Effects of process intensification on homogeneity of an IgG1:κ monoclonal antibody during perfusion culture.

The pharmaceutical industry employs various strategies to improve cell productivity. These strategies include process intensification, culture media improvement, clonal selection, media supplementation and genetic engineering of cells. However, improved cell productivity has inherent risk of impacting product quality attributes (PQA). PQAs may affect the products' efficacy via stability, bioavailability, or in vivo bioactivity. Variations in manufacturing process may introduce heterogeneity in the products by altering the type and extent of N-glycosylation, which is a PQA of therapeutic proteins. We investigated the effect of different cell densities representing increasing process intensification in a perfusion cell culture on the production of an IgG1-κ monoclonal antibody from a CHO-K1 cell line. This antibody is glycosylated both on light chain and heavy chain. Our results showed that the contents of glycosylation of IgG1-κ mAb increased in G0F and fucosylated type glycans as a group, whereas sialylated type glycans decreased, for the mAb whole protein. Overall, significant differences were observed in amounts of G0F, G1F, G0, G2FS1, and G2FS2 type glycans across all process intensification levels. G2FS2 and G2 type N-glycans were predominantly quantifiable from light chain rather than heavy chain. It may be concluded that there is a potential impact to product quality attributes of therapeutic proteins during process intensification via perfusion cell culture that needs to be assessed. Since during perfusion cell culture the product is collected throughout the duration of the process, lot allocation needs careful attention to process parameters, as PQAs are affected by the critical process parameters (CPPs). KEY POINTS: • Molecular integrity may suffer with increasing process intensity. • Galactosylated and sialylated N-glycans may decrease. • Perfusion culture appears to maintain protein charge structure.

Generation of reference cell lines, media, and a process platform for CHO cell biomanufacturing.

Due to the favorable attributes of Chinese hamster ovary (CHO) cells for therapeutic proteins and antibodies biomanufacturing, companies generate proprietary cells with desirable phenotypes. One key attribute is the ability to stably express multi-gram per liter titers in chemically defined media. Cell, media, and feed diversity has limited community efforts to translate knowledge. Moreover, academic, and nonprofit researchers generally cannot study "industrially relevant" CHO cells due to limited public availability, and the time and knowledge required to generate such cells. To address these issues, a university-industrial consortium (Advanced Mammalian Biomanufacturing Innovation Center, AMBIC) has acquired two CHO "reference cell lines" from different lineages that express monoclonal antibodies. These reference cell lines have relevant production titers, key performance outcomes confirmed by multiple laboratories, and a detailed technology transfer protocol. In commercial media, titers over 2 g/L are reached. Fed-batch cultivation data from shake flask and scaled-down bioreactors is presented. Using productivity as the primary attribute, two academic sites aligned with tight reproducibility at each site. Further, a chemically defined media formulation was developed and evaluated in parallel to the commercial media. The goal of this work is to provide a universal, industrially relevant CHO culture platform to accelerate biomanufacturing innovation.

Single in-line biomass probe detects CHO cell growth by capacitance and bacterial contamination by conductivity in bioreactor.

BACKGROUND: Real time process data facilitates timely decisions, enables better process control, and can increase quality assurance. Biological drugs (mol. Wt. ≥ 40 kDa) are manufactured using mammalian cells such as Chinese hamster ovary (CHO) cells in bioreactors and have significant risks of contamination during processing. In such processes, in-line monitoring of biomass can provide real-time cell growth profiles and indications of bioreactor health. METHODS: An in-line conductivity/capacitance probe (Aber Instruments, Aberystwyth, UK) for monitoring CHO cell growth during fed batch cultures for producing an IgG1 monoclonal antibody was employed. Cell growth was measured in real-time using the capacitance probe (pF cm-1 ) while being compared with off-line measurements using a metabolic analyzer (Nova Biomedical, Waltham, MA, USA). Conductivity measurements (mS cm-1 ) detected variations in the solute concentrations in the bioreactor due to nutrient feed, bicarbonate buffer, and cellular metabolism by-products. RESULTS AND CONCLUSION: Abnormal increases in conductivity were found to consistently correspond to bacterial contamination, which was confirmed by orthogonal methods. The contaminated bioreactor runs exhibited sharp increases in conductivity rates hours before dissolved oxygen levels precipitously decreased due to bacterial growth. It is proposed that in-line measurement of conductivity could be employed for early detection of bacterial contaminations. The probe may be adopted in pharmaceutical aseptic aqueous liquid handling processes.

