Journal Pre-proof

Addressing Challenges in the Ultrafiltration of Biomolecules from Complex Aqueous

Environments

Emre Bukusoglu, Harun Koku, P. Zeynep Culfaz-Emecen

PII: S1359-0294(20)30011-X
DOI: https://doi.org/10.1016/j.cocis.2020.03.003
Reference: COCIS 1344

To appear in: Current Opinion in Colloid & Interface Science

Received Date: 13 December 2019

Revised Date: 26 February 2020
Accepted Date: 5 March 2020

Please cite this article as: Bukusoglu E, Koku H, Culfaz-Emecen PZ, Addressing Challenges in the
Ultrafiltration of Biomolecules from Complex Aqueous Environments, Current Opinion in Colloid &
Interface Science, https://doi.org/10.1016/j.cocis.2020.03.003.

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.

© 2020 Elsevier Ltd. All rights reserved.

Addressing Challenges in the Ultrafiltration of Biomolecules from
Complex Aqueous Environments

Emre Bukusoglu*, Harun Koku*, and P. Zeynep Culfaz-Emecen*

Department of Chemical Engineering, Middle East Technical University, Dumlupinar Bulvari No: 1,
Cankaya, Ankara, 06800 Turkey.

*corresponding author(s)
zculfaz@metu.edu.tr
harunk@metu.edu.tr
emrebuk@metu.edu.tr

Abstract
Substantial progress has been made in ultrafiltration processes towards the separation of colloidal
species from aqueous environments. Studies especially in the past decade have advanced the
separations using functional, nanostructured membrane materials, and a thorough understanding of
colloid-membrane and colloid-colloid interactions in aqueous media. In this context, this review
describes the recent advances in ultrafiltration of biomolecular species, most specifically proteins,
from complex environments involving a range of molecular species, ionic, non-ionic substances with
varying concentrations. Ongoing efforts hint that the complexity of the interactions plays a critical
role in the success of the ultrafiltration process that requires system-specific investigation of the
ultrafiltration process and process parameters.

Keywords: ultrafiltration, membranes, proteins, interfaces, interactions.

1. Introduction

Bio-based chemicals ranging from bulk to fine chemicals are ubiquitously in a colloidal size range.
Common examples of high-value bioproducts are pharmaceuticals, neutraceuticals, and other
functional proteins, which are mostly produced by microorganisms and animal cell lines. Although it
is possible to improve the yield, productivity and titer of the target biomolecule through an
integrated approach that also involves the design and development of efficient host strains and
production processes, the recovery and purification of the desired product from the production
medium remains a crucial and inevitably challenging step due to the complex aqueous environment
at the end of the fermentation.

1
Non-glycosylated proteins and enzymes are generally produced either extracellularly or
intracellularly by single-celled microorganisms, whereas glycosylated proteins including monoclonal
antibodies are mostly produced by animal cell-lines. In extracellular protein production, there is no
need to extract the cells and refold the target protein. After separation of producer cells (with size on
the order of microns) from the production medium through centrifugation or conventional filtration
methods, the product remains in an aqueous mixture together with a highly complex variety of other
species. On the other hand, in the case of intracellular protein production, separated cells need to be
lysed prior to the separation and purification of the target molecule, leading to an even more
complex medium in terms of cellular biopolymers. The kinds of species that may be present range
from common salts, alcohols and sugars to species such as proteins and oligopeptides, which are
highly labile with respect to their immediate environment. Thus, the separation of such species
becomes significantly challenging considering their limited stability and the complexity of their
interactions with other species in their medium. A very important recent challenge is to find methods
for the cost-effective separation of sensitive biological species from the product medium.

Ultrafiltration is a favorable approach for the separation of fragile species from the liquid mixtures
since it is a low temperature and relatively low shear process, which can easily be upscaled to a
continuous process. Membranes with pores of ca. 1-100 nm are used which can selectively reject
macromolecular species of ca. 5 kDa and higher using a transmembrane pressure difference (TMP) as
the driving force for permeation through the membrane.

The rate of permeation through an ultrafiltration membrane is typically characterized by the

permeance or the permeate flux, where the permeance is defined as the permeate flux (the amount
of solution permeating through the membrane in unit time per unit membrane area) divided by the
transmembrane pressure difference between the feed and permeate sides of the membrane. The
extent of separation between feed components is expressed in a number of different ways. When
the transmission (transport to the permeate side) or rejection (or retention, in the retentate side) of
a single species from the feed mixture is considered, the rejection (R) or sieving coefficient (S) is
used. Rejection of a certain species i, Ri, is defined as
Ri=1-ci,P/ci,F
Where ci,P and ci,F are the concentration of species i in the permeate and feed sides, respectively. The
sieving coefficient of species i, Si, is defined as Si=1-Ri.
When the transport of two species from solution is to be compared, the selectivity of one species, i,
with respect to the other, j, ψi/j is defined as
ψi/j=Si/Sj

2
The primary separation mechanism in ultrafiltration is size exclusion, where species larger than the
pores of the membrane are totally rejected and those that are smaller are either partially rejected via
steric effects or totally permeate in case they are significantly smaller than the pore dimensions. On
top of this, interfacial interactions between the membrane surface and the species in the feed
mixture affect the rejection of each species either due to preferential adsorption on the membrane
or via electrostatic repulsion of species with like charges to the membrane.

