Cell-line/Process Development - BPI Theater @ BIO 2015

Ray Price (senior director of business development, DiscoveRx) 3:30–3:55 pm

Advances in Research Tools to Accelerate Drug Development

Price introduced the BioSeek drug-discovery platform with examples. The technology is built on three pillars: primary human cells; models that use growth factors or cytokines to model a disease environment and then predict how drugs change biomarker responses in those systems; and comparisons of generated profiles with a reference database of more than 4,000 compounds. DiscoveRx uses that database and informatics tools to predict clinical outcomes.

To be as physiologically relevant as possible, the company generates cultures of endothelial cells that produce factors and peripheral blood mononuclear cells (PBMCs) and that respond to those factors. These human primary cells mimic what happens in vivo, providing for disease-relevant assays. Often this is the first time that a client’s molecule has been tested in a primary cell model. Designed to be quantitative, robust, and reproducible, the assays generally use protein-based biomarker readouts, but the company can build custom versions for microRNA profiling, flow cytometry, and so on. The systems are validated with benchmark drugs and compounds.

DiscoveRx works with different cell types: endothelial cells, immune cells, microphages, fibroblasts, and epithelial cells — many of which can be cocultured. For example, endothelial cells and PBMCs can be stimulated to model an inflammatory environment with TH1 cytokines. Results from a broad panel form a biomap profile. Price showed an example for prednisolone, a synthetic glucocorticoid or steroid. Protein-based biomarkers are used for their physiological and translatable activities. The company also measures cytotoxicity and proliferation. Assays use methionine sulfoximine (MSO) cell treatment as a control.

Pointing to the prednisolone example, Price showed how the profile shows the magnitude of changes in biomarker expression increasing with higher doses as the overall profile shape remains consistent. That’s what he calls a signature profile for prednisolone. Similar drugs might share some aspects of that profile, as when clients’ products are matched to the reference database. He then showed a broader example of P38 MAP kinase inhibitors at different concentrations using Pearson product-moment correlation to measure similarity. That allows the company to identify common mechanisms of action.

The BioSeek platform thus provides drug developers with an ability to distinguish single-target selected molecules, pathway modulators, and broad cell-based activities. Price went through three examples of different applications of this technology in biologics, beginning by comparing anti- TNF α (tumor necrosis factor alpha) antibodies — Enbrel, Humira, and Remicade at 1 µg/mL concentration — as a client might do in positioning a biosimilar or biobetter product. He also compared monovalent and bivalent Fab fragments of Remicade at different concentrations. The profiles differed in illustrated ability to inhibit TNF and to affect T-cell function. Price suggested the conclusion that monovalent TNF inhibitors were less immunosuppressive than bivalent forms. Improving on the selectivity or pharmacology of an existing molecule can be an important aspect of a subsequent clinical development plan.

Another example came from an oncology program. Fibroblasts or endothelial cells are cocultured with immune cells and a HD-29 tumor cell line to simulate a tumor microenvironment. Then a drug (biologic or small molecule) is added and the system stimulated to create a modest inflammatory response. In a recent project, the company tested an anti-PD-1 antibody obtained from a clinical pharmacy at concentrations of from 50 µg/mL down to 1.8 µg/mL. Results are consistent with those seen clinically: increases in biomarkers that reflect inflammatory or immune response.

Price’s third example involved small molecules (nintedanib and pirfenidone) and cystic fibrosis. DiscoveRx used renal or small-airway epithelial cells cocultured with airway fibroblasts and stimulated to cause differentiation into myofibroblasts, then profiled the biomarkers that reflected that transformation. The company then measured how the drugs (and a DMSO control) induced biomarker changes, determining which activities revealed an antifibrotic signature and those that affect inflammation. The hypothesis is that drugs are more likely to be successful if they induce both activities. Price then showed a design of experiments (DoE) matrix approach showing how drugs interact, producing what he called a Combo ELECT profile. It can be used to compare small molecules and biologics, stimuli and test agents, and so on.

An audience member commented on the similarity of the drugs in the example comparisons. “Were you ever surprised that they were structurally divergent or that they didn’t invoke other systems differentially?” Price confirmed that his company has seen just that. It’s common, he said, to find activities that are a surprise or unsuspected by clients especially in broader panels.

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Karen Fallen (vice president and business unit head, clinical development and licensing, Lonza) 1:30–1:55 pm

Using Miniaturization and Automation to Improve Cell-line Development

Fallen spoke about automation for improving cell-line development. The Lonza approach is to set up discrete islands of automation rather than build one large, fully integrated system.

