Low-molecular-weight impurity in Poloxamer 188 responsible for atypical cell culture performance for mAb production.

During a recent manufacturing campaign for a monoclonal antibody using a fed-batch process, poor cell culture performance was observed across two manufacturing sites with similar scales and equipment. Root cause analysis indicated that the poor cell culture performance was linked to the production basal media. Genealogy of the precursor raw materials used in the media revealed that a particular lot of Poloxamer 188 (P188) was the common link to the poor-performing media lots. P188 serves a critical role in protecting cells against shear in cell culture bioprocesses. However, the small-scale studies suggested that the poor cell culture performance was cytostatic in nature rather than being caused due to lack of shear protection. Several P188 lots were tested analytically using SEC-MS and RP-LC-MS methods and a unique low molecular weight species was identified in the suspect lot of poloxamer. The impurity was identified to be polypropylene oxide (PPO), a reaction intermediate in P188 synthesis. Spiking studies with PPO further confirmed its cytostatic nature. This case study highlights yet another scenario where lot-to-lot variability continues to impact bioprocesses and re-emphasizes the need for robust analytical and cell-culture raw material screening methods.

Single-Cell Analysis Reveals that the Enterococcal Sex Pheromone Response Results in Expression of Full-Length Conjugation Operon Transcripts in All Induced Cells.

For high-frequency transfer of pCF10 between E. faecalis cells, induced expression of the pCF10 genes encoding conjugative machinery from the prgQ operon is required. This process is initiated by the cCF10 (C) inducer peptide produced by potential recipient cells. The expression timing of prgB, an "early" gene just downstream of the inducible promoter, has been studied extensively in single cells. However, several previous studies suggest that only 1 to 10% of donors induced for early prgQ gene expression actually transfer plasmids to recipients, even at a very high recipient population density. One possible explanation for this is that only a minority of pheromone-induced donors actually transcribe the entire prgQ operon. Such cells would not be able to functionally conjugate but might play another role in the group behavior of donors. Here, we sought to (i) simultaneously assess the presence of RNAs produced from the proximal (early induced transcripts [early Q]) and distal (late Q) portions of the prgQ operon in individual cells, (ii) investigate the prevalence of heterogeneity in induced transcript length, and (iii) evaluate the temporality of induced transcript expression. Using fluorescent in situ hybridization chain reaction (HCR) transcript labeling and single-cell microscopic analysis, we observed that most cells expressing early transcripts (QL, prgB, and prgA) also expressed late transcripts (prgJ, pcfC, and pcfG). These data support the conclusion that, after induction is initiated, transcription likely extends through the end of the conjugation machinery operon for most, if not all, induced cells.IMPORTANCE In Enterococcus faecalis, conjugative plasmids like pCF10 often carry antibiotic resistance genes. With antibiotic treatment, bacteria benefit from plasmid carriage; however, without antibiotic treatment, plasmid gene expression may have a fitness cost. Transfer of pCF10 is mediated by cell-to-cell signaling, which activates the expression of conjugation genes and leads to efficient plasmid transfer. Yet, not all donor cells in induced populations transfer the plasmid. We examined whether induced cells might not be able to functionally conjugate due to premature induced transcript termination. Single-cell analysis showed that most induced cells do, in fact, express all of the genes required for conjugation, suggesting that premature transcription termination within the prgQ operon does not account for failure of induced donor cell gene transfer.

Recurring genomic structural variation leads to clonal instability and loss of productivity.

Chinese hamster ovary cells, commonly used in the production of therapeutic proteins, are aneuploid. Their chromosomes bear structural abnormality and undergo changes in structure and number during cell proliferation. Some production cell lines are unstable and lose their productivity over time in the manufacturing process and during the product's life cycle. To better understand the link between genomic structural changes and productivity stability, an immunoglobulin G producing cell line was successively single-cell cloned to obtain subclones that retained or lost productivity, and their genomic features were compared. Although each subclone started with a single karyotype, the progeny quickly diversified to a population with a distribution of chromosome numbers that is not distinctive from the parent and among subclones. The comparative genomic hybridization (CGH) analysis showed that the extent of copy variation of gene coding regions among different subclones stayed at levels of a few percent. Genome regions that were prone to loss of copies, including one with a product transgene integration site, were identified in CGH. The loss of the transgene copy was accompanied by loss of transgene transcript level. Sequence analysis of the host cell and parental producing cell showed prominent structural variations within the regions prone to loss of copies. Taken together, we demonstrated the transient nature of clonal homogeneity in cell line development and the retention of a population distribution of chromosome numbers; we further demonstrated that structural variation in the transgene integration region caused cell line instability. Future cell line development may target the transgene into structurally stable regions.