While ultrafiltration is currently being used in several industrial processes for treating biological
mixtures, its major application is either in protein concentration or desalting. Separation of similar-
sized proteins, which is of great technological importance, can hardly be achieved with conventional
ultrafiltration membranes largely due to the wide pore size distribution of most commercial
ultrafiltration membranes and inter-species interactions in the feed mixture as well as the
interactions of the species in the feed with the membrane surface. These phenomena not only
diminish the selectivity of the membrane towards different feed components, but also aggravate
fouling phenomena, which is the deposition of feed species on top of the membrane surface or
within the pores. Resolving these issues requires specialized membranes with finely tuned pore size
and specially designed pore surfaces as well as a thorough understanding of species-species and
species-membrane interactions.

In this context, we first outline the current state-of-the art in the fabrication of novel ultrafiltration
membranes aiming at sharp bioseparations, discussing the effects of membrane properties on
selectivity, permeance and fouling of the membranes during the process. Finally, we illustrate the
type and nature of interactions which affect the ultrafiltration process in the bio-mixtures and
discuss how they relate to the application of these novel membranes.

2. Current state-of-the-art
2.1. Narrow PSD membranes for sharp size selectivity

An ideal membrane for ultrafiltration is the one that exhibits high selectivity and high permeance,
with low and easily cleanable fouling property. There is a natural trade-off between the selectivity
and permeance of a membrane, in ultrafiltration, as well as in many other membrane separations
[1]. However, with advanced membrane materials and fabrication techniques, the trade-off
relationship can be pushed towards an overall increase in selectivity and permeance. High selectivity
and permeance can be achieved in a membrane with a thin selective layer with a narrow pore size
distribution. For decades, ultrafiltration membranes have been prepared using Non-Solvent Induced
Phase Separation (NIPS) or the so-called phase inversion technique, which allows single-step

3
synthesis of integrally asymmetric membranes with a thin selective layer on a supporting layer with
macroporous structure with minimal resistance to mass transfer. The fabrication procedure, in its
simplest description, starts with a homogenous solution of the membrane polymer and its solvent,
which is contacted with a non-solvent for the polymer that is fully miscible with the solvent. The
overall composition moves toward the two-phase region and the system separates into a polymer-
rich phase, eventually forming the membrane, and a polymer-lean phase, which forms the pores
(Figure 1A). In the end, the interface of the polymer that is in contact with the non-solvent becomes
the selective skin layer typically with a thickness of ~100 nm - 1 µm, and below forms a macroporous,
much thicker, supporting layer with interconnected macroscopic pores of size up to >10 µm (Figure
1B). Although the NIPS method has been shown to be a promising route to fabricate ultrafiltration
membranes with varying average pore size and molecular weight cut-off, the low porosity of the skin
layer and the wide distribution of the pore sizes (Figure 1F, obtained by processing the SEM
micrograph of Figure 1B) has limited their use to separations where the species to be separated are
typically at least an order of magnitude different in size [2].

A C diblock co-polymer triblock co-polymer

n
+ N m n
+ m
+ k
OH
PS P4VP PS PHS PS

PS-b-P4VP PS-b-PHS-b-P4VP
D

spheres cylinders gyroid lamellar

E Self-assembly wit h N on-solvent I nduced Phase Separat ion

evaporation nonsolvent
B N on-solvent I nduced Phase Separat ion
cross-sect ion

F cross-sect ion

500 nm 100 nm
nonsolvent

t op surface t op surface

500 nm

Figure 1. (A) a typical ternary phase diagram for a system involving polymer, solvent, and nonsolvent.
The star symbols and the dashed line qualitatively indicates a NIPS process path. (B) A representative
sketch for the NIPS method. The electron micrographs of the cross-section (left) and surface (right) of
an ultrafiltration membrane are shown in the bottom. The two star symbols indicate representative

4
sketches of the microstructures corresponding to the process path shown in A. (C) Two common
amphiphilic diblock and triblock co-polymer architectures used for the synthesis ultrafiltration
membranes through SNIPS method. (D) Common unit cell symmetries for the assembly of block co-
polymers in bulk. (E) A representative sketch for the SNIPS method. The electron micrographs of the
cross-section (left) and surface (right) of an ultrafiltration membrane are shown in the bottom.
Adapted with permission from Ref. [3]. Copyright (2013) American Chemical Society.

Recent trends in macromolecular self-assembly has offered opportunities to extend the NIPS
techniques to a better control over the microstructure of the membranes. Block copolymers (Figure
1C) had also been used for membrane fabrication by their self-assembly. Amphiphilic block
copolymers could self-assemble into useful symmetries such as lamellar, cylindrical, and gyroid
phases depending on the relative fraction of the polymeric blocks comprising the macromolecule
(Figure 2D).