The company is a supplier to the pharmaceutical and biotechnology industries and also makes specialty ingredients. The global organization, headquartered in Basel, Switzerland, has more than 40 sites and about 10,000 employees.

Fallen talked about Lonza’s biologics facilities. The mammalian cell culture facility is in Slough, UK, where the company performs process development and clinical manufacturing. Larger facilities for commercial-scale manufacturing are located in Portsmouth, NH (with bioreactors up to 20,000 L); Porrino, Spain (with 10,000-L bioreactors); and Singapore, where process development, clinical manufacture, and commercial manufacture are performed from very small scale-up to 20,000-L reactors. The Slough and Singapore sites were the focus of Fallen’s presentation about islands of automation.

Automation islands focus on discrete, stand-alone activities that are then linked to form what she referred to as an archipelago. One justification for approaching automation this way is the need to deal with nonlinear work flows, which necessitates performing certain activities in parallel. This saves time and allows the same piece of equipment to be used in different parts of a process — so flexibility is another advantage to this approach. Such a process becomes more resilient and robust because if individual steps along the process have problems, then isolated pieces of automation can be moved — rather than having to destroy an entire platform.

The small islands enable individual components to be changed out more easily than when dealing with one big machine, allowing for maintenance of regulatory compliance and implementation of scientific improvements. System capabilities (such as an additional cell-screening step) can be introduced quickly. And automation reduces the chance of human errors, especially when minor operational changes need to be introduced.

The three archipelagos in this system handle cell- line development, process evaluation, and purification development, all tightly linked by analytics. So automated analytics are also needed to support those processes. Another aspect of flexibility is that individual islands can be housed in different development laboratories but linked by the actual transfer of information and materials. As an example, Fallen detailed the evolution of the company’s cell-line development archipelago, for which automated processes include those for clone picking, culturing in deep-well plates, and operation of a miniature bioreactor system. The system has evolved over the past six or seven years from a single automated imaging platform, with additional time and effort into building the analytical islands to support process development. Previously the overall process time line (which included four rounds of screening) took a labor-intensive 20–23 weeks.

Two years ago, the company moved toward a semiautomated system with the launch of its next-generation GS Xceed gene expression system — providing up to a six-week reduction in cell-line development. It allowed Lonza to increase the number of cell lines that could be generated and screened through the process from 200 to 1,000, reduce the rounds of screening down to three, and shorten the time line from 23 to 17 weeks.

A second productivity screen using a fed-batch culture also increased the number of cell lines evaluated to about nine using the ambr bioreactor system (Sartorius Stedim Biotech). The current cell-line construction workflow uses Lonza’s flexible Light Path services, which streamlines custom material supply by incorporating ambr miniature bioreactors at an earlier point in the process. This then reduced screening rounds to two, shortening the timeline from 17 to 12 weeks, and again, increasing the numbers of cell lines screened. The company has been able to evaluate product-quality characteristics and cell-line performance on more cell lines, resulting in development of high-quality lead cell lines for its customers. The goal is to shorten the time to clinic by identifying the most productive, commercially viable cell lines that achieve the desired product quality and characteristics. The three archipelagos are interlinked with automated analytics for effective cell-line screening of many different types of molecules.

Fallen’s full presentation further illustrates the stepwise evolution of Lonza’s islands of automation and linking of analytical systems.

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Min Zhang (principal scientist and group leader, upstream process development, Fujifilm Diosynth Biotechnologies) 2:00–2:25 pm

The Media Toolbox: A Rapid and Effective Process Development Strategy to Deliver Reliable, High-Quality Biomanufacturing Processes

Fujifilm Diosynth Biotechiologies is committed to continuous innovation of biotechnologies and producing high-quality results for its clients. Last year it launched the Apollo mammalian expression platform. With that single- use platform, the company has its own expression vector, cell line, medium, and feed. Its streamlined approach to analytical and process development makes the entire process more efficient. The company can perform process development and manufacturing; its facilities can house vaccine and gene therapy operations. It also boasts an excellent inspection history with regulatory agencies.

The Media Toolbox is a collection of high-performing basal media and feeds that can support diverse Chinese hamster ovary (CHO) cell lines. Fujifilm Diosynth has screened the basal and feeding media of many different media vendors on a diverse collection of CHO cell lines and rated their performance.

