Brad Snedecor

Application of dynamic flux balance analysis to an industrial Escherichia coli fermentation

We have developed a reactor-scale model of Escherichia coli metabolism and growth in a 1000 L process for the production of a recombinant therapeutic protein. The model consists of two distinct parts: (1) a dynamic, process specific portion that describes the time evolution of 37 process variables of relevance and (2) a flux balance based, 123-reaction metabolic model of E. coli metabolism. This model combines several previously reported modeling approaches including a growth rate-dependent biomass composition, maximum growth rate objective function, and dynamic flux balancing. In addition, we introduce concentration-dependent boundary conditions of transport fluxes, dynamic maintenance demands, and a state-dependent cellular objective. This formulation was able to describe specific runs with high-fidelity over process conditions including rich media, simultaneous acetate and glucose consumption, glucose minimal media, and phosphate depleted media. Furthermore, the model accurately describes the effect of process perturbations--such as glucose overbatching and insufficient aeration--on growth, metabolism, and titer.

Mechanisms of unintended amino acid sequence changes in recombinant monoclonal antibodies expressed in Chinese Hamster Ovary (CHO) cells.

An amino acid sequence variant is defined as an unintended amino acid sequence change and contributes to product heterogeneity. Recombinant monoclonal antibodies (MAbs) are primarily expressed from Chinese Hamster Ovary (CHO) cells using stably transfected production cell lines. Selections and amplifications with reagents such as methotrexate (MTX) are often required to achieve high producing stable cell lines. Since MTX is often used to generate high producing cell lines, we investigated the genomic mutation rates of the hypoxanthine–guanine phosphoribosyltransferase (HGPRT or HPRT) gene using a 6‐thioguanine (6‐TG) assay under various concentrations of MTX selection in CHO cells. Our results show that the 6‐TG resistance increased as the MTX concentration increased during stable cell line development. We also investigated low levels of sequence variants observed in two stable cell lines expressing different MAbs. Our data show that the replacement of serine at position 167 by arginine (S167R) in the light chain of antibody A (MAb‐A) was due to a genomic nucleotide sequence change whereas the replacement of serine at position 63 by asparagine (S63N) in the heavy chain of antibody B (MAb‐B) was likely due to translational misincorporation. This mistranslation is codon specific since S63N mistranslation is not detectable when the S63 AGC codon is changed to a TCC or TCT codon. Our results demonstrate that both a genomic nucleotide change and translational misincorporation can lead to low levels of sequence variants and mistranslation of serine to asparagine can be eliminated by substituting the TCC or TCT codon for the S63 AGC codon without impacting antibody productivity. Biotechnol. Bioeng. 2010;107: 163–171.

Fast identification of reliable hosts for targeted cell line development from a limited-genome screening using combined φC31 integrase and CRE-Lox technologies

The use of targeted integration (TI) in cell line development (CLD) usually introduces one copy of a recombinant gene into a predetermined transcriptionally active locus. This reduces the heterogeneity typically associated with traditional random integration (RI) CLD with regards to varied productivity and instability, resulting from diverse chromosomal influences, varied copy numbers, and repeat‐induced rearrangement. As such, TI CLD offers the hope of a predictable and consistent CLD process for establishing stable clones. However, given the low copy number, cell lines established from a TI CLD process tend to exhibit low productivity. Here, we describe our nonviral‐based approach for quickly establishing and identifying TI hosts from a limited genome screening. Importantly, the TI hosts identified are consistent and reliable in supporting the production of diverse antibodies regardless of antibody subclass (IgG1 vs. IgG4) or prior traditional CLD performance (relatively easy vs. difficult to express antibodies). Moreover, an approximately twofold increase in titer can be achieved by using a CRE recombinase‐mediated cassette exchange (RMCE) strategy with an exchange vector carrying two units of the antibody gene. Two RMCE hosts that were established were able to produce up to ∼1.7 and 2 g/L of antibodies in nonoptimized fed‐batch shake flask production cultures with chemically defined media. Potentially, this strategy may be applied to the production of bispecific antibodies with a fast turnaround time.

