Recombinant adeno-associated virus production evaluation in Chinese hamster ovary cells.

The gene therapy field has advanced in recent years with five recombinant adeno-associated virus (rAAV) based products winning Food and Drug Administration (FDA) approval. As the number of therapeutic applications and overall production demands for rAAV increase, it is valuable to evaluate rAAV production in different production cells. Chinese hamster ovary (CHO) cells have been a robust host for biomolecule manufacturing for more than 35 years. However, there is no report to our knowledge describing the use of CHO cells for rAAV production. In this study, we examined the ability of CHO cells to produce rAAV using a transient plasmid transfection approach. Our results demonstrated that CHO is capable of producing rAAV with detectable viral fundamental components including viral RNAs, proteins, and rAAV viral particles. We identified the expression of cap proteins as one of the limiting factors for rAAV production in CHO cells. We therefore added an additional cytomegalovirus (CMV)-Cap plasmid to the CHO transfection. After increasing cap protein expression, we detected rAAV titers as high as 3 × 108 viral genomes for every 2 × 109 capsids in CHO cells using a quintuple transfection method (standard AAV2 Rep/Cap, helper, gene of interest plasmids, plus CMV-E1, and CMV-Cap plasmids) with comparable full particle percent (average 15%) to that of human embryo kidney (HEK)-derived rAAV. Our study provides a foundation for potential rAAV production in CHO cells.

A high cell density transient transfection system for therapeutic protein expression based on a CHO GS-knockout cell line: process development and product quality assessment.

Transient gene expression (TGE) is a rapid method for the production of recombinant proteins in mammalian cells. While the volumetric productivity of TGE has improved significantly over the past decade, most methods involve extensive cell line engineering and plasmid vector optimization in addition to long fed batch cultures lasting up to 21 days. Our colleagues have recently reported the development of a CHO K1SV GS-KO host cell line. By creating a bi-allelic glutamine synthetase knock out of the original CHOK1SV host cell line, they were able to improve the efficiency of generating high producing stable CHO lines for drug product manufacturing. We developed a TGE method using the same CHO K1SV GS-KO host cell line without any further cell line engineering. We also refrained from performing plasmid vector engineering. Our objective was to setup a TGE process to mimic protein quality attributes obtained from stable CHO cell line. Polyethyleneimine (PEI)-mediated transfections were performed at high cell density (4 × 10(6) cells/mL) followed by immediate growth arrest at 32 °C for 7 days. Optimizing DNA and PEI concentrations proved to be important. Interestingly, found the direct transfection method (where DNA and PEI were added sequentially) to be superior to the more common indirect method (where DNA and PEI are first pre-complexed). Moreover, the addition of a single feed solution and a polar solvent (N,N dimethylacetamide) significantly increased product titers. The scalability of process from 2 mL to 2 L was demonstrated using multiple proteins and multiple expression volumes. Using this simple, short, 7-day TGE process, we were able to successfully produce 54 unique proteins in a fraction of the time that would have been required to produce the respective stable CHO cell lines. The list of 54 unique proteins includes mAbs, bispecific antibodies, and Fc-fusion proteins. Antibody titers of up to 350 mg/L were achieved with the simple 7-day process. Titers were increased to 1 g/L by extending the culture to 16 days. We also present two case studies comparing product quality of material generated by transient HEK293, transient CHO K1SV GS-KO, and stable CHO K1SV KO pool. Protein from transient CHO was more representative of stable CHO protein compared to protein produced from HEK293.

Transcriptional and post-transcriptional targeting for enhanced transient gene expression in CHO cells.