The self-assembly and non-solvent-induced phase separation (SNIPS, Figure 1E) technique has
offered an elegant and simple method to fabricate isoporous membranes (Figure 1F, obtained by
processing the SEM micrograph of Figure 1E) made of block copolymers where the thin isoporous
selective layer is formed on a macroporous supporting layer which forms in a single step, thereby
yielding an intrinsically asymmetric membrane with a highly porous and selective skin layer. A skin
layer with a thickness of ~200-300 nm and uniform pore diameter of ca. 40 nm was obtained as
viewed from SEM images. During the formation of this microscopic structure, it was shown that there
are two competing mechanisms, non-solvent induced phase separation and the self-assembly of
block copolymers, that result in the formation of a hexagonally packed, cylindrical surface pores with
a uniform diameter [4]. The porosity was exceptionally high (ca. 2.4 x 1014 pores/m2) and the
permeance was an order-of-magnitude higher than membranes of similar average pore diameter
that would be obtained by regular NIPS [5].

Variations of the SNIPS methods via the use of block copolymers with different architectures,
different symmetries, within blends of different compositions, or additional post modifications offer
a range of possible routes that lead to control over the pore sizes and their structures. As an example
study, Clodt et al. employed PS-b-P4VP with various molecular weights ranging between 76 to 300
kg/mol and 17% to 26% P4VP wt% to synthesize ultrafiltration membranes with pore sizes in the
range 17 to 86 nm (Figure 2A)[6]. In a different approach, a triblock copolymer polystyrene-b-poly(4-
hydroxystyrene)-b-polystyrene (PS40k-b-PHS27.3k-b-PS40k), which would self-assemble to form a
lamellar symmetry with a periodicity of 50 nm in bulk, was used in the synthesis of the membranes
The SNIPS method allows combination of additives with polymers, such as 11% wt PS40k-b-PHS27.3k-b-

5
PS40k solution in dioxane, that result in open pores with sizes 9 ± 2 nm by controlled hydrogen
bonding. Further reduction of the pore size and control over the surface pore structures were
possible through the use of imidazole or pyridine, where the former promotes stronger interactions
with -OH groups. Varying the relative concentrations of such hydrogen bonding additives, the pore
sizes of the membranes could finely be tuned between 3 ± 1 nm to 5 ± 1 nm. In a different approach,
Yu et al. achieved a control over the pore size via controlled, electroless growth of gold at the
surfaces of the pores of the membranes made from block copolymers PS175k-b-P4VP65k (Figure 2B)[7].
A precise control over the pore sizes in the range 3.0 nm to 20 nm of pore sizes. By using a blend of
two block copolymers, PS1807-b-P4VP609 and PS144-b-PAA22 where the P4VP and PAA blocks interact
through hydrogen bonding, the pore size could be driven down to 1.5 nm, which is extremely
attractive as it corresponds to the nanofiltration range where many significant potential separations
can be carried out (Figure 2C)[8]. On the other hand, binary blends of different pairs of PS-b-P4VP
diblock copolymers were shown to influence the pore size of the membranes. It was also shown that
the resulting pore size of the membranes can be predicted via a linear relationship to the
composition of the blend [9]. Modifying the hydrophobic PS block of the membrane with graphene
oxide, the antifouling properties of the membranes was also improved, thereby tackling the most
important bottleneck of ultrafiltration processes [10].

A
low MW BCP
pore pore

B
gold deposition

C
Addition of
low MW BCP

pore

pore

Figure 2. Schematic illustration of three recent approaches for the reduction of the membrane pore
sizes. (A) The method of synthesis with low molecular weight block copolymers developed by Clodt
et al. [6], (B) The method of electroless gold deposition developed by Yu et al. [7], and (C) the
method of addition of low molecular weight block co-polymers developed by Yu et al. [8].

6
The ultrafiltration membranes fabricated by SNIPS were tested in protein rejection from aqueous
media, with synthetic single protein and binary protein solutions at different ionic strength and pH
values, in a number of studies. Qiu et al. have shown, through diffusion experiments at physiological
pH (7.4) using a BSA (MW=67 kDa, Dh=6.8 nm) - IgG (MW=150 kDa, Dh=14 nm) binary mixture, where
the size difference is too small for selective permeation through regular NIPS membranes, a
separation factor of 87 for BSA over IgG [3]. This selectivity could not be explained by the size
difference alone and the high rejection of IgG was attributed to electrostatic interactions and
adsorption on pore walls. The membranes were also selective for BSA over BHb (65 kDa) which had
the same size yet different pI values (4.7 and 7.0 for BSA and BHb, respectively) [3]. When the
pyridine groups on the membranes were quaternized to impart a positive charge to the membranes,
these proteins permeated preferentially when the pH was equal to their pI, but were rejected when
they were charged. Rejection of the positively charged protein was attributed to electrostatic
repulsion, while rejection of the negatively charged protein was attributed to its adsorption on the
pore walls. In a later study, isoporous PS membranes fabricated by degrading the P4VP group of the
PS-b-P4VP in alkaline medium showed selectivity for BSA and lysine over IgG, which was essentially
attributed to size differences [11].

Separation of BSA and IgG has also been reported using nanocrystalline silicon membranes fabricated
via high precision silicon deposition, annealing and subsequent etching making use of spontaneous
pore formation during the rapid thermal annealing step [12]. With these membranes as well, the
separation performance was affected not only by the pore size but also the negative charge of the
native silicon oxide layer on the pores which gave rise to electrostatic exclusion as well as adsorption
effects.

