High Performance Tangential Flow Filtration

Ultrafiltration and microfiltration have traditionally been limited to separating species that differ in size by at least 10-fold. In contrast, HPTFF enables the separation of solutes without limit to their relative size. HPTFF is able to obtain the high selectivity required for effective protein purification by exploiting several recent developments. Firstly, HPTFF is operated in the pressure-dependent regime, with the filtrate flux and device fluid mechanics chosen to minimize fouling and exploit the effects of concentration polarization. Since optimal separation in HPTFF is obtained at a specific filtrate flux, the membrane module should be designed to maintain a nearly uniform flux and transmembrane pressure throughout the module. This can be done using a co-current filtrate flow to balance the feed-side pressure drop through the module. Secondly, the buffer pH and ionic strength are adjusted to maximize differences in the effective volume of the different species. The effective volume of a charged protein (as determined by size exclusion chromatography) accounts for the presence of the diffuse electrical double layer surrounding the protein. Protein transmission through the membrane can be reduced by increasing the effective protein volume, e.g. by increasing the net protein charge (by adjusting the pH) or by increasing the double-layer thickness (by reducing the solution ionic strength). Thirdly, the electrical charge of the membrane is chosen to increase the electrostatic exclusion of all species with like charge. Thus, a positively charged membrane will provide much greater rejection of a positively charged protein than will a negatively charged membrane of the same pore size. Fourthly, protein separations in HPTFF are accomplished using a diafiltration mode to wash the impurity (or product) out of the retentate. The diafiltration maintains an appropriate protein concentration in the retentate throughout the separation, and it allows one to obtain purification factors for products collected in the retentate that are much greater than the membrane selectivity due to the continual removal of impurities in the filtrate.

Although HPTFF is still a new membrane technology, a number of recent studies have clearly demonstrated the potential of this separation technique. Several of these results are summarized in Table 2. Purification factors for the separation of bovine serum albumin (BSA) from an antigen-binding fragment (Fab) were greater than 800-fold with either protein collected in the retentate depending upon the choice of solution pH and membrane surface charge. BSA and haemoglobin have essentially identical molecular weight but different surface charge characteristics. In this case, operation at pH 7 caused a strong electrostatic exclusion of the negatively charged BSA from the negatively charged membrane. The separation of BSA monomer and dimer occurs primarily because of the difference in protein size, with the

Table 2 Purification factors and yields for HPTFF processes3

Product (MW)

Impurity (MW)

Purification factor

Yield

BSA (68 000)

Fab (45 000)

990

94%

Fab (45 000)

BSA (68 000)

830

69%

BSA (68 000)

Hb (67 000)

100

68%

IgG (155 000)

BSA (69 000)

30

84%

BSA (68 000)

BSA dimer (136 000)

9

86%

aBSA, Bovine serum albumin; Fab, antigen-binding fragment from recombinant DNA antibody; Hb, bovine haemoglobin; IgG, human immunoglobin.

aBSA, Bovine serum albumin; Fab, antigen-binding fragment from recombinant DNA antibody; Hb, bovine haemoglobin; IgG, human immunoglobin.

smaller monomer collected in the filtrate. However, electrostatic interactions are also important in this system due to the combined effects of size and charge on protein transmission and to possible differences in the charge-pH profiles for the monomer and dimer.

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