OBJECTIVE: To develop a simple approach to increase titers of transient gene expression in CHO cells without relying on host cell line engineering as recent reports suggest that for PEI-mediated transfections, under optimized conditions, DNA delivery into cells and nuclei is not the limiting factor. RESULTS: N, N-Dimethyl acetamide (DMA) was utilized to enhance transcription. To target post-transcriptional events, we evaluated the co-expression of various genes involved in the unfolded protein response, namely XBP1S, ATF4, CHOP and HSPA5. XBP1S overexpression led to a 15-85 % increase in titer for multiple therapeutic proteins. Mechanistic studies confirmed that addition of 0.125 % DMA increased transgene mRNA levels as expected. However, overexpression of XBP1S had no effect on transgene mRNA levels, indicating that it influenced post-transcriptional events. Since DMA and XBP1S targeted different pathways, the combination of the two approaches led to an additive improvement in protein titer (150-250 % titer increase). CONCLUSION: Transcriptional and post-transcriptional pathways of transient gene expression can be targeted to increase titers without resorting to host cell line engineering in a simple, short, 7 day production process.

Gene copy number, gene configuration and LC/HC mRNA ratio impact on antibody productivity and product quality in targeted integration CHO cell lines.

The augmentation of transgene copy numbers is a prevalent approach presumed to enhance transcriptional activity and product yield. CHO cell lines engineered via targeted integration (TI) offer an advantageous platform for investigating the interplay between gene copy number, mRNA abundance, product yield, and product quality. Our investigation revealed that incrementally elevating the gene copy numbers of both IgG heavy chain (HC) and light chain (LC) concurrently resulted in the attainment of plateaus in mRNA levels and product titers, notably occurring beyond four to five gene copies integrated at the same TI site. Furthermore, maintaining a fixed gene copy number while varying the position of genes within the vector influenced the LC/HC mRNA ratio, which subsequently exerted a substantial impact on product titer. Moreover, manipulation of the LC/HC gene ratio through the introduction of surplus LC gene copies led to heightened LC mRNA expression and a reduction in the levels of high molecular weight species. It is noteworthy that the effects of excess LC on product titer were dependent on the specific molecule under consideration. The strategic utilization of PCR tags enabled precise quantification of transcription from each expression slot within the vector, facilitating the identification of highly expressive and less expressive slots. Collectively, these findings significantly enhance our understanding of stable antibody production in TI CHO cell lines.

Utilizing targeted integration CHO pools to potentially accelerate the GMP manufacturing of monoclonal and bispecific antibodies.

Monoclonal antibodies (mAbs) are effective therapeutic agents against many acute infectious diseases including COVID-19, Ebola, RSV, Clostridium difficile, and Anthrax. mAbs can therefore help combat a future pandemic. Unfortunately, mAb development typically takes years, limiting its potential to save lives during a pandemic. Therefore "pandemic mAb" timelines need to be shortened. One acceleration tool is "deferred cloning" and leverages new Chinese hamster ovary (CHO) technology based on targeted gene integration (TI). CHO pools, instead of CHO clones, can be used for Phase I/II clinical material production. A final CHO clone (producing the mAb with a similar product quality profile and preferably with a higher titer) can then be used for Phase III trials and commercial manufacturing. This substitution reduces timelines by ~3 months. We evaluated our novel CHO TI platform to enable deferred cloning. We created four unique CHO pools expressing three unique mAbs (mAb1, mAb2, and mAb3), and a bispecific mAb (BsAb1). We then performed single-cell cloning for mAb1 and mAb2, identifying three high-expressing clones from each pool. CHO pools and clones were inoculated side-by-side in ambr15 bioreactors. CHO pools yielded mAb titers as high as 10.4 g/L (mAb3) and 7.1 g/L (BsAb1). Subcloning yielded CHO clones expressing higher titers relative to the CHO pools while yielding similar product quality profiles. Finally, we showed that CHO TI pools were stable by performing a 3-month cell aging study. In summary, our CHO TI platform can increase the speed to clinic for a future "pandemic mAb."

Strategies to improve CHO cell culture performance: Targeted deletion of amino acid catabolism and apoptosis genes paired with growth inhibitor supplementation.