Recently, Zhang et al. prepared membranes with nanospheres of PS15k-b-PAA1.6k and depositing them
on polycarbonate membranes of pore sizes ~600 nm to form selective membranes with effective
pore sizes around 6.5 – 10 nm [13]. Since the pores of the membrane corresponded to the voids
between the deposited nanospheres, of which the outer surfaces were PAA, the charging of the
pores could be varied by changing the solution pH, thus helping the separation of the species with
similar molecular weights based on their charges. They have shown this property with the separation
of BSA (66 kDa) and bovine hemoglobin (BHb, 65 kDa).

Amphiphilic molecules at smaller scales have also provided opportunities for designing separation
membranes through their liquid crystalline phases. In liquid crystals (LCs), the constituent molecules
possess significant ordering whereas maintaining their fluidic properties. Liquid crystalline phases can

7
be obtained through their two classes, the so called lyotropic and thermotropic liquid crystals. In
thermotropic LCs, the ordering transitions can be driven by temperature only, whereas it is a
function of both temperature and the composition in lyotropic LCs. Reactive, crosslinkable
counterparts (LC monomers) of LCs were employed in the synthesis of membranes. The challenge in
these studies was to synthesize membranes with pores that span through the thickness of the films.
Small amphiphilic molecules, such as the monomer shown in Figure 3A, have been shown to self-
assemble into structures of inverted hexagonal (Hıı) phase symmetries with cylindrical channels
where the hydrophilic head groups of the monomers were forming the hydrophilic channels [14].
Polymeric films were obtained following photopolymerization of the solutions of monomer, leading
to ~1.2 nm in diameter pores leading to a sharp rejection of the species by size. Further studies with
similar monomer structures (Figure 3B) have also demonstrated that films with different unit cell
symmetries, for example cubic bicontinuous or lamellar phases, could be obtained [15]. A more
recent study has employed the thermotropic LCs to design polymeric films with nanopores [16]. The
method relied on the use of hydrogen-bridged smectic LC monomer with crosslinkable acrylate
groups to form the layered structure of the lamellar phase. With these methods, mechanically robust
films with d-spacing of 2.7 nm (dependent on the sizes of the constituent molecules) were
synthesized with selectivity of species of the sizes in the order of nm based on their charges. More
recent examples of the LC-templated synthesis of polymeric membranes have combined the use of
the mixtures of reactive and non-reactive mesogens in the thermotropic nematic phases (Figure 3C),
that allow templating the nanostructure of the polymeric films. Following photopolymerization and
removal of the unreacted LCs, polymeric films with polydisperse mesoscale pores was obtained that
were in the order of 10 nm in diameter [17]. Although synthesis of membranes with narrowed pore
sizes are yet to be discovered using this method, it allows easy means to manipulate the
directionality of the pores via tuning the LC ordering before polymerization. Studies have shown that
the pure water permeances could be increased by two orders of magnitude, from 1.6 ± 0.1
L/m2.h.bar to 658.5 ± 211.1 L/m2.h.bar, when the films were synthesized with parallel and
perpendicular alignment of the films. The rejections up to 78% were obtained when the filtrations
were performed from solutions of 1 g/L of BSA in PBS.

8
Figure 3. Examples of lyotropic (A, B) and thermotropic (C) liquid crystalline molecules. (A) The
monomer used to synthesize membranes with inverse hexagonal symmetries. Adapted with
permission from Ref. [14]. Copyright © 2005 WILEY-VCH Verlag GmbH & Co. (B) the monomer used
to synthesize membranes with bicontinuous cubic phase symmetries. Adapted with permission from
Ref. [15]. Copyright (2011) American Chemical Society. (C) the reactive and nonreactive mesogen
mixture used for the nematic LC-templated synthesis of polymeric membranes. Adapted with
permission from Ref. [17]. Copyright (2018) American Chemical Society.

In isoporous membranes, size selectivity is often not the only mechanism in play, as in the size range
relevant to ultrafiltration, interfacial interactions between the proteins and the membrane are
significant and at times may dominate the separation behavior, as shown in many previous studies
mentioned above. Efforts have also been devoted for the design of membranes which employ the
use of size selectivity together with charging of the membranes, therefore to take advantage of the
electrostatic interactions for enhanced selectivity.

While these membranes offer significantly increased selectivity and permeance, care should be taken
to avoid fouling in the form of a gel or cake layer formation on the membrane, as in such a case the
permeance of the membrane will be dominated by the resistance of the gel layer and the selectivity
could likely be compromised.

9
2.2. Membranes with designed surface charge for selectivity based on electrostatic
interactions

Conventional ultrafiltration membranes prepared via phase inversion (or NIPS) can typically separate
molecules differing by at least an order of magnitude in size. As mentioned in the previous section,
isoporous membranes can greatly enhance this selectivity, effectively fractionating proteins much
closer in dimensions. An additional parameter that is exploited along this line is the charge on the
membrane and protein surfaces. By designing the charge on the membrane surface and tuning the
net charge on proteins through varying the pH, high selectivities were demonstrated between
proteins of similar size.

