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


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

Pressure Range Study

Figure 8. Concentration and diafiltration time profiles versus ΔP
The permeate flux rate can decrease significantly, especially during the diafiltration step because of gradual precipitation of host cell contaminants from concentrated cell culture supernatant. Therefore, range studies of feed and retentate pressures are essential for smooth TFF operation. The experiments examined the effects from changes of transmembrane pressure (TMP) as well as ΔP, which is the pressure difference between feed and retentate (Figures 8 and 9).

Figure 9. Concentration and diafiltration time profiles versus transmembrane pressure
When TMP was kept constant (15 psi), the increase of ΔP from 20 psi to 30 psi prolonged the concentration time very moderately, while the diafiltration time was reduced significantly from 4 to 2.5 hours.

Figure 10. Cost comparison of disposable versus reusable tangential flow filtration membrane
However, under constant ΔP (20 psi), elevating TMP reduced the concentration time and had very little impact on diafiltration time.

By optimizing ΔP and TMP ranges, an additive effect on overall TFF process time reduction can be obtained.

Recycling of TFF Membrane

Fouling of TFF membrane by gel layer formation from complex composition of cell culture is common during the diafiltration stage. Normalized water permeability (NWP) is monitored at the beginning and end of each cycle to evaluate the cleanliness of the membrane and performance consistency. Typically, NWP decreases sharply during the first couple of cycles (10–20%) and stays in the range of 60–80% in subsequent runs. Using NWP as the sole evaluation criteria can terminate membrane lifetime earlier than necessary. NWP can be used as an early indication of membrane modification, and is not necessarily directly related to process performance.5 Further, membrane performance can be measured by monitoring fluxversus-time curve, process recovery, process time, and product purity.6 The TFF membrane life can be at least 10 cycles in a typical HuMAb process as assessed by the flux curve, process time, and product recovery in a scale-down TFF system. In this context, an alternate approach can be to use a single-use membrane, which can reduce or eliminate cleaning and cleaning validation.7,8 Fully disposable crossflow systems are available in the market, which require extra costs for disposable adapter plates, pressure gauges, a flow meter, and a conductivity meter.7 But savings are more from eliminating cleaning validation and reductions in cleaning solutions, labor cost, membrane storage, process waste disposal, and analytical requirements. Disposable units will have significant cost advantage, especially when the membrane cycle usage is limited (Figure 10).

Comparison of Affinity Versus Nonaffinity Schemes

Table 1. Comparison of operation cycles for nonaffinity and affinity schemes
Even though primary recovery TFF adds a unit operation to the whole process, this step results in significant batch volume and overall capture cycletime reduction. Loading time reduction is advantageous for high-binding resins where bed volumes can be relatively smaller. For example, for a 5,000-L batch, the unit operation includes one TFF and two cycles of cation exchange versus six cycles of Protein A (Table 1).

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