FX knockout CHO hosts can express desired ratios of fucosylated or afucosylated antibodies with high titers and comparable product quality.

During antibody dependent cell cytotoxicity (ADCC) the target cells are killed by monocytes and natural killer cells. ADCC is enhanced when the antibody heavy chains core N‐linked glycan lacks the fucose molecule(s). Several strategies have been utilized to generate fully afucosylated antibodies. A commonly used and efficient approach has been knocking out the FUT8 gene of the Chinese hamster ovary (CHO) host cells, which results in expression of antibody molecules with fully afucosylated glycans. However, a major drawback of the FUT8‐KO host is the requirement for undertaking two separate cell line development (CLD) efforts in order to obtain both primarily fucosylated and fully afucosylated antibody species for comparative studies in vitro and in vivo. Even more challenging is obtaining primarily fucosylated and FUT8‐KO clones with similar enough product quality attributes to ensure that any observed ADCC advantage(s) can be strictly attributed to afucosylation. Here, we report generation and use of a FX knockout (FXKO) CHO host cell line that is capable of expressing antibody molecules with either primarily fucosylated or fully afucosylated glycan profiles with otherwise similar product quality attributes, depending on addition of fucose to the cell culture media. Hence, the FXKO host not only obviates the requirement for undertaking two separate CLD efforts, but it also averts the need for screening many colonies to identify clones with comparable product qualities. Finally, FXKO clones can express antibodies with the desired ratio of primarily fucosylated to afucosylated glycans when fucose is titrated into the production media, to allow achieving intended levels of FcγRIII‐binding and ADCC for an antibody. Biotechnol. Bioeng. 2017;114: 632–644.

It's time to regulate: Coping with product-induced nongenetic clonal instability in CHO cell lines via regulated protein expression

Clonal instability and titer loss during Chinese hamster ovary (CHO) cell line development (CLD) has several underlying causes, the most prominent of which are DNA copy number loss and DNA silencing. However, in some cases, clonal instability is due to the toxicity of the therapeutic protein(s) that clones express. Unlike DNA copy number loss, which may occur in some clones or DNA silencing that is prevalent in certain regions of the genome, the hallmark of product induced clonal instability is its manifestation in all the selected clones. To circumvent such product induced clonal instability, we have developed a vector construct that utilizes a regulated protein expression system in which the constitutive expression of the target protein(s) is prevented unless doxycycline is added to the culture. We have then successfully used this system to express, at high titers, an antibody for which constitutive expression results in clonal instability perhaps due to intracellular accumulation of the antibody. Our data shows that unlike the constitutively expressed or continuously induced clones, uninduced clones do not display instability. Furthermore, maintaining the uninduced clones in culture for months or subjecting them to freeze‐thaws did not have any effects on their titers. All together, our findings suggest that a regulated expression system could be suitable for production of difficult proteins that trigger instability.

Slashing the timelines: Opting to generate high-titer clonal lines faster via viability-based single cell sorting.

Chinese hamster ovary (CHO) cell line development (CLD) is a long and laborious process, which requires up to 5 − 6 months in order to generate and bank CHO lines capable of stably expressing therapeutic molecules. Additionally, single cell cloning of these production lines is also necessary to confirm clonality of the production lines. Here we introduce the utilization of viability staining dye in combination with flow cytometer to isolate high titer clones from a pool of selected cells and single cell deposit them into the wells of culture plates. Our data suggests that a stringent selection procedure along with viability dye staining and flow cytometry‐based sorting can be used to isolate high expressing clones with titers comparable to that of traditional CLD methods. This approach not only requires less labor and consumables, but it also shortens CLD timelines by at least 3 weeks. Furthermore, single cell deposition of selected cells by a flow sorter can be regarded as an additional clonality assurance factor that in combination with Day 0 imaging can ensure clonality of the production lines.