The pH and the ionic strength of the ultrafiltration feed solution due to the presence of salts causes
critical changes in the charging of the colloidal species (detailed in the next section), therefore
affecting their permeation in ultrafiltration. One of the key properties influencing the outcome is the
isoelectric point of the protein molecules. Proteins at their isoelectric point have zero net charge, and
positive or negative net charge when the medium pH is above or below their isoelectric points,
respectively, and this can be exploited for practical operations. Nyström et al. have shown that
separation of proteins from their mixtures could be accomplished by tuning the solution pH to the
isoelectric point of one of the protein species in the mixture, which in turn permeates more readily
through the membrane compared to the other protein species that are relatively more charged at
that pH, leading to their electrical repulsion by the membrane surface [18].

Charged membranes and suitably adjusted feed pH and ionic strength were used to increase
selectivity and throughput in separations of proteins, typically from synthetic mixtures of controlled
composition [19,20]. In High Performance Tangential Flow Filtration (HPTFF) process output is further
optimized by operating in the pressure-dependent flux regime, i.e. below the limiting flux where no
gel formation on the membrane takes place, and by using modules with enhanced hydrodynamics to
minimize concentration polarization as well as transmembrane pressure gradients across the
module. With this approach van Reis et al. fractionated BSA (68 kDa, pI = 4.7) and antigen binding
fragment (Fab) of a recombinant DNA antibody (45 kDa, pI = 8.5) [19]. They used negatively and
positively charged membranes in a pH range of 4.6 - 8.7 covering the pI values of both proteins and
reported that the highest sieving coefficients were obtained when the protein was neutral and the
highest rejections when the protein has the same charge as the membrane. The effect of protein
charge in increasing rejection is both due to electrostatic exclusion when the membrane is of the
same charge and to the increased effective volume. Later, Ebersold et al. showed that electrostatic
exclusion can be used to fractionate protein variants that differ by only a single amino acid residue
using myoglobin (17.9 kDa, pI = 6.9) and a negatively charged variant with a negatively charged

10
membrane at pH values above the pI of both variants [20]. In both studies it was also demonstrated
that increasing ionic strength decreases selectivity due to its shielding effect on electrostatic
interactions. Charged membranes were also investigated for fractionation of whey proteins [21].
Overall, the highest selectivities were observed under conditions where one protein is neutral and
the other bears the same charge as the membrane. The selectivity using charged membranes could
also be increased to some extent through the use of electrical potential as an additional driving force
to transmembrane pressure [22].

Charged membranes were also used in a few studies with actual feed streams. In one such study,
positively charged cellulose membranes were used to fractionate a recombinant DNA derived
antibody fragment (99 kDa, pI = 9.1, 2.3 mg/mL in mixture) from host cell proteins (0.003 mg/mL in
mixture) expressed in Eschericia coli using a HPTFF procedure optimizing the buffer pH, ionic strength
and the permeate flux for high selectivity and high throughput [23]. By optimizing process
parameters, a purification factor and yield comparable to the conventional purification process of
hydrophobic interaction chromatography followed by ultrafiltration/diafiltration was reported.

Overall, charged membrane surfaces have shown promising potential for separation of proteins of
different charge at optimized buffer compositions. While two proteins of similar size have been
sharply separated in synthetic mixtures, high selectivities in real mixtures where there are multiple
proteins as well as ionic and nonionic species is more challenging due to the variety in protein
species’ properties and interspecies interactions.

2.3. Effect of interfacial interactions on the membrane separation

A regular ultrafiltration process concentrates the colloidal species at the feed side of the membrane.
This feed contains not only the target molecules but also other colloidal species, salts and small
organic molecules that need to be separated using multiple steps. Although ultrafiltration can
selectively separate the target species from this complex mixture, high colloidal concentrations are
frequently observed to cause aggregation, leading in turn to complications such as membrane
fouling, reduced activity due to loss of protein conformation, and an increase in the solution
viscosity. Aggregation is a consequence of the interactions between the target molecules among
themselves and also with other constituent species of the liquid medium.

Electrostatic interactions due to charged moieties including groups on the protein, hydrogen
bonding, van der Waals forces and hydrophobic interactions in combination with each other play a
critical role in determining whether a protein is folded or denatured, in solution or a precipitate.
These events usually lead to solution conditions that may be hard to recover from, for example due

11
to irreversible aggregation of protein [24,25]. Thus, there have been attempts and some progress
towards decreasing such effects at high concentrations. The practical conclusion of these studies is
that solubility depends on many variables including temperature, pH, the identities and
concentrations of the dissolved salts or organic agents. Depending on the desired outcome, these
variables can be controlled and manipulated to stabilize the target proteins, to induce precipitation
of the unwanted proteins, or to obtain a certain target crystal.