Stochasticity in the enterococcal sex pheromone response revealed by quantitative analysis of transcription in single cells.

In Enterococcus faecalis, sex pheromone-mediated transfer of antibiotic resistance plasmids can occur under unfavorable conditions, for example, when inducing pheromone concentrations are low and inhibiting pheromone concentrations are high. To better understand this paradox, we adapted fluorescence in situ hybridization chain reaction (HCR) methodology for simultaneous quantification of multiple E. faecalis transcripts at the single cell level. We present direct evidence for variability in the minimum period, maximum response level, and duration of response of individual cells to a specific inducing condition. Tracking of induction patterns of single cells temporally using a fluorescent reporter supported HCR findings. It also revealed subpopulations of rapid responders, even under low inducing pheromone concentrations where the overall response of the entire population was slow. The strong, rapid induction of small numbers of cells in cultures exposed to low pheromone concentrations is in agreement with predictions of a stochastic model of the enterococcal pheromone response. The previously documented complex regulatory circuitry controlling the pheromone response likely contributes to stochastic variation in this system. In addition to increasing our basic understanding of the biology of a horizontal gene transfer system regulated by cell-cell signaling, demonstration of the stochastic nature of the pheromone response also impacts any future efforts to develop therapeutic agents targeting the system. Quantitative single cell analysis using HCR also has great potential to elucidate important bacterial regulatory mechanisms not previously amenable to study at the single cell level, and to accelerate the pace of functional genomic studies.

Mechanisms of peptide sex pheromone regulation of conjugation in Enterococcus faecalis.

In many gram positive bacteria, horizontal transfer and virulence are regulated by peptide-mediated cell-cell signaling. The heptapeptide cCF10 (C) activates conjugative transfer of the Enterococcus faecalis plasmid pCF10, whereas the iCF10 (I) peptide inhibits transfer. Both peptides bind to the same domain of the master transcription regulator PrgX, a repressor of transcription of the prgQ operon encoding conjugation genes. We show that repression of prgQ by PrgX tetramers requires formation of a pCF10 DNA loop where each of two PrgX DNA-binding sites is occupied by a dimer. I binding to PrgX enhances prgQ repression, while C binding has the opposite effect. Previous models suggested that differential effects of these two peptides on the PrgX oligomerization state accounted for their distinct functions. Our new results demonstrate that both peptides have similar, high-binding affinity for PrgX, and that both peptides actually promote formation of PrgX tetramers with higher DNA-binding affinity than Apo-PrgX. We propose that differences in repression ability of PrgX/peptide complexes result from subtle differences in the structures of DNA-bound PrgX/peptide complexes. Changes in the induction state of a donor cell likely results from replacement of one type of DNA-bound peptide/PrgX tetramer with the other.

A comparative genomic hybridization approach to study gene copy number variations among Chinese hamster cell lines.

Chinese Hamster Ovary (CHO) cells are aneuploid in nature. The genome of recombinant protein producing CHO cell lines continuously undergoes changes in its structure and organization. We analyzed nine cell lines, including parental cell lines, using a comparative genomic hybridization (CGH) array focused on gene-containing regions. The comparison of CGH with copy-number estimates from sequencing data showed good correlation. Hierarchical clustering of the gene copy number variation data from CGH data revealed the lineage relationships between the cell lines. On analyzing the clones of a clonal population, some regions with altered genomic copy number status were identified indicating genomic changes during passaging. A CGH array is thus an effective tool in quantifying genomic alterations in industrial cell lines and can provide insights into the changes in the genomic structure during cell line derivation and long term culture. Biotechnol. Bioeng. 2017;114: 1903-1908. © 2017 Wiley Periodicals, Inc.

Advancement in bioprocess technology: parallels between microbial natural products and cell culture biologics.

The emergence of natural products and industrial microbiology nearly eight decades ago propelled an era of bioprocess innovation. Half a century later, recombinant protein technology spurred the tremendous growth of biologics and added mammalian cells to the forefront of industrial producing cells in terms of the value of products generated. This review highlights the process technology of natural products and protein biologics. Despite the separation in time, there is a remarkable similarity in their progression. As the new generation of therapeutics for gene and cell therapy emerges, its process technology development can take inspiration from that of natural products and biologics.

Augmenting Chinese hamster genome assembly by identifying regions of high confidence.

