Development of a recovery process for a fed-batch mammalian cell culture product involves several objectives: process scalability, robustness, maximizing product yield, elimination of subsequent purification steps, and low cost of goods. In an effort to achieve those objectives, we developed a three-stage primary recovery process to remove biomass and clarify the feed stream for downstream column chromatography (Figure 1). The initial stage involves removal of whole cells and larger cellular debris using a continuous disc-stack centrifuge. Depth filtration is the second stage, removing smaller particulates based on size exclusion and adsorption. The third stage consists of 0.2-µm filtration, which removes potential bioburden. To assist in process development, we are investigating innovative approaches to achieve better process control and maximize processing efficiency. Monitoring pressure in the depth filtration process step is one potential area for improving throughput and efficiency.

PRODUCT FOCUS: ALL BIOLOGICS

PROCESS FOCUS: DOWNSTREAM PROCESSING

WHO SHOULD READ: PROCESS DEVELOPMENT AND MANUFACTURING

KEYWORDS: HARVEST, FILTRATION, PRESSURE DIFFERENTIAL, PROCESS CONTROL, DATA MANAGEMENT

LEVEL: INTERMEDIATE

Depth filtration coupled with 0.2-µm filtration adequately removes cellular debris and contaminants from a feed stream. It offers two different methods for removing biomass and other contaminants. The depth filter medium is a porous mix of diatomaceous earth and cellulosic fibers that removes small particles (<1 µm) by size exclusion. That medium may also contain positively charged adsorptive binding sites. They can also effectively remove smaller charged particles that are too small to be removed by size exclusion but can impair subsequent column chromatography operations.

Because of inherent differences in cell lines, a purification process needs to be developed individually for each mammalian cell culture. There is a shortage of relevant data on process feed streams that can accurately predict the reliability of depth filtration. But pressure differential (pressure drop across a depth filter) is an important means of monitoring the overall performance of a depth filter during use. After initial sizing for a particular clarification process, depth filter performance still needs to be closely monitored to prevent premature fouling, which can be caused by unforeseen impurities or less-than-optimal process parameters, such as a high flow rate. This would require additional depth filter area to clarify the remaining cell culture harvest.

No correlation currently exists between the characteristics of a cell culture (e.g., cell viability, cell density, viscosity) and the ability of a depth filter to successfully clarify it. Changes in media feeding rates and other growth parameters play a role in the characteristics of a cell culture to be clarified. So even with proper filter sizing for a given volume of a particular culture, depth filter performance can still suffer if key variables are not monitored closely. Monitoring the pressure differential is a way to ensure that a cell culture harvest is not prematurely plugging the porous medium of a depth filter.

Traditional analog, stainless steel pressure gauges have been used to record both pre- and post-depth filter pressure. A major drawback of such gauges is their need for frequent calibration and cleaning verification and validation. Another drawback is the need for an in-line stainless steel sanitary “tee” to install each pressure gauge into a flow path. That causes a short deadleg with a hold-up volume, which can lead to inaccurate readings. For mammalian cell culture operations, pressure readings have to be taken by operators and recorded manually. Frequent data collection can put a strain on limited manpower and may also lead to transcription errors.

Single-Use Pressure Sensors and Data Collection

At Centocor, we were interested in PendoTECH single-use pressure sensors as an alternative to stainless steel pressure gauges to be used in conjunction with the company's PressureMAT monitor, alarm, and transmitter system. The combination can be used to record and transmit pressure information to a data collection system. Each pressure sensor has an in-line, flow-through design, and sizes are available from Luer to 1-in. hose barb fittings (Photo 1), eliminating the need for sanitary tees and their associated hold-up volumes. This reduces the number of process components (e.g., gaskets, clamps, tubing adapters), and the disposability of the pressure sensors reduces demand on cleaning verification/validation.