While a fully predictive theory of how or when a protein will precipitate is not available, the forces
and interactions that govern the solubility and precipitation of proteins, and the associated kinetic
and thermodynamic principles are now fairly well-understood [24–26]. This knowledge has
traditionally guided separations e.g. to explore the ranges of ionic strength or isoelectric point (pI) to
induce precipitation in a protein [27]. For instance, it is known that increasing the concentration of a
salt present in the medium may induce the precipitation of a protein (salting-out) or conversely
increase its solubility (salting-in). Thus, aggregation of a protein may occur within its salting-in range
upon dilution, or within its salting-out range by increasing the salt concentration. The concentration
of salt at which the protein transitions from salting-in to salting-out, as well as the extent of the
variation of solubility with salt concentration, depend on the salt and protein used, although some
patterns have been identified. Hofmeister's pioneering study, published more than thirteen decades
ago, tested salts with respect to their potential to precipitate protein (i.e. salting-out). The result of
this and subsequent studies is the Hofmeister series in which, based on their salting-out tendencies,
anions could be ordered as SO42->HPO42->Acetate>Citrate>Cl->NO3->ClO3->I->ClO4->SCN- and cations as
NH4+>K+>Na+>Li+>Mg2+>Ca2+>Guanidium.

Although such empirical guidelines are useful, the physicochemical properties of the biomolecular
species are usually complex, in that they do not change monotonously (as their hard-colloid
counterparts) and do not demonstrate a simple dependence on the pH or ionic strength of the
aqueous medium. Instead, protein interactions are strongly dependent on the identities and
concentration of salts and other co-solutes in the environment, as well as the concentrations of the
protein species themselves. Furthermore, the heterogeneous, anisotropic distribution of charges and
hydrophobic regions is influenced by the mixture environment, and plays a profound role. The
distribution of charge for example, depends on pH and it is now relatively straightforward to study
and visualize the details of the pH-dependent charge of a protein using free software. For example,
Figure 4 shows the change in the charge distribution of a human growth hormone (hGH) as a
function of the solution pH (note that the pI of hGH is around 5.1). Thus, it is possible to move
beyond the basic concept of net charge, and investigate anisotropic distributions of localized charges,
which may even dominate the interactions of a protein. It has been shown, for example, that

12
proteins may, somewhat counter-intuitively, attach to an adsorption surface with the same sign of
net charge, by virtue of oppositely charged patches locally associating with each other [28]. A striking
example is given by Leckband et al., in which the researchers show that the forces between the
protein pair biotin-streptavidin can be switched from repulsive to attractive by changing the charge
state of only two amino acids [29]. Thus, a simplified description of protein structure is often
insufficient to predict effects that may be governed by local interactions that can dominate overall
behavior.

Figure 4. Variation of charge distribution with pH of a human growth hormone molecule (PDB access
file 1HGU). Regions colored deep red indicate relatively negative electrostatic potentials, the deeper
blue regions positive, and white indicates charge states close to neutral. Obtained using the APBS
biomolecular solvation software suite with the default options [30].

To address the complexity of protein interactions and establish a better understanding of the
phenomena leading to the association of proteins with each other as well as other moieties, more
rigorous measures and methods of analysis are increasingly used. In particular, attempts to predict
the association of a protein with itself or with other protein species have become widespread. This is
important for ultrafiltration since aggregation or solubilization of the protein molecules in the
medium directly affects critical properties such as viscosity or the apparent sizes of the molecular
species in the solutions. One useful quantity for studying self-association is the osmotic second virial
coefficient, B22, which has a well-defined basis in mixture molecular thermodynamics. This coefficient
is typically interpreted as a measure of the overall attractive or repulsive tendency between
molecules of the same protein for a given set of conditions - a highly negative value of B22 implies
strong attractive forces between the molecules of the protein, and thereby a propensity to
aggregate. It is possible to determine B22 experimentally, using methods such as static light
scattering, small angle neutron scattering and self-interaction chromatography [31,32]. In some

13
cases, experimental B22 values have been used to identify distinctive trends such as a 'crystallization
slot', i.e. a range in which crystallization may be observed, or simply as a way to characterize and
interpret protein-protein interactions under varying conditions [33–35]. Dumetz et al. analyzed the
self-interaction trends of seven different proteins by observing the variation of B22 with salt
concentration, using several different salts [34]. They observed that the B22 values could be positive
or negative at low concentrations, and increase or decrease with increasing salt concentration
depending on the type of protein or salt. The B22 values of proteins in sodium chloride varied slightly,
if at all for salt concentrations as high as 4 M, whereas for ammonium sulfate, the B22 values of all
proteins were found to eventually decrease with increasing salt concentration to negative values
despite the different initial trends. However, the onset of the drop varied from one protein to the
other. The authors also studied the precipitation behavior, characterized the precipitates as
aggregate or crystal, and noted that the B22 decrease observed for the ammonium sulfate solutions
coincided with protein crystallization followed by aggregate formation.

