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


Partial Purification of Host Cell Contaminants


Figure 3. Reduction of CHO host cell DNA during primary recovery (PR) of different HuMAbs
Primary recovery TFF is also a partial purification step. As the pH moves lower during diafiltration, Chinese hamster ovary (CHO) host cell contaminants, especially DNA, gradually precipitate out. As the pH passes through the wide pI range of host cell proteins, it causes gradual precipitation of these contaminants. CHO DNA is significantly reduced as the diafiltration progresses, from starting ~106 pg/mg to ~103 pg/mg at the end of the primary recovery step, while CHO host cell proteins (HCP) are reduced at a modest level (Figure 2). Figure 3 shows a robust purification step for CHO DNA reduction for different Hu-MAbs. DNA partial purification at TFF generally benefits non-Protein A purification schemes, especially for high cell density and high titer cell cultures. This also prolongs capture cycle lifetime because of cleaner and uniform feed.


Figure 3. Reduction of CHO host cell DNA during primary recovery (PR) of different HuMAbs
HuMAbs in the diafiltered buffer condition at relatively lower pH also exhibit better stability than the clarified cell culture supernatant (Figure 4). TFF bulk typically has a three- to four-week storage time, while clarified bulk can last only several days. The prolonged stability could be because of the removal of proteases from the cell culture supernatant during buffer exchange. This could be beneficial because the cell densities tend to increase for hightiter cell culture processes and more proteases are produced during the cell culture and harvest operations. Also, the secondary modifications of proteins, such as deamidation and isomerization of antibodies, are better controlled at the lower pH as a result of diafiltration compared to the high pH of cell culture supernatant.4 This provides a convenient in-process hold point, which can potentially benefit flexible manufacturing schedules in commercial scales.

Optimization of Primary Recovery TFF


Figure 4. Monoclonal antibody stability in clarified cell culture bulk versus primary recovery (PR) tangential flow filtration bulk
The primary recovery TFF processes complex, clarified cell culture bulk, and diafiltration can result in enhanced precipitation and gel layer formation, thereby influencing the flux rate. Optimizing the operating conditions to maximize the flux and minimize the operating time should consider several process parameters. Choosing a suitable diafiltration buffer and a TFF membrane is essential to decrease operating time. Testing load protein amount and concentration fold of the feed, as well as optimal combination of inlet and retentate pressures, is important to define the maximized flux rate and optimized throughput.


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