The first advantage of using the Media Toolbox collection is speed. Media vendors launch new products every few years, but no one has time to test them all — and yet, choosing the wrong media and feed limits cell growth. When the company receives a client’s cell line in its original medium, a team places the CHO cells into the new medium and measures their performance (three weeks). Next, they perform a media/feed screening using shake flasks to identify the top three or four media/ feed combinations and measures elements including metabolism, cell growth, and titer. The top combinations will be confirmed using benchtop bioreactors (four weeks). Finally, the team looks at cell growth, metabolism, titers, and productivity, then identifies the top combination and performs process optimization work. It takes three months or less to identify the top combination after implementation of the toolbox.

The second advantage is efficacy. Seven years ago, the company took on a project in phase 1 and 2 with a complicated process involving CHO cells. After scale up using 2,000-L stainless steel bioreactors, it achieved a titer of about 2.5 g/L. But in returning to the process for phase 3 manufacturing, the company was recently challenged by an inability to reproduce those same titers, instead getting 1 g/L. The investigation into the root cause of the low titer suggested that a material, specifically the media–feed combination, could be at fault. With some trepidation, the client agreed to try the Media Toolbox approach. The resultant improvement in titer (5 g/L) and cell viability continued throughout the duration of the cell growth. Data from different scales – 2 L, 10 L, and 200 L – revealed titers similar to each other, showing good scalability and reproducibility.

In a second case study involving clones, a client wanted a process for CGMP (current good manufacturing practices) manufacturing. The titer was low at 200–300 mg/L, and the client needed to get to a titer of 600 mg/L, nearly double. Fujifilm Diosynth used the Media Toolbox to reach a titer of 1.5 g/L with good productivity. One combination was moved from the process development laboratory into manufacturing. Cell growth, titer, and cell density in single-use 2-L, 10-L, and 200-L bioreactors in that laboratory as well as in a 1,000 L bioreactor for manufacturing remained nearly the same, with very good scalability and reproducibility, illustrating the robust performance of the Media Toolbox.

Clients have three choices at Fujifilm Diosynth Biotechnologies. If a client has a good process, it can be transferred for scale up to a manufacturing level. If a client wants to start from the beginning, Fujifilm Diosynth can use the Apollo platform and perform a clone selection. After the company identifies one or two clones, the development process will be optimized and then transferred for CGMP manufacturing. If a client wants to improve the performance of its own cell line, the Media Toolbox can identify the best media–feed combination and optimize the development process before moving the client into manufacturing.

The Media Toolbox delivers high performance and a reliable process reflected by speed, quality, and robustness.

One question was asked during the Q&A period: How long will the Media Toolbox take to get to manufacturing?

Zhang replied that it takes about three months to identify the top media–feed combination, one to two months using the bench bioreactors, and one to two months scaling up to manufacturing size, with another month to complete manufacturing, and so it takes about a half year to generate material for clinical testing.

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Steven Hager (director of business development, Catalent Biologics) 4:00–4:25 pm

Rapid Development of High-Performance Cell Lines Using GPEx

Hager (a late addition to the program) spoke about Catalent’s GPEx technology for engineering mammalian cell lines. Catalent acquired the techttps://bioprocessintl.com/wp-admin/post.php?post=46723&action=edithnology from Redwood Bioscience for developing antibody–drug conjugates (ADCs). The company’s experience with GPEx now comprises over 400 different antibody-expressing cell lines as well as over 60 recombinant protein-expressing cell lines. In addition, he noted that about 40% of all marketed biosimilar antibodies are made using GPEx cell lines.

The rest of his talk focused on this proprietary technology. The first of several advantages is that it does not use antibiotic selection or typical gene amplification. It simply repeats the process of inserting single copies of the genes into the active region of the genome, allowing for higher expression of an antibody or recombinant protein than from typical transfection-based technologies. GPEx also works with any cell line without the need to modify it.

Not having an antibiotic-resistant gene in the construct prevents having an extraneous protein that could end up harming the cells and slowing cell growth. Not using antibiotic selection enables insertion of multiple different genes to achieve the desired end product. As Hager explained, “for example, if a client needs a processing enzyme to activate its protein, we can insert additional copies of that enzyme into the cell pool and then insert the gene of interest expressing the protein, and you end up with a very efficient mechanism for activating your particular protein of interest. And we take advantage of this with our ADC technology, which requires an enzyme to convert a cysteine into a formylglycine. So we started out by creating a pool of cells that overexpress the formylglycine-generating enzyme.

“Then we can put in the antibody sequence that we’re engineering, and we end up getting full conversion of our target cysteine into formylglycine. We get conversion rates of over 95% using this process. It also works for a system from which companies need to obtain more than one protein.”