For a deeper understanding of the local interactions between molecules of the same species, it is
possible to compute B22 values based on theory, starting from the crystal structure of the protein and
calculating the potential of mean force over the relative orientations of two molecules of the protein.
Although limited by the underlying simplifications, atomic-level approaches are especially
informative when studying local effects due to the anisotropy of the molecule. Such detail can be
utilized, for example, to identify relative orientations of a protein molecule that result in a high
degree of complementarity [36]. Similar fine-grained studies have also shed light on the effects of
ionic species and protein, for example by proposing a molecular level interpretation of the
Hofmeister series [37]. The detailed treatment by Okur et al. analyzes the interactions of cationic and
anionic species with the protein backbone and its charged, polar or hydrophobic side chains based on
data from molecular dynamics simulations, spectroscopic methods and thermodynamics principles.
The authors confirmed the order of the Hofmeister series for the interactions of cationic salt species
with the anionic side chains, however, they also identified cases of deviations from and even a
reversal of the order, and attributed them mainly to interactions with the positively charged side-
chains of the biomolecular species. The study highlights the importance of avoiding simple rules in
favor of system-specific investigation based on the architecture of the biomolecules of interest, in
order to predict the effects of the salts on the stabilities of the proteins.

Here we note that beyond pH and salt ions in the separation medium, protein stability can also be
influenced by the presence of additional molecules in the aqueous environment such as amino acids,
sugars, carbohydrates and surfactants. Such agents are frequently used in pharmaceutical industries
for the stabilization of the proteins, thus, have been studied extensively in the literature. There have

14
been several review articles published that present the details on the mechanisms of such species on
the stability of the proteins [38]. Although their effects are more critical at high concentrations of
proteins, they are also effective at the intermediate concentrations. For example; Tween 20 and
Tween 80 have shown to prevent the proteins from adsorbing to the hydrophobic surface as well as
to protect from shear-induced aggregation during processing [39]. It should be noted that the
influence of co-solutes such as poly(ethylene glycol) depends not only on the direct interactions of
these substances with protein, but also on the effects of other species such as salts, which may
modify these interactions [40].

Although many studies focus on the quantification of the interaction of like protein molecules, the
study of cross-species interactions can be useful in investigating heterogeneous aggregation of
proteins. A parameter analogous to B22 is the osmotic second virial cross coefficient (B23), which
serves as a measure of the overall interaction between two different protein species, The B23 can also
be estimated experimentally, using cross-interaction chromatography (CIC) [41–43]. In complex
mixtures that might contain hundreds of proteins at widely varying concentrations, determining B23
values may not be as practical, but CIC in combination with other analytical tools can still be used to
obtain insight into the interaction behavior of a target molecule with other proteins [44,45].

Cross-interactions between proteins have been studied using ultrafiltration directly as well; Filipe and
Ghosh describe their investigation of protein-protein association for selected protein pairs, where
one of the proteins (e.g. lysozyme) is readily transmitted by the membrane while the other (e.g. BSA)
is retained [46]. Using pulsed injection ultrafiltration, the effect of the retained protein on the sieving
coefficient of the transmitted one is used as a measure of the interaction of the two species. Their
analysis showed that associative interactions between proteins forming co-precipitates or soluble
aggregates can decrease the transmission of a protein, which is transmitted to a large extent when
alone in solution. Increased transmission due to the presence of a second protein was also shown
under other conditions, which was attributed to Donnan effects causing the transmission of an
otherwise rejected protein to maintain the transmembrane charge balance. Using similar proteins in
single, binary and ternary mixtures, Nyström et al. showed how protein transmission, selectivity and
fouling are affected by the presence of more than one protein in the feed solution [18]. They carried
out filtrations at different pH values, compared to the pI of the proteins and with membranes of
different hydrophilicity and charge, and showed that protein aggregates not only influenced
individual protein rejections and selectivities but also the extent of fouling on the membrane [18].

Protein-protein and protein-membrane interactions are also a major reason for fouling on the
membrane surface. Preventing protein adsorption on the membrane can greatly relieve detrimental
effects of fouling on the process, both by decreasing the performance loss during the filtration stage

15
and by facilitating flux recovery after fouling has occurred. While cellulose membranes, which are
prevalent in ultrafiltration of biological mixtures, show good antifouling performance due to their
inherent hydrophilic character [47], surface modifications of other membrane materials with
poly(ethylene glycol) or zwitterionic groups were also shown to resist fouling [48]. In recent years,
zwitterionic membrane surfaces attracted increased interest as they were shown to resist fouling of
species of varying chemistries, including proteins [49,50], and remain stable at a wide pH range,
enabling both operation and cleaning in this range [51].

In conclusion, interactions between species in the feed mixture and those with the membrane
surface may pose obstacles to the process performance, such as when they lead to fouling on the
surface or a decrease in selectivity due to aggregation in the solution, and yet it may also be possible
to exploit these interactions for increased selectivity. A fully predictive, general approach to identify
and address these challenges and opportunities is currently unavailable; theoretical studies cannot
capture the full complexity of the multitude of possible interactions between the species of interest
and their environment, while experimental studies focus mainly on specific systems of interest.
However, advances brought about by high-performance computing, coupled to high-throughput
experimentation and sophisticated analysis techniques increasingly enable more in-depth study of
real systems, and guide the analysis and design of the ultrafiltration process.

3. Concluding Remarks and Future Outlook

While ultrafiltration is defined as a membrane separation focusing on colloidal species, the

separation of such is not possible based merely on size selectivity. As highlighted above, the
complexity of the interactions within the feed mixtures and the membranes requires a system-
specific analysis for the choice of the conditions for an ultrafiltration process. As discussed with
exemplifying studies, the choice of the membranes, their pore size distribution and functionality,
solution pH and ionic strength requires a comprehensive thought that is critical for the feasible
design of an ultrafiltration process.

