Designing a Strategy for pH Control to Improve CHO Cell Productivity in Bioreactor

Background: Drastic pH drop is a common consequence of scaling up a mammalian cell culture process, where it may affect the final performance of cell culture. Although CO2 sparging and base addition are used as common approaches for pH control, these strategies are not necessarily successful in large scale bioreactors due to their effect on osmolality and cell viability. Accordingly, a series of experiments were conducted using an IgG1 producing Chinese Hamster Ovary (CHO-S) cell culture in 30 L bioreactor to assess the efficiency of an alternative strategy in controlling culture pH. Methods: Factors inducing partial pressure of CO2 and lactate accumulation (as the main factors altering culture pH) were assessed by Plackett-Burman design to identify the significant ones. As culture pH directly influences process productivity, protein titer was measured as the response variable. Subsequently, Central Composite Design (CCD) was employed to obtain a model for product titer prediction as a function of individual and interaction effects of significant variables. Results: The results indicated that the major factor affecting pH is non-efficient CO2 removal. CO2 accumulation was found to be affected by an interaction between agitation speed and overlay air flow rate. Accordingly, after increasing the agitation speed and headspace aeration, the culture pH was successfully maintained in the range of 6.95-7.1, resulting in 51% increase in final product titer. Similar results were obtained during 250 L scale bioreactor culture, indicating the scalability of the approach. Conclusion: The obtained results showed that pH fluctuations could be effectively controlled by optimizing CO2 stripping.

Multi-omics profiling of a CHO cell culture system unravels the effect of culture pH on cell growth, antibody titer and product quality

A robust monoclonal antibody (mAb) bioprocess requires physiological parameters such as temperature, pH, or dissolved oxygen (DO) to be well-controlled as even small variations in them could potentially impact the final product quality. For instance, pH substantially affects N-glycosylation, protein aggregation and charge variant profiles, as well as mAb productivity. However, relatively less is known about how pH jointly influences product quality and titer. In this study, we investigated the effect of pH on culture performance, product titer and quality profiles by applying longitudinal multi-omics profiling, including transcriptomics, proteomics, metabolomics and glycomics, at three different culture pH set points. The subsequent systematic analysis of multi-omics data showed that pH set points differentially regulated various intracellular pathways including intracellular vesicular trafficking, cell cycle, and apoptosis, thereby resulting in differences in specific productivity, product titer and quality profiles. In addition, a time-dependent variation in mAb N-glycosylation profiles, independent of pH was identified to be mainly due to the accumulation of mAb proteins in the endoplasmic reticulum (ER) over culture time, disrupting cellular homeostasis. Overall, this multi-omics-based study provides an in-depth understanding of the intracellular processes in mAb-producing CHO cell line under varied pH conditions and could serve as a baseline for enabling the quality optimization and control of mAb production.

Nisin Synthesis, Relationship with the Growth of the Producing Strain, Culture pH and Nitrogen Consumption

Nisin, an antibacterial compound produced by Lactococcus lactis strains, has been approved by the US Food and Drug Administration to be used as a safe food additive to control the growth of undesirable pathogenic bacteria. Nisin is commonly described as a pH-dependent primary metabolite, since its production depends on growth and culture pH evolution. However, the relationship between bacteriocin synthesis and the consumption of the limiting nutrient has not been described until now. Therefore, this study aimed to develop a competitive four-dimensional Lotka Volterra-like equation to describe the relationships between culture pH, limiting nutrient (total nitrogen: TN) consumption and production of biomass (X) and nisin (BT) in four series of batch fermentation with L. lactis CECT 539 in diluted whey (DW)-based media. The developed four-dimensional LV-like equation (with a unique set of parameters) could not be used to describe all cultures belonging to each fermentation series. However, the four-dimensional LV-like equation described accurately each individual culture, providing a good description of the relationships between pH, TN, X and BT, higher values for R2 and F-ratios, lower values (< 10%) for the mean relative percentage deviation modulus, with bias and accuracy factor values approximately equal to one.