Chinese hamster ovary (CHO) cells are the predominant host of choice for recombinant monoclonal antibody (mAb) expression. Recent advancements in gene editing technology have enabled engineering new CHO hosts with higher growth, viability, or productivity. One approach involved knock out (KO) of BCAT1 gene, which codes for the first enzyme in the branched chain amino acid (BCAA) catabolism pathway; BCAT1 KO reduced accumulation of growth inhibitory short chain fatty acid (SCFA) byproducts and improved culture growth and titer when used in conjunction with high-end pH-controlled delivery of glucose (HiPDOG) technology and SCFA supplementation during production. Accumulation of SCFAs in the culture media is critical for metabolic shift toward higher specific productivity and hence titer. Here we describe knocking out BCKDHa/b genes (2XKO), which act downstream of the BCAT1, in a BAX/BAK KO CHO host cell line background to reduce accumulation of growth-inhibitory molecules in culture. Evaluation of the new 4XKO CHO cell lines in fed-batch production cultures (without HiPDOG) revealed that partial KO of BCKDHa/b genes in an apoptosis-resistant (BAX/BAK KO) background can achieve higher viabilities and mAb titers. This was evident when SCFAs were added to boost productivity as such additives negatively impacted culture viability in the WT but not BAX/BAK KO cells during batch production. Altogether, our findings suggest that SCFA addbacks can significantly increase productivity and mAb titers in the context of apoptosis-attenuated CHO cells with partial KO of BCAA genes. Such engineered CHO hosts can offer productivity advantages for expressing biotherapeutics in an industrial setting.

SPEED-MODE cell line development (CLD): Reducing Chinese hamster ovary (CHO) CLD timelines via earlier suspension adaptation and maximizing time spent in the exponential growth phase.

Chinese hamster ovary (CHO) cells are the preferred system for expression of therapeutic proteins and the majority of all biotherapeutics are being expressed by these cell lines. CHO expression systems are readily scalable, resistant to human adventitious agents, and have desirable post-translational modifications, such as glycosylation. Regardless, drug development as a whole is a very costly, complicated, and time-consuming process. Therefore, any improvements that result in reducing timelines are valuable and can provide patients with life-saving drugs earlier. Here we report an effective method (termed SPEED-MODE, herein) to speed up the Cell line Development (CLD) process in a targeted integration (TI) CHO CLD system. Our findings show that (1) earlier single cell cloning (SCC) of transfection pools, (2) speeding up initial titer screening turnaround time, (3) starting suspension adaptation of cultures sooner, and (4) maximizing the time CHO cultures spend in the exponential growth phase can reduce CLD timelines from ~4 to ~3 months. Interestingly, SPEED-MODE timelines closely match the theoretical minimum timeline for CHO CLD assuming that CHO cell division is the rate limiting factor. Clones obtained from SPEED-MODE CLD yielded comparable titer and product quality to those obtained via a standard CLD process. Hence, SPEED-MODE CLD is advantageous for manufacturing biotherapeutics in an industrial setting as it can significantly reduce CLD timelines without compromising titer or product quality.

Chromatin Accessibility Plays an Important Epigenetic Role on Antibody Expression From CMV Promoter and DNA Elements Flanking the CHO TI Host Landing-Pad.

Targeted integration (TI) Chinese hamster ovary (CHO) platforms are commonly used for protein expression. However, the impact of epigenetic modifications on protein expression in TI cell lines remains elusive since almost all the epigenetic studies focus on random integration (RI) of the gene of interest and only within the promoter region. To address the impact of epigenetic modifications on TI CHO cells, we utilized a standard mAb-1 to identify and characterize TI clones with the same transgene copy numbers but different levels of transgene transcription and titer. Surprisingly, while CMV promoters were not methylated and histone acetylation/methylation was present, these epigenetic markers did not trend with mRNA transcription and protein expression in our TI model. Instead, ATAC-seq data analysis revealed that differences in chromatin accessibility within the TI site could be a major factor impacting these observed differences. However, neither chromatin accessibility nor histone acetylation/methylation profiles in early cultures were predictive of high-expressing clones early during the CLD process. Finally, modulation of the histone profiles (H3K27ac and H3K4me3) at the CMV promoters within the TI integration site using dCas9 fusion proteins was not effective in further increasing mAb titers which could have been likely due to interference of the dCas9 fusion proteins with transcription from the CMV promoters. Overall, our data suggests increasing chromatin accessibility at the TI site is the most effective way to increase mRNA transcription and hence, productivity in TI cell lines.