He stressed the importance of demonstrating a cell line’s genetic stability and expression stability to the FDA. Catalent’s process leads to very stable inserts for creating master cell banks. (A slide depicted studies performed on 17 different cell lines expressing 17 different proteins, with stability studied out to 60 generations.)

To emphasize the proven nature of the GPex technology, Hager pointed out that his company has more than 40 current customers and more than 80 active projects. It has generated more than 400 antibody-expressing cell lines. It has 22 biosimilar programs, with five commercial biosimilar products made using GPEx technologies around the world and more than 30 clinical trials under way using GPEx cell lines. He added that the typical time from DNA to drug-product release is about 14 months.

A slide depicting the construct showed its lack of an antibiotic resistant gene. As he explained it, “basically, we’re using a sequence that contains a self-activating one and two from a murine leukemia virus. We have an extended packaging region and a promoter, and we use a nonhuman primate promoter to help express the genes in our construct. Then we have the gene of interest and an RNA export and stability element because our message goes in as RNA rather than DNA, and then the end is the self-activating sequence two. The process that we use is a two-step process based on using a retrovector particle to insert the genes into the cells. We need to make the retrovector particles, and then we use them to insert the genes.” Hager’s diagram shows the initial production process to cotransfect a VSV-G gene that expresses a unique protein for the coat of the particle. A plasmid with the gene of interest is cotransfected into a packaging cell line.

The human cell line used contains some components for making the retrovector particles. The retrovector manufacturing takes place in the human cell. Catalent concentrates the retrovector particles by centrifugation and uses them to transfect the CHO or human cells. The retrovector process is efficient because it exploits the fact that the G-protein “recognizes phosphatidylserine on the cell surface of the CHO cell or human cell, so it’s a very specific mechanism. Once the particle binds, the integration process takes place, and we see integration of the gene.” The resulting pool of cells expressing the protein can be used to manufacture protein for initial in vitro studies or can go straight to limiting dilution cloning and clone selection. For antibodies, the company performs a two-step process that inserts the light-chain retrovector and then inserts the heavy chain using a heavy-chain retrovector to create a final cell pool that expresses the fully formed antibody.

Hager emphasized the speed, reproducibility, and straightforward nature of the process. (His slides offered examples of expression levels achieved). He concluded that GPEx cell-line engineering creates cell lines that express at high levels — even very difficult-to-express recombinant proteins are expressed at levels that a company can use to progress. The cells are responsive to upstream process development, and the technology is easy to scale up.

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Hartmut Tintrup (chief business officer, Cevec) 2:30–2:55 pm

CAP Go Cell-Expression System: The New Standard in Authentic Human Glycosylation

Cevec offers individual glycooptimized CAP Go cell lines (cultured from human amniocyte cells) for N- and O-glycolated plasma proteins, complex glycoproteins, and antibodies. Each glycooptimized cell line provides tailor- made glycosylation patterns. After describing his company’s operating units and facilities, Tintrup offered more detail about the new expression platform.

Most expression systems are optimized for antibody production. But when it comes to more complex proteins (e.g., plasma proteins and coagulation factors), companies often get low yields and missing or incorrect posttranslational modifications. With Chinese hamster ovary (CHO) cells, the results can even be nonhuman/ immunogenic on rare occasions (e.g., blood factor IX). Tintrup explained that is why his company is convinced that human cells are the best production hosts for biopharmaceuticals.

Robust CAP Go cells grow in single-cell suspension with high (~90%) viability in any type of bioreactor using a specially designed, chemically defined, serum-free medium for current good manufacturing practice (CGMP) production of plasma proteins and others at high titers. Difficult-to-express protein titers can be up to 10-fold higher than from CHO cells — partly because of cell viability levels and a corresponding lack of protein degradation. Companies that have worked with CAP Go cells report a low degree of aggregation in culture (routinely >5 × 10⁷ cell density). Once engineered, the cells are stable for at least 100 passages.

Cevec offers a portfolio of CAP cell lines that are individually glycooptimized to address certain glycosylation patterns. Complex glycoproteins present a number of different glycan structures on their structural surfaces: e.g., biantennary and triantennary structures. With these cell lines, users choose the isoform they want, and Cevec enriches a CAP Go cell culture for targeted production of that isoform: N-linked glycans, O-linked glycans, and others. This provides pure protein products, simplifying purification and reducing manufacturing time and cost overall.

Cevec has fully documented this cell line’s history with regulatory agencies in both Europe (through the Paul- Ehrlich Institute) and the United States. The company is currently scheduling meetings with the FDA to deposit a cell substrate biological master file that could be referenced by user filings in the future.