Improving Influenza HA-Vlps Production in Insect High Five Cells via Adaptive Laboratory Evolution

The use of non-standard culture conditions has proven efficient to increase cell performance and recombinant protein production in different cell hosts. However, the establishment of high-producing cell populations through adaptive laboratory evolution (ALE) has been poorly explored, in particular for insect cells. In this study, insect High Five cells were successfully adapted to grow at a neutral culture pH (7.0) through ALE for an improved production of influenza hemagglutinin (HA)-displaying virus-like particles (VLPs). A stepwise approach was used for the adaptation process, in which the culture pH gradually increased from standard 6.2 to 7.0 (ΔPh = 0.2–0.3), and cells were maintained at each pH value for 2–3 weeks until a constant growth rate and a cell viability over 95% were observed. These adapted cells enabled an increase in cell-specific HA productivity up to three-fold and volumetric HA titer of up to four-fold as compared to non-adapted cells. Of note, the adaptation process is the element driving increased specific HA productivity as a pH shift alone was inefficient at improving productivities. The production of HA-VLPs in adapted cells was successfully demonstrated at the bioreactor scale. The produced HA-VLPs show the typical size and morphology of influenza VLPs, thus confirming the null impact of the adaptation process and neutral culture pH on the quality of HA-VLPs produced. This work strengthens the potential of ALE as a bioprocess engineering strategy to improve the production of influenza HA-VLPs in insect High Five cells.

Conservation of an Ancient Oxidation Response That Controls Archaeal Epigenetic Traits Through Chromatin Protein Networks

Epigenetic variants of the archaeon Sulfolobus solfataricus called SARC have evolved heritable traits including extreme acid resistance, enhanced genome integrity and a conserved “SARC” transcriptome related to acid resistance. These traits appear to result from altered chromatin protein function related to the heritable hypomethylation of chromatin proteins Cren7 and Sso7D. To clarify how this might occur, ChIPseq and Affinity Purification Mass Spectrometry (AP-MS) were used to compare Cren7 and Sso7D genome binding sites and protein networks between lineages (wild type and SARC) and culture pH (pH 1 and 3). All SARC transcriptome loci were bound by these chromatin proteins but with invariant patterns indicating binding alone was insufficient to mediate the SARC traits. In contrast, chromosome association varied at other loci. Quantitative AP-MS was then used to identify protein interaction networks and these included transcription and DNA repair proteins implicated in the evolved heritable traits that varied in abundance between SARC and wild type strains. Protein networks included most of the S-adenosylmethionine (SAM) synthesis pathway including serine hydroxymethyltransferase (SHMT), whose abundance varied widely with culture pH. Because epigenetic marks are coupled to SAM pools and oxidative stress in eukaryotes, occurrence of a similar process was investigated here. Archaeal SAM pools were depleted by treatment with SAM pathway inhibitors, acid or oxidative stress and, like eukaryotes, levels were raised by vitamin B12 and methionine supplementation. We propose that in archaea, oxidation-induced SAM pool depletion acting through an SHMT sensor, drove chromatin protein hypomethylation and thereby protein network changes that established the evolved SARC epigenetic traits.Significance StatementArchaea and eukaryotes share many molecular processes, including chromatin-mediated epigenetic inheritance of traits. As with eukaryotes, archaeal protein complexes were formed between trait-related proteins and chromatin proteins, subject to chromatin protein methylation state. Oxidation-induced depletion of S-adenosylmethionine (SAM) pools likely resulted in chromatin protein hypomethylation. Subsequent chromatin enrichment of serine hydroxymethyltransferase as a response to oxidative stress could modulate methylation at specific genomic loci. The interplay between archaeal metabolism and chromatin appear consistent with patterns observed in eukaryotes and indicate the existence of an ancient oxidation signal transduction pathway controlling epigenetics.