Combining regulated and constitutive protein expression significantly boosts protein expression by increasing productivity without affecting CHO cell growth.

Chinese hamster ovary (CHO) cells are commonly used for the expression of therapeutic proteins. To increase the titer output of CHO production cultures either specific productivity (Qp), growth, or both need to be increased. Generally, Qp and growth are inversely correlated and cell lines with high Qp have slower growth and vice versa. During the cell line development (CLD) process, the faster-growing cells tend to take over the culture and represent the majority of the isolated clones post single cell cloning. In this study, combinations of regulated and constitutive expression systems were used to supertransfect targeted integration (TI) cell lines expressing the same antibody either constitutively or under-regulated expression. Clone screening with a hybrid expression system (inducible + constitutive) allowed identification and selection of higher titer clones under uninduced conditions, without a negative impact on cell growth during clone selection and expansion. Induction of the regulated promoter(s) during the production phase increased the Qp without negatively affecting growth, resulting in approximately twofold higher titers (from 3.5 to 6-7 g/L). This was also confirmed using a 2-site TI host where the gene of interest was expressed inducibly from Site 1 and constitutively from Site 2. Our findings suggest that such a hybrid expression CLD system can be used to increase production titers, providing a novel approach for expression of therapeutic proteins with high titer market demands.

Expressing antigen binding fragments with high titers in a targeted integration CHO host by optimizing expression vector gene copy numbers and position: A case study.

Antigen binding fragments (Fab) are a promising class of therapeutics as they maintain high potency while having significantly smaller size relative to full-length antibodies. Because Fab molecules are aglycosylated, many expression platforms, including prokaryotic, yeast, and mammalian cells, have been developed for their expression, with Escherichia coli being the most commonly used Fab expression system. In this study, we have examined production of a difficult to express Fab molecule in a targeted integration (TI) Chinese Hamster Ovary (CHO) host. Without a need for extensive host or process optimization, as is usually required for E. coli, by simply using different vector configurations, clones with very high Fab expression titers were obtained. In this case, by increasing heavy chain (HC) gene copy numbers, clones with titers of up to 7.4 g/L in the standard fed-batch production culture were obtained. Our findings suggest that having a predetermined transgene integration site, as well as the option to optimize gene copy number/dosage, makes CHO TI hosts an effective system for expression of Fab molecules, allowing Fab expression using platform process and without significant process development efforts.

High-throughput screening and selection of yeast cell lines expressing monoclonal antibodies.

The methylotrophic yeast Pichia pastoris has recently been engineered to express therapeutic glycoproteins with uniform human N-glycans at high titers. In contrast to the current art where producing therapeutic proteins in mammalian cell lines yields a final product with heterogeneous N-glycans, proteins expressed in glycoengineered P. pastoris can be designed to carry a specific, preselected glycoform. However, significant variability exists in fermentation performance between genotypically similar clones with respect to cell fitness, secreted protein titer, and glycan homogeneity. Here, we describe a novel, multidimensional screening process that combines high and medium throughput tools to identify cell lines producing monoclonal antibodies (mAbs). These cell lines must satisfy multiple selection criteria (high titer, uniform N-glycans and cell robustness) and be compatible with our large-scale production platform process. Using this selection process, we were able to isolate a mAb-expressing strain yielding a titer (after protein A purification) in excess of 1 g/l in 0.5-l bioreactors.

Development of a high cell density transient CHO platform yielding mAb titers greater than 2 g/L in only 7 days.

We developed a simple transient Chinese Hamster Ovary expression platform. Titers for a random panel of 20 clinical monoclonal antibodies (mAbs) ranged from 0.6 to 2.7 g/L after 7 days. Two factors were the key in obtaining these high titers. First, we utilized an extremely high starting cell density (20 million cells/ml), and then arrested further cell growth by employing mild hypothermic conditions (32°C). Second, we performed a 6-variable Design of Experiments to find optimal concentrations of plasmid DNA (coding DNA), boost DNA (DNA encoding the XBP1S transcription factor), transfection reagent (polyethylenimine [PEI]), and nutrient feed amounts. High coding DNA concentrations (12.5 mg/L) were found to be optimal. We therefore diluted expensive coding DNA with inexpensive inert filler DNA (herring sperm DNA). Reducing the coding DNA concentration by 70% from 12.5 to 3.75 mg/L did not meaningfully reduce mAb titers. Titers for the same panel of 20 clinical mAbs ranged from 0.7 to 2.2 g/L after reducing the coding DNA concentration to 3.75 mg/L. Finally, we found that titer and product quality attributes were similar for a clinical mAb (rituximab) expressed at very different scales (volumes ranging from 3 ml to 2 L).

