## 2 Advancements in Ultrafiltration for Protein and Peptide Separation
### High-Performance Tangential Flow Filtration (HPTFF)  
Tangential velocity is used to prevent the foulant deposition on and in membrane pore. HPTFF basically belongs to the category of dynamic UF, except it also separates proteins based on net charge besides molecular size. Not only does it improve the performance, but HPTFF can also cater a new process design that is more economically practical yet discharges minor wastewater. Exploration of HPTFF has been used to simplify isolation methods by joining hydrophobic interaction chromatography and subsequent UF/DF in one unit. From this research, it was found that the performance of HPTFF was similar to conventional process, but the yield of Fab'2 from recombinant _E. coli_ with 100 L m\({}^{-2}\) h\({}^{-1}\) was 12% higher than the UF/DF process, and there was no loss of selectivity after 10 times regeneration of novel positively charged 100-kDa RC, modified by bromo-propyl-trimethylammonium bromide [153].  
In HPTFF, high selectivity can be achieved by (1) minimizing fouling and concentration polarization with operation in pressure-dependent regime just below the transition point, (2) increasing disparities of hydrodynamic volume or effective size (core dimension and its extended electric double layer) between products and by adjusting pH far from the IEP and optimum ionic strength (low value inhibits buffer ions generating the shielding of charges), (3) enhancing electrostatic repulsion of compounds with alike size, (4) signifying purification and yield by diafiltration, and (5) decreasing TMP with co-current filtrate flow configuration [154; 155; 156; 157]. For molecules exhibiting radically different charge and/or size, HPTFF was proven as a promising advancement in UF for protein separation of lysozyme and ovalbumin from chicken egg white [158], BSA and hemoglobin [159], and lactose from pretreated casein whey [160].  
For similar charges, HPTFF can hardly perform with notable yield and selectivity. For instance, size-based removal of peptide dimer or oligomer from monoclonal antibody alemluzumum only showed monomer purification factor of four for one-staged diafiltration [86]. Recent strategy of improving purity and yield was by engineering molecular charge in order to arrange the electrostatic interaction and protein transport. One finding to obtain pure myoglobin demonstrated that retention of lysozyme was increased by the present of BSA (completely rejected by 30 kDa MWCO) as dual-facilitating agent since negatively charged lysozyme was attracted by positively charged BSA (IEP 4.9) at pH 9.0 and myoglobin migration to permeate will follow the Donnan Effect [161]. Small charge affinity ligand like Cibacon Blue has been used to manipulate BSA net charge [162; 154; 163]. In another research, small-stirred UF cell and negatively-charge regenerate cellulose (with covalent attachment of sulfonic acids) were employed to separate BSA and ovalbumin, and reached high resolution: 30-fold selectivity, 90-fold improvement of purification factor, and more than 90% yield of BSA [162].  
By replicating the process for HPTFF (two stages diafiltration) with Pellicon XL tangential flow module which can be scaled-up linearly for membrane area above 80 m\({}^{2}\), the result attained more than 80% yield and 15-fold purification factor of BSA in retentate, whereas ovalbumin was of 95% yield with 50 L m\({}^{-2}\) h\({}^{-1}\) permeate. In this research, they suggested that DV number can be manipulated to elevate the yield and purification factor [154]. Despite those distinguished features, HPTFF is vulnerable to bigger MWCO membrane. This trend was confirmed by [PERSON] et al. that testing UF rotating disk membrane (UF-RDM)for separation and concentration of leaf protein from alfalfa juice [23; 145; 146]. Higher MWCO and temperature was attributed to aggravating irreversible fouling and separation performance during VRF = 6 and VRF = 12, but also to leveraging better filtration behavior and productivity. However, HPTFF requires a high energy cost [71].