Time of harvest affects the cells ability to be lysed during the homogenization step. This is due to the cell’s fragility during the exponential phase, where cell division is rapidly happening, causing weaker cell walls compared to the cells in the stationary phase. Low cell viability and extended batch or fed batch results in more cell-related impurities compared to short batch or fed batch harvested during an early stationary phase. Processes using TFF as an initial clarification step are directly affected by such variations due to the inconsistency of feed material.
Homogenization can create variability in feed for centrifuge and TFF depending on the number of passages and pressure applied for cell rupture (Figure 3). Multiple passages with high pressure results in particulate load that has fine cell debris with a complex size and density distribution. Integrating homogenization with centrifugation and finding the ideal window of operation optimizes the efficiency. This includes obtaining the target product recovery with a higher centrifuge flow at a minimum number of passages with an efficient rupture pressure.
In the event that this is not done, variability from homogenizer output can affect not only the centrifuge efficiency but also TFF flux due to the inability of the centrifuge to capture fine particulate impurities present in supernatant.
Another approach would be to integrate the latest technologies available, such as production of bacterial ghosts by protein E-mediated lysis (lysis tunnel formed by the expression of PhiX174 lysis gene). As a result, the expensive, time-consuming, and risk-associated homogenization process could be avoided.
This method of lysis could also ease the primary recovery process as the ghost cells are still intact, and the particulate material is minimal compared to other physical methods of lysis. The contaminants, such as DNA and RNA, could be degraded during the process as the respective enzymes that catalyze the degradation could also release during the process.
For extracellular products, centrifugation becomes a direct target to create a window for subsequent NFF (normal flow filtration) or TFF optimization.
Integrating process development with GMP is advantageous because it reduces the uncertainty of scale-up issues. It can also help by speeding up the validation process because most critical parameters are defined during process development. Speed to market is achieved by meaningful process development. In addition, it eases the tech transfer from the process development lab to the manufacturing facility.
Effective process development needs to keep large-scale processes in mind while developing the process and defining the critical process parameters. One example is the scale-up of process parameters such as dissolved oxygen (DO) control. The process development lab uses pure oxygen or higher agitation/impeller revolution per minute for DO control, which might be necessary to reach higher cell densities and product yield. Such control measures may not be available at large scale.
Another example of the benefit of integrated process development and manufacturing is the avoidance of issues associated with heat transfers at small scale, which could be problematic as the scale increases.
Similarly, for downstream operations, designing filtration process must take into account filter geometry, protein binding, pump requirements, linear scalability, and batch-to-batch variations. When designing concentration and diafiltration steps one must consider the effectiveness and impact at large scale. For example, it might be possible to do several buffer exchanges at a small scale without any volume consideration, but at a large scale this may become nearly impossible. Likewise, when designing a chromatography process several column volumes of equilibration, washing, and elution may be common but this must be minimized during the final stage of process development. The amount of buffer used by chromatography and filtration processes can be high if the process is developed without consideration of scale. It can also impact WFI, buffer preparation area, and buffer storage capacity.
The discovery phase influences the course of process development in different areas such as selecting the host organism, protein expression, and consequently, purification. These are extremely important factors in late phases of biopharmaceutical manufacturing.
Process development is a vital part of GMP manufacturing and must meet the expectations of developing scalable, cost-effective, and robust processes. Methods and techniques used at process development scale to achieve purity and yield must be feasible and practical at large scale.
Integrated process design is of utmost importance. Integration of all stages early on in process development allows companies to steer clear of bottlenecks in the overall manufacturing process. Using integrated process design, an optimal window of operation is found for the process at large scale and thereby efficiencies realized.