Generation of High Expressing Chinese Hamster Ovary Cell Pools Using the Leap-In Transposon System.

Clonally derived cell lines (CDCL) from Chinese Hamster Ovary (CHO) host cell lines, remain the most popular method to manufacture therapeutic proteins. However, CHO cell pools are increasingly being used as an alternate method to produce therapeutic proteins for preclinical drug development in an effort to shorten the time required for new drug development. It is essential that these CHO pools exhibit the desired attributes of CHO CDCLs such as high protein titers and consistent product quality attributes (PQAs). In this study the authors evaluated the Leap-In Transposase®, for the expression of four different proteins (three mAbs and one Bispecific mAb). The resultant pool titers ranges from 2.0 to 5.0 g L-1 for the four proteins compared to 1.5-3.3 g L-1 from the respective control pools (generated by random gene integration). The resultant cell pools are a homogeneously expressing cell population. The average gene copy numbers are similar or lower in the evaluation pools relative to the control pools. The higher titers in the evaluation pools are attributed to higher levels of both IgG-LC and IgG-HC mRNA. In conclusion, the Leap-In transposase generates high titer, homogeneous CHO pools in a short time-period without introducing any undesired PQAs.

Polymer-mediated flocculation of transient CHO cultures as a simple, high throughput method to facilitate antibody discovery.

Most biopharmaceutical drugs, especially monoclonal antibodies (mAbs), bispecific antibodies (BsAbs) and Fc-fusion proteins, are expressed using Chinese Hamster Ovary (CHO) cell lines. CHO cells typically yield high product titers and high product quality. Unfortunately, CHO cell lines also generate high molecular weight (HMW) aggregates of the desired product during cell culture along with CHO host cell protein (HCP) and CHO DNA. These immunogenic species, co-purified during Protein A purification, must be removed in a multi-step purification process. Our colleagues have reported the use of a novel polymer-mediated flocculation step to simultaneously reduce HMW, HCP and DNA from stable CHO cell cultures prior to Protein A purification. The objective of this study was to evaluate this novel "smart polymer" (SmP) in a high throughput antibody discovery workflow using transiently transfected CHO cultures. SmP treatment of 19 different molecules from four distinct molecular categories (human mAbs, murine mAbs, BsAbs and Fabs) with 0.1% SmP and 25 mM stimulus resulted in minimal loss of monomeric protein. Treatment with SmP also demonstrated a variable, concentration-dependent removal of HMW aggregates after Protein A purification. SmP treatment also effectively reduced HCP levels at each step of mAb purification with final HCP levels being several fold lower than the untreated control. Interestingly, SmP treatment was able to significantly reduce high concentrations of artificially spiked levels of endotoxin in the cultures. In summary, adding a simple flocculation step to our existing transient CHO process reduced the downstream purification burden to remove impurities and improved final product quality. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1393-1400, 2017.

Evaluation of piggyBac-mediated CHO pools to enable material generation to support GLP toxicology studies.

