Cell line development, animal-free media, disposables, and the implications of process change were among the issues discussed at SAFC Biosciences’ (www.safcbiosciences.com) “Modern Aspects of Cell Culture and Bioproduction” seminars held in Cambridge and Edinburgh, U.K., recently.
Recent developments within SAFC include the acquisition of Pharmorphix, a solid-form research company based in the U.K. The solid-form of a small molecule pharmaceutical can determine its physical properties and is an important element in regulatory submissions. Pharmorphix was scheduled to set up a new lab within the SAFC Madison, WI, high-potency API facility in January.
By 2007, the market size for biopharmaceuticals could be $32.5 billion (mammalian) and $17.5 billion (microbial). “The proportion of mammalian production has been going up since 1998, and we are at the start of a flood of production of biomolecules,” said Avinoam Kadouri, Ph.D., CEO of Rainbow Biotechnologies (www.rainbowbiotech.com). “There are 371 biopharmaceuticals in 425 indications, with an increasing dominance of monoclonal antibodies. Therefore production of proteins on the hundred-kilogram scale is needed.” He added that competition in the industry is increasing too—with many new virtual or semivirtual companies starting up. All of this means that the speed at which a biopharmaceutical gets to market is increasingly important.
“The initial response to these demands was to go for the bigger bioreactors and from continuous to fed-batch processes,” said Dr. Kadouri, “but the predicted capacity disaster of 2005 did not happen. The issue is not bioreactor size but the productivity of the clone. If this goes up by a factor of ten, then the bioreactor can shrink. Investment in process development and optimization, with emphasis on upstream and downstream processing, is also important.”
Volker Sandig, M.D., Ph.D., vp of molecular biology and virology at ProBioGen (www.probiogen.de), described approaches to improve clone productivity that use ProBioGen starter cell lines and SAFC custom media. “Getting a high-producing cell line generally depends upon multiple, well-optimized steps, some of which are not yet well understood,” Dr. Sandig said. The presence of a foreign gene in the producing clone takes up the cell’s metabolic resources, especially during transcription. This issue has been addressed by optimizing the gene and the signal peptide.
Over half of cell line development projects use the human CMV promoter, but there is a need to prevent the rapid inactivation of this promoter, said Dr. Sandig. This can be done by combining it with protective elements that prevent DNA methylation. High-producing clones resulting from these experiments are selected by automated methods. After this, their actual value in terms of robustness and product quality is realized during fermentation, which is done in miniature bioreactors in the form of 50-mL spin tubes.
Linda Somerville, Ph.D., technical manager at SAFC Biosciences, described the development of an animal component-free (ACF) medium for CHO cell-lines. A design of experiment (DOE) matrix-based approach was used; amino acids, plant hydrolysates, iron, selenium, lipids, and vitamins were added to a baseline medium, which was then tested on three recombinant CHO cell-lines. The resulting ACF cloning medium is comparable to a traditional serum-containing medium in supporting cell growth and survival. It also supports transfection and selection and can be scaled-up.
Jonathon Dempsey, Ph.D., head of process development at Cambridge Antibody Technology (CAT; www.cambridgeantibody.com), added that an understanding of cellular metabolism and biochemistry are essential in medium development. Metabolic fingerprinting of cell lines allows for rational medium design, and provision of nutrients in response to cellular demands has been shown to enhance productivity. Analysis of amino acid use has generated data that has been used to design a fed-batch medium for antibody production in NSO and CHO cells. Bovine serum albumin is replaced by cyclodextrins, transferrin with ferric ammonium citrate, thereby making the medium protein-free. Process optimization has led to antibody titers of over 3 g per liter in NSO and over 2.4 per liter in CHO. There are some limits, Dr. Dempsey added, in using commercial media, but it can be useful to develop one in association with a medium vendor.