Resilient immortals, characterizing and utilizing Bax/Bak deficient Chinese hamster ovary (CHO) cells for high titer antibody production

Cell death due to apoptosis is frequently observed in large‐scale manufacturing of therapeutic proteins, and can reduce product accumulation in bioreactors. Several different strategies that involve overexpression of antiapoptotic or downregulation of proapoptotic proteins have been designed in attempt to curb this problem in Chinese hamster ovary (CHO) cell culture. However, each of these designs has their own shortcomings and limits, rendering them ineffective for large‐scale protein production. Recently, we have reported generation of a Bax and Bak deficient dhfr−/− CHO cell line using zinc‐finger nucleases. Here we demonstrate that puromycin, but not methotrexate, selection can be used to generate antibody‐expressing Bax and Bak deficient clones that are not only resistant to apoptosis, but that can also achieve higher titers relative to parental CHO cells due to higher cell density. Additionally, we show that Bax and Bak deficient cells have more mitochondria with healthy membrane potential, an attribute that perhaps contributes to their more potent growth compared to parental cells. Bax and Bak deficient cells do not readily apoptose, as shown by the ability to withstand high concentrations of apoptosis inducing agents, such as sodium butyrate, without a reduction in viability, growth, or titer. These traits render Bax and Bak deficient cells a potentially attractive host for production of therapeutic proteins at industrial scale.

Cell culture efforts to reduce glycation in recombinant humanized antibody

Background Glycation is a common post-translational modification of proteins, resulting from the chemical reaction between reducing sugars such as glucose and the primary amino groups on protein [1]. This non-enzymatic glycosylation reaction generates structural heterogeneity in recombinant IgG1 antibodies produced by cell culture processes [2]. Recent analytical characterization of a full-length humanized antibody secreted by Chinese Hamster Ovary (CHO) cells revealed that glycation of this protein occurs predominantly at lysine 49 on the light chain of the antibody [3]. This finding contrasts with historical data that have suggested that glycation sites are typically located randomly at all accessible lysine residues distributed over the entire molecule [2,3].

Probing the importance of clonality: Single cell subcloning of clonally derived CHO cell lines yields widely diverse clones differing in growth, productivity, and product quality

In the past few decades, a large variety of therapeutic antibodies and proteins have been expressed in Chinese hamster ovary (CHO) cells. This mammalian expression system is robust, scalable, relatively inexpensive, and importantly allows for post‐translational modifications that are important for some therapeutic proteins. Historically, CHO cell lines were derived from colonies of cells grown in semi‐solid or liquid plates using either serum‐containing or serum‐free media. Current advancements in cell sorting and imaging technologies have allowed for isolating and imaging single cell progenitors at the seeding step, significantly increasing the probability of isolating clonally derived cell lines. However, it is debatable how much population heterogeneity can be eliminated when clonally derived cell lines, originated from a single cell progenitor, are scaled up. To further investigate this phenomenon, we subcloned two different clonally derived (day 0 imaged and visually inspected) cell lines expressing antibody‐X. The results showed that when six randomly chosen subclones of each line were evaluated in a production assay, these subclones displayed a range of variation in titer, specific productivity, growth, and product quality attributes. Some subclones displayed variations in transgene copy numbers. Additionally, clonal derivation did not assure stability of the derived cell lines. Our findings show that cell heterogeneity exists in a population even when derived from a single cell progenitor.

Development and characterization of an automated imaging workflow to generate clonally-derived cell lines for therapeutic proteins

In the development of biopharmaceutical products, the expectation of regulatory agencies is that the recombinant proteins are produced from a cell line derived from a single progenitor cell. A single limiting dilution step followed by direct imaging, as supplemental information, provides direct evidence that a cell line originated from a single progenitor cell. To obtain this evidence, a high‐throughput automated imaging system was developed and characterized to consistently ensure that cell lines used for therapeutic protein production are clonally‐derived. Fluorescent cell mixing studies determined that the automated imaging workflow and analysis provide ∼95% confidence in accurately and precisely identifying one cell in a well. Manual inspection of the images increases the confidence that the cell line was derived from a single‐cell to >99.9%.