Generating purified protein for GLP toxicology studies (GLP-Tox) represents an important and often rate limiting step in the biopharmaceutical drug development process. Toxicity testing requires large amounts of therapeutic protein (>100 g), typically produced in a single 500-2,500 L bioreactor, using the final CHO clonally derived cell line (CDCL). One approach currently used to save time is to manufacture GLP-Tox material using pools of high-producing CHO CDCLs instead of waiting for the final CDCL. Recently, we reported CHO pools producing mAb titers >7 g/L using piggyBac-mediated gene integration (PB CHO pools). In this study, we wanted to leverage high titer PB CHO pools to produce GLP-Tox material. A detailed product quality attribute (PQA) assessment was conducted comparing PB CHO pools to pooled Top4 CDCLs. Four mAbs were evaluated. First, we found that PB CHO pools expressed all four mAbs at high titers (2.8-4.4 g/L in shake flasks). Second, all four PB CHO pools were aged to 55 generations (Gen). All four PB CHO Pools were found to be suitable over 55 Gen. Finally, we performed bioreactor scale-up. PB CHO pool titers (3.7-4.8 g/L) were similar or higher than the pooled Top 4 CDCLs in 5 L bioreactors (2.4-4.1 g/L). The PQAs of protein derived from PB CHO pools were very similar to pooled Top 4 CHO CDCLs according to multiple orthogonal techniques including peptide mapping analysis. Taken together, these results demonstrate the technical feasibility of using PB CHO pools to manufacture protein for GLP-Tox. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1436-1448, 2017.

Transient and stable CHO expression, purification and characterization of novel hetero-dimeric bispecific IgG antibodies.

IgG bispecific antibodies (BsAbs) represent one of the preferred formats for bispecific antibody therapeutics due to their native-like IgG properties and their monovalent binding to each target. Most reported studies utilized transient expression in HEK293 cells to produce BsAbs. However, the expression of biotherapeutic molecules using stable CHO cell lines is commonly used for biopharmaceutical manufacturing. Unfortunately, limited information is available in the scientific literature on the expression of BsAbs in CHO cell lines. In this study we describe an alternative approach to express the multiple components of IgG BsAbs using a single plasmid vector (quad vector). This single plasmid vector contains both heavy chain genes and both light chain genes required for the expression and assembly of the IgG BsAb, along with a selectable marker. We expressed, purified, and characterized four different IgG BsAbs or "hetero-mAbs" using transient CHO expression and stable CHO minipools. Transient CHO titers ranged from 90 to 160 mg/L. Stable CHO titers ranged from 0.4 to 2.3 g/L. Following a simple Protein A purification step, the percentage of correctly paired BsAbs ranged from 74% to 98% as determined by mass spectrometry. We also found that information generated from transient CHO expression was similar to information generated using stable CHO minipools. In conclusion, the quad vector approach represents a simple, but effective, alternative approach for the generation of IgG BsAbs in both transient CHO and stable CHO expression systems. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:469-477, 2017.

Bioreactor scale up and protein product quality characterization of piggyBac transposon derived CHO pools.

Chinese hamster ovary (CHO) cells remain the most popular host for the production of biopharmaceutical drugs, particularly monoclonal antibodies (mAbs), bispecific antibodies, and Fc-fusion proteins. Creating and characterizing the stable CHO clonally-derived cell lines (CDCLs) needed to manufacture these therapeutic proteins is a lengthy and laborious process. Therefore, CHO pools have increasingly been used to rapidly produce protein to support and enable preclinical drug development. We recently described the generation of CHO pools yielding mAb titers as high as 7.6 g/L in a 16 day bioprocess using piggyBac transposon-mediated gene integration. In this study, we wanted to understand why the piggyBac pool titers were significantly higher (2-10 fold) than the control CHO pools. Higher titers were the result of a combination of increased average gene copy number, significantly higher messenger RNA levels and the homogeneity (i.e. less diverse population distribution) of the piggyBac pools, relative to the control pools. In order to validate the use of piggyBac pools to support preclinical drug development, we then performed an in-depth product quality analysis of purified protein. The product quality of protein obtained from the piggyBac pools was very similar to the product quality profile of protein obtained from the control pools. Finally, we demonstrated the scalability of these pools from shake flasks to 36L bioreactors. Overall, these results suggest that gram quantities of therapeutic protein can be rapidly obtained from piggyBac CHO pools without significantly changing product quality attributes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:534-540, 2017.

The metabolic effects of GDF15 are mediated by the orphan receptor GFRAL.