Adventitious agent detection methods in bio-pharmaceutical applications with a focus on viruses, bacteria, and mycoplasma.

Adventitious agents present significant complications to biopharmaceutical manufacturing. Adventitious agents include numerous lifeforms such as bacteria, fungi, viruses, mycoplasma, and others that are inadvertently introduced into biological systems. They present significant problems to the stability of cell cultures and the sterility of manufacturing products. In this review, detection methods for bacteria, viruses, and mycoplasma are comprehensively addressed. Detection methods for viruses include traditional culture-based methods, electron microscopy studies, in vitro molecular and antibody assays, sequencing methods (massive parallel or next generation sequencing), and degenerate PCR (polymerase chain reaction). Bacteria, on the other hand, can be detected with culture-based approaches, PCR, and biosensor-based methods. Mycoplasma can be detected via PCR (including specific kits), microbiological culture methods, and enzyme-linked immunosorbent assays (ELISA). This review highlights the advantages and weaknesses of current detection methods while exploring potential avenues for further development and improvement of novel detection methods. Additionally, a brief evaluation of the transition of these methods into the gene therapy production realm with a focus on viral titer monitoring will be presented.

An ABCG Transporter Functions in Rab Localization and Lysosome-Related Organelle Biogenesis in Caenorhabditis elegans.

ABC transporters couple ATP hydrolysis to the transport of substrates across cellular membranes. This protein superfamily has diverse activities resulting from differences in their cargo and subcellular localization. Our work investigates the role of the ABCG family member WHT-2 in the biogenesis of gut granules, a Caenorhabditis elegans lysosome-related organelle. In addition to being required for the accumulation of birefringent material within gut granules, WHT-2 is necessary for the localization of gut granule proteins when trafficking pathways to this organelle are partially disrupted. The role of WHT-2 in gut granule protein targeting is likely linked to its function in Rab GTPase localization. We show that WHT-2 promotes the gut granule association of the Rab32 family member GLO-1 and the endolysosomal RAB-7, identifying a novel function for an ABC transporter. WHT-2 localizes to gut granules where it could play a direct role in controlling Rab localization. Loss of CCZ-1 and GLO-3, which likely function as a guanine nucleotide exchange factor (GEF) for GLO-1, lead to similar disruption of GLO-1 localization. We show that CCZ-1, like GLO-3, is localized to gut granules. WHT-2 does not direct the gut granule association of the GLO-1 GEF and our results point to WHT-2 functioning differently than GLO-3 and CCZ-1 Point mutations in WHT-2 that inhibit its transport activity, but not its subcellular localization, lead to the loss of GLO-1 from gut granules, while other WHT-2 activities are not completely disrupted, suggesting that WHT-2 functions in organelle biogenesis through transport-dependent and transport-independent activities.

Metabolic engineering of Chinese hamster ovary cells towards reduced biosynthesis and accumulation of novel growth inhibitors in fed-batch cultures.

