Optimizing the Primary Recovery Step in Nonaffinity Purification Schemes for HuMAbs - An alternative approach to traditional Protein A schemes is comparable in overall process efficiency, product reco

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Optimizing the Primary Recovery Step in Nonaffinity Purification Schemes for HuMAbs
An alternative approach to traditional Protein A schemes is comparable in overall process efficiency, product recovery, and quality.


BioPharm International Supplements


Choosing the Diafiltration Buffer


Figure 5. Diafiltration flux curves from different tangential flow filtration membranes
The foremost step in optimization is to identify a suitable buffer system for TFF because the chemical composition of the buffer plays a key role in product recovery. Several aspects should be considered when choosing an optimal buffer system. First, the diafiltration buffer should be compatible with the product during exchange without affecting product recovery. Second, the buffer needs to be compatible with the capture load conditions. For instance, in the case of a HuMAb process, the best binding condition for CEX was found to be sodium phosphate buffer with sodium chloride. However, the process performance at TFF stage was compromised by using this CEX binding buffer as diafiltration buffer, which resulted in poor product recovery. To accommodate best conditions for both TFF and CEX, diafiltration can be performed in a phosphate buffer without NaCl to prevent product loss, and added before binding to CEX.

Evaluation of TFF Membranes


Figure 6. Concentration and diafiltration time profiles versus load volume
Performance differences could exist in membranes from various manufacturers probably because of variation in pore size distribution even when they are made up of the same material and with similar molecular weight cut-off (MWCO). To identify the most suitable membrane for HuMAbs, evaluation of three membrane sources and two different pore sizes, 30 kD versus 50 kD, with similar process streams were performed (Figure 5). Better recyclability and consistent flux were noted with 50 kD membranes, whereas with 30 kD, the second cycle performance was compromised by decreased flux rate. However, similar product recoveries were obtained by both 50 kD and 30 kD membranes. Among three different membranes from different suppliers, one of them outperformed for process consistency during two cycles in terms of process flux rate.

Determining Load Amount and Concentration Fold


Figure 7. Concentration and diafiltration time profiles versus concentration fold
A series of studies on the load volume were performed and the results are plotted in Figure 6. At constant concentration factor, as the load volume increases from 40 L/M2 to 200 L/M2 , the concentration and diafiltration times increase. Though the overall processing time is increased as load volume is changed from 40 L/M2 and 200 L/M2 , even at the highest load amount, TFF operation could be completed by 6.5 hours. This translates into a five-fold lower membrane cost for processing the same amount of material.

As concentration fold increases from 5X to 15X, there is a moderate increase in concentration time. However, the major impact is seen on diafiltration step, which occupies maximum TFF operation time (Figure 7). In fact, the diafiltration time at 10X to 15X is less than half of 5X concentration fold. This results in decreasing membrane and buffer usage and processing time, affecting overall raw material and labor costs.


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