Growth/differentiation factor 15 (GDF15), also known as MIC-1, is a distant member of the transforming growth factor-ß (TGF-ß) superfamily and has been implicated in various biological functions, including cancer cachexia, renal and heart failure, atherosclerosis and metabolism. A connection between GDF15 and body-weight regulation was initially suggested on the basis of an observation that increasing GDF15 levels in serum correlated with weight loss in individuals with advanced prostate cancer. In animal models, overexpression of GDF15 leads to a lean phenotype, hypophagia and other improvements in metabolic parameters, suggesting that recombinant GDF15 protein could potentially be used in the treatment of obesity and type 2 diabetes. However, the signaling and mechanism of action of GDF15 are poorly understood owing to the absence of a clearly identified cognate receptor. Here we report that GDNF-family receptor α-like (GFRAL), an orphan member of the GFR-α family, is a high-affinity receptor for GDF15. GFRAL binds to GDF15 in vitro and is required for the metabolic actions of GDF15 with respect to body weight and food intake in vivo in mice. Gfral-/- mice were refractory to the effects of recombinant human GDF15 on body-weight, food-intake and glucose parameters. Blocking the interaction between GDF15 and GFRAL with a monoclonal antibody prevented the metabolic effects of GDF15 in rats. Gfral mRNA is highly expressed in the area postrema of mouse, rat and monkey, in accordance with previous reports implicating this region of the brain in the metabolic actions of GDF15 (refs. 4,5,6). Together, our data demonstrate that GFRAL is a receptor for GDF15 that mediates the metabolic effects of GDF15.

Generation of stable Chinese hamster ovary pools yielding antibody titers of up to 7.6 g/L using the piggyBac transposon system.

Chinese hamster ovary (CHO) cells remain the default production host for many biopharmaceutical drugs, particularly monoclonal antibodies (mAb). Production of gram and kilogram quantities of protein typically requires the generation of stable CHO clones. Unfortunately, this process takes several months, significantly slowing down the drug discovery and development process. Therefore, improved technologies are needed to accelerate biopharmaceutical drug discovery and final drug substance manufacturing. In this study, we describe the generation of stable CHO pools using the piggyBac transposon system. We evaluated the system using four model antibody molecules (3 mAbs and 1 bispecific Ab). Stable CHO pools were isolated in 7-12 days. Using a simple 16-day fed-batch process, we measured titers ranging from 2.3 to 7.6 g/L for the four model antibodies. This represented a 4- to 12-fold increase relative to the controls. Additionally, we isolated stable CHO clones. We found that the stable CHO clones isolated from the piggyBac transposon pools yielded titers two to threefold higher relative to the control clones. Taken together, these results suggest that stable CHO pool and clone generation can be significantly improved by using the piggyBac transposon system. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1301-1307, 2016.

High-throughput mAb expression and purification platform based on transient CHO.

A high-cell-density transient transfection system was recently developed in our laboratory based on a CHO-GS-KO cell line. This method yields monoclonal antibody titers up to 350 mg/L from a simple 7-day process, in volumes ranging from 2 mL to 2 L. By performing transfections in 24-deep-well plates, a large number of mAbs can be expressed simultaneously. We coupled this new high-throughput transfection process to a semiautomated protein A purification process. Using a Biomek FX(p) liquid handling robot, up to 72 unique mAbs can be simultaneously purified. Our primary goal was to obtain >0.25 mg of purified mAb at a concentration of >0.5 mg/mL, without any concentration or buffer-exchange steps. We optimized both the batch-binding and the batch elution steps. The length of the batch-binding step was important to minimize mAb losses in the flowthrough fraction. The elution step proved to be challenging to simultaneously maximize protein recovery and protein concentration. We designed a variable volume elution strategy based on the average supernatant titer. Finally, we present two case studies. In the first study, we produced 56 affinity maturation mAb variants at an average yield of 0.33 ± 0.05 mg (average concentration of 0.65 ± 0.10 mg/mL). In a second study, we produced 42 unique mAbs, from an early-stage discovery effort, at an average yield of 0.79 ± 0.31 mg (average concentration of 1.59 ± 0.63 mg/mL). The combination of parallel high-yielding transient transfection and semiautomated high-throughput protein A purification represents a valuable mAb drug discovery tool.