Chinese hamster Ovary (CHO) cell lines are the dominant industrial workhorses for therapeutic recombinant protein production. The availability of genome sequence of Chinese hamster and CHO cells will spur further genome and RNA sequencing of producing cell lines. However, the mammalian genomes assembled using shot-gun sequencing data still contain regions of uncertain quality due to assembly errors. Identifying high confidence regions in the assembled genome will facilitate its use for cell engineering and genome engineering. We assembled two independent drafts of Chinese hamster genome by de novo assembly from shotgun sequencing reads and by re-scaffolding and gap-filling the draft genome from NCBI for improved scaffold lengths and gap fractions. We then used the two independent assemblies to identify high confidence regions using two different approaches. First, the two independent assemblies were compared at the sequence level to identify their consensus regions as "high confidence regions" which accounts for at least 78 % of the assembled genome. Further, a genome wide comparison of the Chinese hamster scaffolds with mouse chromosomes revealed scaffolds with large blocks of collinearity, which were also compiled as high-quality scaffolds. Genome scale collinearity was complemented with EST based synteny which also revealed conserved gene order compared to mouse. As cell line sequencing becomes more commonly practiced, the approaches reported here are useful for assessing the quality of assembly and potentially facilitate the engineering of cell lines.

Antagonistic Donor Density Effect Conserved in Multiple Enterococcal Conjugative Plasmids.

UNLABELLED: Enterococcus faecalis, a common causative agent of hospital-acquired infections, is resistant to many known antibiotics. Its ability to acquire and transfer resistance genes and virulence determinants through conjugative plasmids poses a serious concern for public health. In some cases, induction of transfer of E. faecalis plasmids results from peptide pheromones produced by plasmid-free recipient cells, which are sensed by the plasmid-bearing donor cells. These plasmids generally encode an inhibitory peptide that competes with the pheromone and suppresses self-induction of donors. We recently demonstrated that the inhibitor peptide encoded on plasmid pCF10 is part of a unique quorum-sensing system in which it functions as a "self-sensing signal," reducing the response to the pheromone in a density-dependent fashion. Based on the similarities between regulatory features controlling conjugation in pAD1 and pAM373 and those controlling conjugation in pCF10, we hypothesized that these plasmids are likely to exhibit similar quorum-sensing behaviors. Experimental findings indicate that for both pAD1 and pAM373, high donor densities indeed resulted in decreased induction of the conjugation operon and reduced conjugation frequencies. This effect was restored by the addition of exogenous inhibitor, confirming that the inhibitor serves as an indicator for donor density. Donor density also affects cross-species conjugative plasmid transfer. Based on our experimental results, we propose models for induction and shutdown of the conjugation operon in pAD1 and pAM373. IMPORTANCE: Enterococcus faecalis is a leading cause of hospital-acquired infections. Its ability to transfer antibiotic resistance and virulence determinants by sharing its genetic material with other bacteria through direct cell-cell contact via conjugation poses a serious threat. Two antagonistic signaling peptides control the transfer of plasmids pAD1 and pAM373: a peptide pheromone produced by plasmid-free recipients triggers the conjugative transfer in plasmid-containing donors, and an inhibitor peptide encoded on the plasmid and produced by donor cells serves to modulate the donor response in accordance with the relative abundance of donors and recipients. We demonstrate that high donor density reduces the conjugation frequency of both of these plasmids, which is a consequence of increased inhibitor concentration in high-donor-density cultures. While most antibiotic strategies end up selecting resistant strains and disrupting the community balance, manipulating bacterial signaling mechanisms can serve as an alternate strategy to prevent the spread of antibiotic resistance.

Transcriptomic analysis of cobalt stress in the marine yeast Debaryomyces hansenii.

The yeast Debaryomyces hansenii overproduces riboflavin upon exposure to subtoxic levels of cobalt (Co(+2)). However, mechanisms for survival have yet to be studied and have been hindered by D. hansenii's high genetic heterogeneity among strains. In this study, we used transcriptomic analyses and RNA-seq in order to identify differentially expressed genes in D. hansenii in response to cobalt exposure. Highly upregulated genes under this condition were identified to primarily comprise DNA damage and repair genes, oxidative stress response genes, and genes for cell wall integrity and growth. The main response of D. hansenii to heavy metal stress is the activation of non-enzymatic oxidative stress response mechanisms and control of biological production of reactive oxygen species. Our results indicate that D. hansenii does not seem to be pre-adapted to survive high concentrations of heavy metals. These organisms appear to possess genetic survival and detoxification mechanisms that enable the cells to recover from heavy metal stress.