivity although in a cell line specific context. To overcome challenges of sponge technology we also implemented the recently developed CRISPR/Cas9 system to target miRNAs. When phenotypes after sponge and CRISPR/Cas9 mediated depletion of members of the miR-23 cluster were assessed, it was demonstrated that even enhanced properties were exhibited using CRISPR/Cas9 in case of miR-24 and miR-27 regarding productivity and longevity. Furthermore, we implemented a CRISPR/Cas9 library for genome wide recessive knockout screens to identify proteins involved in high productivity phenotypes or are important for survival of stress conditions i.e. hyperosmolality. Mixed populations expressing the sgRNA-library were sorted for high productivity using low temperature stains and were adapted to high salt conditions. Enrichment or depletion of sgRNAs was subsequently analyzed using Next-Generation Sequencing. SgRNA abundance analysed after low temperature stain showed enrichment of distinct populations. Functional annotation of enriched genes showed no evidence in relation to productivity. Exploiting miRNAs and genome-wide knockout studies to improve the bioprocess phenotype highlights these methods as interesting tools for further investigation regarding applications within biopharmaceutical industry

Production of bioengineered outer membrane vesicles as a vaccine platform

Development of new vaccines based on vaccine platforms forms an interesting opportunity to significantly reduce the development time. New vaccines are required to keep up with newly emerging diseases that spread quickly in the interconnected global world. The traditional development of new vaccines is a lengthy process, as a new production process must be developed for each vaccine. A vaccine platform allows the use of the existing production process for new vaccine targets. Bacterial outer membrane vesicles (OMVs) produced by Neisseria meningitidis are highly suitable candidates to form a vaccine platform. N. meningitidis OMVs have been safely used as meningococcal vaccines. OMVs are non-replicative nanoparticles derived from the bacterial membrane, that can display heterologous antigens. N. meningitidis OMVs have been produced by extraction of vesicle like structures from bacterial cells. However, OMVs spontaneously released from bacteria have advantages over extracted OMVs as they can be directly purified from the supernatant of the bacterial culture, have enhanced quality, and trigger broader immune responses. On the downside, the yields of spontaneously released OMVs are low. The aim of this thesis was to obtain a better understanding of outer membrane vesicle formation by Neisseria meningitidis and OMV quality, and use this to develop improved OMV production processes that can become a cost-effective basis for an OMV-based vaccine platform. A vaccine platform should be versatile and adaptable for the addition of heterologous antigens onto the OMV. In Chapter 2 we explored existing methods for antigen decoration of OMVs through a comprehensive literature review. We distinguished two approaches of OMV platforms, based on either separate production of antigen and OMV followed by coupling or production of the antigen directly by the OMV producing bacterium. Separate antigen production seems more suitable for viral and therapeutic targets as it allows coupling of complex glycosylated targets to the OMV. Production of antigens directly by the OMV producing bacterium is probably more suited for microbial targets and allows for the most straightforward production process. To optimize OMV production processes, a new method for OMV quantification was needed as current quantification methods of OMVs were indirect and elaborate. In Chapter 3 we successfully used nanoparticle tracking analysis to quantify OMVs directly from sterile filtered culture samples, in a high-throughput manner. Now that we had a more reliable method available to quantify OMV release, our next step was to improve the OMV yields by studying the release of OMVs from the bacterium. A previous study had shown that cysteine depletion causes a stationary growth phase in which OMVs are released. In Chapter 4 we show that sulfur depletion in general resulted in OMV release of N. meningitidis cultures and found that sulfate depltion results in an even higher level of OMV release. Mechanistically, OMVs were enriched in phospholipids following sulfate depletion, suggesting that enrichment of phospholipids is an important factor in the OMV release process. A second parameter is oxidative stress that had been previously observed in cysteine depleted cultures, as well as in the sulfate depleted cultures described in Chapter 4. We found that high dissolved oxygen tension could mimick this situation and trigger increased OMV release. Because dissolved oxygen tension is a well-controlled process parameter, high dissolved oxygen concentrations could be conveniently used to stimulate OMV release. This was demonstrated in Chapter 5 where we showed that sulfur depletion and high dissolved oxygen tension stimulate the OMV release per bacterium and can be applied in batch production processes. Chapter 6 presents a proof of concept of the OMV-based vaccine platform in which the findings from the previous chapters were combined. We expressed outer surface protein A and outer surface protein C of Borrelia burgdorferi, the cause of Lyme disease, on N. meningitidis OMVs. These OMVs with heterologous model antigens were produced in a batch production process. In this process, sulfur depletion and high-dissolved oxygen concentrations were combined to establish high OMV yields. Purification based on scalable unit-operations resulted in a recovery of 90 mg OMV associated protein per liter culture. This production proces could be used as a basis for the development of novel Lyme disease vaccines. Lastly, in chapter 7 we suggest that OMV production can be further improved by adopting continuous production processes. Continuous production results in increased volumetric productivities, enhanced process control, and reduced variability. However, before continuous OMV production can be used for OMV vaccines, a method to assure OMV quality in the lengthy cultivations needs to be developped. This thesis shows that high yields of spontaneously released N. meningitidis OMVs can be obtained by stimulating release of OMVs from the bacterium by process parameters. These OMVs show potential as modular production platform and could boost future vaccine development. OMV based vaccine platforms will reduce the time required to develop new vaccines, which is urgently needed to meet the demand for new vaccines.</p