Considerations for Successful Upstream Process Development

BioPharm International speaks with Robin Ng, senior bioengineer in process development at Shire Pharmaceuticals, about the factors to consider when setting up the upstream portion of a biomanufacturing process.

BioPharm: What parameters influence the choice of a production system?

Ng: In general, an upstream process in biopharmaceutical production refers to the process of manufacturing the products of interest using biological entities, such as bacterial or animal cells. Because this process is aimed towards manufacturing, the properties or attributes of the desired products dictate the selection of a production system. In practice, the production system includes the selection of host cells, cell lines, and culture media, as well as the bioreactor system and its operating parameters. It is important to keep in mind that the development of the process is highly tied to the business needs, available resources, regulatory constraints, and project timeline. In addition, appropriate risk assessments need to be conducted and well documented throughout the development process to support rational decisions. Therefore, the development of each process is unique because of the specific constraints encountered during development, as well the advancement of the available technologies. Last but not least, it is always important to remember that process safety should be a top priority.

BioPharm: Can you briefly describe the process of choosing a cell line for production? What are the criteria for selecting a cell line?

Ng: In the cell-culture process, genetically-engineered cells are used to manufacture the proteins of interest. It is desirable that the commercial manufacture be initiated from a single Master Cell Bank (MCB). In the initial stage of development, cell cloning and subcloning are conducted following transfection and amplification. With recent advances in automation, the selection process is mostly conducted through a high throughput screening (HTS) system where millions of clones are screened and only a few clones are selected to move forward for further development.

The initial selection criteria for a cell line are typically high titer and robust growth. Obtaining a high-producing cell line can require additional time and resources. Therefore, it is important for the process developer to understand the needs and demands for the process. Whenever possible, the target market demand should be revisited to ensure reasonable and balanced process development efforts.

The secondary criteria in cell-line development include cell stability and product quality attributes. What a process developer should look for in cell stability is the ability for cells to produce the comparable protein levels (i.e., phenotypic stability) and maintain genetic integrity as the cells are continuously propagated into the production phase. In most development cases, many product quality attributes are still unknown during cell-line development. This remains a challenge in cell-culture process development because clinical data cannot be correlated with various product quality attributes during most of the development stage. Therefore, appropriate risk assessments based on scientific justification, process understanding, and similar processes are very important during early development.

It is also important to perform the final selection of candidate cell lines in a representative bioreactor system. The use of a representative bioreactor system or process is important to help predict process performance in a large-scale bioreactor system. In most companies, a baseline process will be used to screen the few final candidates cell lines.

It is always helpful to start with a good candidate cell line. Changing cell lines during development is usually undesirable because it leads to extensive comparability studies between materials generated. Knowing your endpoint is always a good start for developing your process.

BioPharm: Selection of media is also important to a successful cell-culture process. What are the key considerations when choosing cell culture media? Can you discuss approaches to developing or selecting media?

Ng: The cell-culture medium serves two purposes in cell culture: it functions as the medium (with a buffering capacity) where the cells grow, and it provides nutrients for cell growth and production. As mentioned previously, each process is unique. The selection of the media depends on the process, business, or regulatory constraints. In some cases, these constraints arise downstream of the cell culture process. When selecting for culture media, a process developer should always consider the business and regulatory needs, the presence of unstable materials, lot-to-lot variation, media compatibility with the production system, available grades of the culture media, ease of formulation, and the impact on downstream purification.

With advances in culture media development, several protein-free chemically defined (CD) media are readily available for off-the-shelf use. The use of these protein-free CD media helps in several aspects of development: they reduce regulatory risk from animal-derived components and lot-to-lot variation, and they increase the developability of culture media (because all of the components are known). In addition, they can reduce cost in some cases.