Chinese hamster ovary (CHO) cells in fed-batch cultures are known to consume large amounts of nutrients and divert significant portion of them towards the formation of byproducts, some of which, including lactate and ammonia, are known to be growth inhibitory in nature. A major fraction of these inhibitory metabolites are byproducts or intermediates of amino acid catabolism. Limiting the supply of amino acids has been shown to curtail the production of corresponding inhibitory byproducts resulting in enhanced growth and productivities in CHO cell fed-batch cultures (Mulukutla et al., 2017). In the current study, metabolic engineering of CHO cells was undertaken in order to reduce the biosynthesis of these novel growth inhibitors. Phenylalanine-tyrosine (Phe-Tyr) and branched chain amino acid (BCAA) catabolic pathways were engineered as part of this effort. Four genes that encode enzymes in the Phe-Tyr pathway, which were observed to be minimally expressed in CHO cells, were in turn overexpressed. Metabolically engineered cells were prototrophic to tyrosine and had reduced production of the inhibitory byproducts from Phe-Tyr pathway including 3-phenyllactate and 4-hydroxyphenyllactate. In case of BCAA catabolic pathway, branched chain aminotransferase 1 (BCAT1) gene, which encodes the enzyme that catalyzes the first step in the catabolism of BCAAs, was knocked out in CHO cells. Knockout (KO) of BCAT1 function completely eliminated production of inhibitory byproducts from BCAA catabolic pathway, including isovalerate, isobutyrate and 2-methylbutyrate, resulting in significantly enhanced cell growth and productivities in fed-batch cultures. This study is first of its kind to demonstrate that metabolic engineering of essential amino acid metabolism of CHO cells can significantly improve cell culture process performance.

Function and regulation of the Caenorhabditis elegans Rab32 family member GLO-1 in lysosome-related organelle biogenesis.

Cell type-specific modifications of conventional endosomal trafficking pathways lead to the formation of lysosome-related organelles (LROs). C. elegans gut granules are intestinally restricted LROs that coexist with conventional degradative lysosomes. The formation of gut granules requires the Rab32 family member GLO-1. We show that the loss of glo-1 leads to the mistrafficking of gut granule proteins but does not significantly alter conventional endolysosome biogenesis. GLO-3 directly binds to CCZ-1 and they both function to promote the gut granule association of GLO-1, strongly suggesting that together, GLO-3 and CCZ-1 activate GLO-1. We found that a point mutation in GLO-1 predicted to spontaneously activate, and function independently of it guanine nucleotide exchange factor (GEF), localizes to gut granules and partially restores gut granule protein localization in ccz-1(-) and glo-3(-) mutants. CCZ-1 forms a heterodimeric complex with SAND-1(MON1), which does not function in gut granule formation, to activate RAB-7 in trafficking pathways to conventional lysosomes. Therefore, our data suggest a model whereby the function of a Rab GEF can be altered by subunit exchange. glo-3(-) mutants, which retain low levels of GLO-3 activity, generate gut granules that lack GLO-1 and improperly accumulate RAB-7 in a SAND-1 dependent process. We show that GLO-1 and GLO-3 restrict the distribution of RAB-7 to conventional endolysosomes, providing insights into the segregation of pathways leading to conventional lysosomes and LROs.

Behavioral correlates of the distributed coding of spatial context.

Hippocampal place cells respond heterogeneously to elemental changes of a compound spatial context, suggesting that they form a distributed code of context, whereby context information is shared across a population of neurons. The question arises as to what this distributed code might be useful for. The present study explored two possibilities: one, that it allows contexts with common elements to be disambiguated, and the other, that it allows a given context to be associated with more than one outcome. We used two naturalistic measures of context processing in rats, rearing and thigmotaxis (boundary-hugging), to explore how rats responded to contextual novelty and to relate this to the behavior of place cells. In experiment 1, rats showed dishabituation of rearing to a novel reconfiguration of familiar context elements, suggesting that they perceived the reconfiguration as novel, a behavior that parallels that of place cells in a similar situation. In experiment 2, rats were trained in a place preference task on an open-field arena. A change in the arena context triggered renewed thigmotaxis, and yet navigation continued unimpaired, indicating simultaneous representation of both the altered contextual and constant spatial cues. Place cells similarly exhibited a dual population of responses, consistent with the hypothesis that their activity underlies spatial behavior. Together, these experiments suggest that heterogeneous context encoding (or "partial remapping") by place cells may function to allow the flexible assignment of associations to contexts, a faculty that could be useful in episodic memory encoding.