Tintrup listed several application examples on a slide and highlighted two of them. One is the C1 esterase inhibitor used to treat hereditary angioedema. Most treatments are currently plasma-purified molecules, making a recombinant form desirable. Another example is human alkaline phosphatase, which is in several clinical trials for treating a number of inflammatory indications (e.g., sepsis, colitis, and arthritis).

If the C1 inhibitor is structurally defective, it can lead to blood vessel damage in patients’ upper airways with recurring attacks of edema and even tetanus 5–12 times a year, which can be life threatening. Tintrup compared pharmacokinetic data for a recombinant C1 inhibitor produced by CAP Go cells and a plasma-purified version. He said this was the first time a recombinant version has been shown to match the serum-purified material. Cevec has produced two recombinant versions of the C1 inhibitor: one with an increased plasma half-life and one with a decreased plasma half-life. Both have the same specific activity as the serum-derived molecule. Tintrup also showed comparable results from other analytical methods: isoelectric focusing, Western blot, gas chromatography, and glycan structure analysis.

Next he showed pharmacokinetics of alkaline phosphatases produced by CAP Go cells, which were improved over those produced through other expression systems. Isoelectric focusing showed differences in their posttranslational modifications that provided those improved pharmacokinetics.

At its facility in Köln, Germany, Cevec provides cell-line development services. “You could give us your molecule and we would apply our CAP GO technology to perform cell development for you,” said Tintrup. As a contract manufacturer, the company performs process development (upstream production and downstream processing) and manufacturing up to clinical phase 2 materials. “Cevec provides CGMP material with its technology.” The company can also generate a cell line (within two to four months) based on a single clone with good performance, then guarantee a smooth technology transfer to any other facility. Services also include GMP master cell banking and drug-product fill–finish as well as support in filing dossiers with regulatory authorities.

Tintrup said that his company has signed two agreements recently in the field of coagulation factors with major pharmaceutical companies. “We are on track to accept a variety of different challenging molecules.”

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Guillaume Plane (development and marketing manager, EMD Millipore) 3:00–3:25 pm

Implementing Clone Generation in Fast-Track Manufacturing of Proteins

Plane began by explaining that EMD Millipore is the life sciences division of the Merck KGaA Group — the German Merck. It acquired Millipore five years ago, creating the life sciences division in the process. Plane’s presentation introduced the company’s clone generation service.

He emphasized the importance of early decision-making regarding of cell lines and production media on the success of later clinical and marketing stages. He said that it is critical for a company to know what type of product it will be making and what will be critical to controlling its cost of goods (CoG) once that product is on the market. No matter what those choices are, he stressed that companies also need to control how they develop their drugs and protect their intellectual property (IP).

EMD Millipore’s clone generation is conducted in a good manufacturing practices (GMP) facility that also provides support for process development and GMP production. The goal is to reduce CoG by increasing protein expression, reducing a customer’s time line by using a good template for cell-line development, and complying with GMPs.

Following initial transfection, the company performs a first and a second selection step to ensure that the pools of cells are transfected with the DNA of interest. The next step is to demonstrate the clonality of the desired cell line — a critical step, defined in ICH Q5D. This service benefits from use of the CloneSelect imager (Molecular Devices), which in his words “allows us to see one clone, one single cell, to ensure that the cell bank that we will generate afterward and that all of the process that is going to be developed — and even the GMP and commercial production afterward — is based on one single clone that was isolated one time during clone generation.”

Then after performing genome purification, the company can provide customers with material for generating its master cell bank, the first step in GMP process development.

Process Development Support: Additional services encompass many different steps in upstream and downstream process development. The company can provide drug substance for preclinical studies, formulation, creation of a working cell bank for clearance studies, and other GMP-related requirements for submission of an investigational new drug application (IND). Service offerings include bioreactors from 50 to 2,000 L for production from phase 1 through phase 3 and state-of-the-art downstream-processing suites for drug purification and formulation. Clients are provided with a cell line and production process and materials for clinical studies.

CHRIS JOHNSON (WWW.BIOPROCESSINTL.COM)

If a client wants to build its own facility to control costs, it can take advantage of Millipore’s Provantage Services, a facility design and implementation program that offers end-to-end solutions using the company’s own GMP facility near Martillac, France, as a template.

Plane concluded by emphasizing that decisions made at the very beginning of bioprocess development — choosing a good cell line, selecting the best culture medium, and optimizing yield — are critical to the success of every eventual biopharmaceutical marketing campaign and represent crucial steps toward ensuring success.

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