Another important trend in cell culture is the growing use of disposables. Denise DeTommaso, marketing manager of biodisposables at SAFC Biosciences, said that single-use systems are desirable because they are flexible, light, transportable, and allow issues associated with outsourcing, space, cleaning, validation, and contamination risk to be addressed. They are now being used at all stages of production. At SAFC, there are four facilities where liquid is manipulated, and one of these has been adapted from hard pipe to disposables.
DeTommaso described some of the challenges in implementation, which is a stepwise process. “You must get the engineering and validation people involved early on,” she said. Other important steps include identifying vendors, describing the system, gathering technical data, making a prototype, and finally, full implementation. SAFC has thus developed a 100% disposable manifold, which is flexible and requires no CIP/SIP. It is now looking at a disposable aseptic bag-filling machine. Drawbacks of disposables include lead times, the economics of large bags, and logistics when multiple bags and multiple locations are involved.
SAFC has looked at a hybrid manifold that is stainless steel with a disposable connection. There is a significant difference in clean time between stainless steel (6.5 hours) and hybrid/disposables (4 hours each).
Scale-up is another key challenge in cell culture. Allen Gross, SAFC Biosciences’ marketing manager, described how the company faces these by integrating and overlapping key scale-up components: cell-line selection, medium optimization, process development, and downstream development. “When these are segmented, it increases overall development time,” Gross pointed out.
SAFC uses laser-enabled analysis and process (LEAP) for clonal selection, which is otherwise a bottleneck. This uses high-throughput cell imaging and laser-mediated cell manipulation, which allows failures to be discarded earlier.
Another cell line development technology, CellXpress™, capitalizes on the tracking capacity of LEAP and allows high producing clones to be chosen for medium optimization. The DOE/matrix approach to medium optimization gives a better focus on the components for cell growth and for cell productivity, which tend to be different. Meanwhile, it should always be remembered that even minor modifications in process can have major downstream implications. “That is why we strongly believe that the purification group should be involved from the start,” said Gross.
Putting all this together requires professional project management; it is also important to try to understand the synergies between the different components of a cell culture process. “Integrating improvements decreases the development cycle by incorporating a multifactorial approach that capitalizes on the synergies between each development segment,” concluded Gross.
Process Changes in the Clinic
It is often necessary to make process changes during clinical development, and these will require the attention of the regulatory agencies. Graham Roberts, Ph.D., senior regulatory and product development manager at Constella Group (www.constellagroup.com), said that the agencies will accept the need for process changes, but safety will always be their top priority, and companies will need to satisfy them on this point.
A research-based cell culture process is usually the quickest to get proof-of-concept, but cost and timeline issues will require changes that will help optimize this process. Technical improvements, such as the move toward serum-free media, and an increase in regulatory hurdles often drive process changes. The impact of such changes may vary. They may, in fact, have minimal impact on safety or efficacy, and in such cases, the regulatory authorities will require little extra data. At the other extreme, a process change may have a major impact on the characteristics of the product and there will be a need to repeat both clinical and nonclinical tests.
Getting the demands of process change right will mean minimal impact on the clinical development program and lead to a state-of-the-art process and also a good relationship with the regulatory authority, said Dr. Roberts. Getting it wrong will delay the clinical program, add to costs, and lead to an uncertain relationship with the regulatory authority.
Dr. Roberts recommends planning ahead and introducing changes early. The rationale for process changes needs to be transparent. “Ensure that you have the appropriate tools and data, sensitive and robust analytical methods, and in-process controls for both the old and new processes,” Dr. Roberts advised.
When it comes to comparability packages, the regulatory agency will require both in vitro and in vivo comparability and additional clinical and nonclinical data on aspects such as stability and immunogenicity.
It is a good idea to present your comparability strategy to the agency in advance, manage expectations on both sides, and get the agency’s input into the protocol. Assessors tend to work on a case-by-case basis and are data-driven, and one is unlikely to get a definitive decision in advance of IND/CTA amendment submission.
“Know your product, know your process—after all, you are the expert,” was Dr. Roberts’ final tip.