Monoclonal antibody purification processes have been challenged recently to recover 5 g/L cell culture titers in existing manufacturing facilities. This requires the modification of a platform purification process comprising Protein A chromatography as the robust capture step followed by two ion exchange chromatography steps. Because buffer and pool tank volumes are the primary facility bottlenecks, process development efforts have focused primarily on the order of unit operations, the dynamic binding capacity of current and new resins, reducing pool manipulations and buffer consumption, and elution buffer selection. This article presents examples in which new operating conditions or purification technologies can help to accommodate increases in titer without extensive changes to the manufacturing facility or equipment requirements.
Over the past five years, there has been a dramatic increase in the titers of recombinant proteins, particularly in the production of IgG monoclonal antibodies (MAbs) using Chinese hamster ovary (CHO) cells. Improvements in cell lines, media composition, and cell culture operating conditions have all contributed to higher expression levels. In most cases, these changes have had little or no impact on manufacturing equipment and facilities. However, as titers have increased by an order of magnitude or more using existing cell culture bioreactors, there has been a concomitant increase in the starting mass of protein entering downstream purification. This development has resulted in a shift in emphasis from increasing volumetric productivity for low-titer processes to handling significant increases in the amount of protein during downstream processing, particularly in existing facilities. Although incremental increases in titers can be handled by increasing the scale of purification unit operations (e.g., using larger chromatography columns and filters), at some point linear scaling exceeds the physical limits of existing facilities.
To address the purification challenges of high antibody titers, it is imperative to determine the specific limitations of a company's manufacturing facility or facilities. This can best be accomplished by using a model that performs a facility fit using an existing process. Such a model can identify the bottlenecks for each unit operation at a specific manufacturing facility as a function of antibody titer. In some cases, it may be necessary to use multiple model iterations to find a bottleneck, model a change to the process or facility, and then determine where the next bottleneck may be. For the purposes of this article, we assume that one cell culture batch is purified to produce one bulk. Alternative process options such as split batch processes may be considered, and these could potentially allow higher recoverable titers from one cell culture batch.
Lonza, Ltd., Basel, Switzerland
Platform Process Bottlenecks
Almost all current antibody purification platform processes use Protein A affinity chromatography as the capture step because it is robust, widely applicable, and facilitates the implementation of a successful standard purification process.1,2 Figure 1 shows two platform purification processes that we have implemented. After cell and product separation during harvest operations, Protein A affinity chromatography is used as the initial capture step with low-pH viral inactivation in the Protein A pool. Depending on the platform, this is followed by either the cation exchange chromatography step in bind-and-elute mode3 or the anion exchange chromatography step in flow-through mode. The virus filtration operation is placed between the two ion exchange chromatography steps. After the third chromatography step, ultrafiltration–diafiltration is performed using tangential flow filtration (TFF) for final product formulation.
Potential volume bottlenecks caused by increased titers are included for each unit operation of each process shown in Figure 1. Underneath each potential bottleneck are process options that should be considered for optimization to avoid facility bottlenecks, and those having the greatest impact are shown in red. We have found that the primary bottlenecks in existing facilities are buffer and pool tank volumes, whereas secondary bottlenecks include pump capacities or pipe diameters that limit flow rates. For these reasons, the use of larger chromatography columns or filter surface areas is not sufficient to handle higher titers.