The approach of cell culture media development can generally be divided into two main groups: a top-down approach and bottom-up approach. In the top-down approach, a developer seeks an effective basal media or additive that can improve protein production without the need to know each of the components or how the components work. This top-down approach is usually conducted through a series of mixture experiments as well as through properly designed experiments to screen different additives. In contrast, the bottom-up approach relies heavily on understanding at the molecular, cellular, and process levels. In this approach, a measurable amount of additives are added because a particular component is either depleted or serves key functions at the molecular or cellular level. One way to find out the depleted component in the bottom-up approach is to analyze the spent media from the cell culture. Once identified, the depleted components can be added back to the media through front loading in the basal media, or feed enhancements can be introduced to the culture at different culture duration times. The recent advancement of "omics"—metabolomics, proteomics, and genomics—has shed light on the identification of key components in culture media, as well.

BioPharm: What factors should be considered when choosing a bioreactor system?

Ng: Once the cell line and culture media have been identified, a process can be further developed through various bioreactor culture systems. There are two main categories in selecting a bioreactor system: bioreactor operating mode and scale-independent, as well as scale-dependent, operating parameters.

Additionally, there are several operational modes that can be considered when developing a cell culture process: the fed-batch process, perfusion process, perfusion with cell bleeding, and a chemostat process. Although a fed-batch process is frequently used in the current biopharmaceutical industry, the long residence time of products in the bioreactor at an elevated temperature might not be desirable for certain proteins. For such proteins, perfusion-based or continuous cultures (where products are continuously being removed) are favored. Again, each process offers its own advantages and disadvantages and needs to be properly assessed.

The selection of scale-independent parameters also depends on properties of the proteins of interest and the growth characteristics of the cells being cultured. During the various process development stages, these parameters are characterized to maximize the process productivity and robustness. One caveat to the development is that improving productivity should not compromise the quality attributes of the product. The effects of these scale-independent parameters on product quality attributes should be critically assessed during development. In practice, the selection of relevant product quality attributes can make a difference in development efforts. Similarly, proper risk assessment tools should be used and well documented to guide the development efforts in achieving the desired end points.

Scale-dependent parameters are usually addressed during the latter stage of development. The selection of these parameters can affect process scalability. Several considerations for scale-independent parameters include bioreactor scales and dimensions, and other engineering considerations of the bioreactor design, such as bioreactor dimensions, sparger size, rate, and location; impeller type, size, position, and agitation rate; and other considerations, including pumping direction, working volume, nutrient injection point, base injection point, and heat transfer.

In most cases, the selection of one parameter or operational condition can affect the other parameters.

BioPharm: Can you discuss the concept of manufacturability as it applies to a biological product? When developing a cell-culture process, what parameters should a developer be considering to ensure manufacturability?

Ng: The end goal of cell culture process development is a robust manufacturing process that generates quality products through the cultivation of cells to meet the market demand. The keywords in that statement are 'quality products' and 'market demand,' and there are multiple ways to deliver the end goal. Nevertheless, considerations for manufacturability should be heavily assessed during process development. The concept of manufacturability will ensure that the process being developed can in fact be safely and appropriately applied in a large-scale manufacturing facility.

A developer should always keep the process simple and consider the operational limitations and equipment required or involved in large-scale manufacturing. Facility fit is a key aspect in manufacturability. A process should be developed with the final state in mind and the developer should also be prepared for the expected differences in large-scale manufacturing. Those differences may include osmolality, pCO₂ level, some metabolite levels, shear level, and hydrodynamics across scales. Another factor to consider is the availability of GMP-grade raw materials for culture media additives. Any of these expected differences should be properly assessed, documented, and addressed.

BioPharm: What advice would you offer to someone who is starting out in cell culture process development?

Ng: The cell-culture process is a fairly complex process that includes several activities and development constraints from the business and regulatory sides, as well as timeline considerations. Consideration for process safety should always be the top priority. One piece of advice I have for a process developer is to know the end point and know the development system well. Among the end points are target yield, desired product quality attributes, timeline, business, or regulatory needs. The development system will include not only the upstream process development, but also downstream, analytical, as well as formulation development. The developer needs to use reasonable risk assessment tools that are scientifically based to guide the development process and decision making. Last but not least, a developer should consider the extended effect of the process as far forward as possible and as early as possible during development. Remember that each process is unique with its own requirements as well as constraints.

Robin Ng, PhD is a senior bioengineer in process development at Shire Pharmaceuticals in Lexington, MA.