To date, studies show that isoporous membranes enhance selectivity between protein species that
are close in size to values that cannot be achieved with conventional ultrafiltration membranes. The
pore size, on the other hand, is almost never the only determining parameter with these
membranes, as in the size range relevant to ultrafiltration, protein-membrane interactions are
significant and can affect or even dominate the separation performance. Combined effects of narrow
pore size distribution and charged or functional pore surfaces can further enhance selectivity.

16
Furthermore, using responsive groups on the pore surfaces may allow precise tuning of permeance
and rejection, thereby a more controlled and selective separation.

Using membranes with charged surfaces and pH control in relationship to the pI of protein species in
the mixture, high selectivities between proteins of similar size can be achieved. On the other hand,
electrostatic attraction between the charged membrane and oppositely charged species in the feed
can potentially aggravate fouling phenomena. Hindering deposition of proteins while maintaining the
charged nature of the surface and the charge-based selectivity may be achieved through patches of
charged and antifouling regions on the membrane. Another approach could be to have fouling-
release surfaces in response to a certain stimulus, from which foulants can be removed easily.

Protein-protein and protein-membrane interactions become more pronounced at highly

concentrated solutions, which is the case when concentration polarization and/or fouling occurs on
the membrane surface during an ultrafiltration process. While aggregation of a single protein can
increase its rejection, aggregation of two or more different proteins or their coupled transport will
typically decrease the selectivity between these species. This may or may not be advantageous
depending on the separation desired, but certainly need to be considered specifically for overcoming
the challenge of separating complex biomolecular mixtures. Attractive interactions between different
proteins or proteins and smaller species can also be employed to remove species smaller than
ultrafiltration range using ultrafiltration membranes.

Studies to integrate molecular modeling, high precision measurements of interaction parameters and
detailed observations and profiling of macroscopic behavior are expected to intensify, to formulate a
unified, multi-scale description of protein interactions and thereby guide separation procedures.

The amphiphilic small molecular species or mesogenic monomers appears to be a possible candidate
design tool for the synthesis of membranes with tunable internal structuring. Although past studies
have demonstrated the possibility of the membranes to be used for ultrafiltration purposes, a more
focused approach is required for the ultrafiltration processes to benefit from the inherent ordering of
the liquid crystalline phases. Such approaches may involve either, for example, combining the
current techniques with the molecular species exhibiting liquid crystalline phases or post
modification of the currently developed techniques with liquid crystals to incorporate responsive
characteristics towards the external stimuli. Beyond these, the literature developed for the assembly
of the colloidal species in liquid crystals through elastic interactions, formation of the defects in liquid
crystals, or even the liquid crystalline phases with additional complexity beyond nematics or
cholesterics may offer additional levels of control over the structures of the ultrafiltration
membranes.

17
With all that said, membranes with high selectivity and permeance will only be practically useful in a
predictable and durable ultrafiltration process if fouling can be controlled. Preventing the occurrence
of concentration polarization through control of process parameters can avoid deposit formation on
the membrane. Preventing adsorptive fouling requires an antifouling surface, which is typically one
that forms a hydration layer around and hence prevents adsorption of foulants, such as surfaces with
poly(ethylene glycol) groups, zwitterionic groups or the cellulose membrane surface itself In addition
to avoiding the formation of fouling layers on the surface, it is also possible and necessary, as fouling
at some stage of the process is typically unavoidable, to employ strategies that aim at removing
foulant layers easily once they form.

Overall, the species that are to be purified or concentrated are usually present in extremely complex
environments comprised of a range of biomacromolecular species with different molecular weights,
and charges. As we have identified in this Article, during treatment of such complex environments,
the solution properties such as pH, colloidal size range, charge distribution, ionic effects, interfacial
interactions, etc. should be considered to design an efficient separation process (Figure 5). It has
been accepted that there would be no single-step solution for the purification of such species,
instead, cascade or hybrid processes would be a good alternative design for the purification. The
current state-of-the-art in the literature has reached to a level that can resolve the purification issues
of the biomolecules through designing membranes with specific pore size distribution and charges.
Although such approaches have made significant progress, their use has been limited to simple
aqueous environments, sometimes applicable to binary protein mixtures. Thus, further progress is
required in order to apply ultrafiltration to the purification of complex aqueous environments.

18
 +
+ -
+ -
-
+
-
-

- +
- -
+ -
+

+ - +
- - - - - - - - -
-

Figure 5. A sketch representing the complexity of the aqueous environments involving the
biomolecular species.

Acknowledgments

The authors thank Prof. Pınar Çalık and Prof. Halil Kalıpçılar for their insightful discussion.

This research did not receive any specific grant from funding agencies in the public, commercial, or
not-for-profit sectors.

Conflict of Interest

The authors declare no conflict of interest.

Corresponding authors

To whom the correspondence should be addressed. zculfaz@metu.edu.tr, harunk@metu.edu.tr,

emrebuk@metu.edu.tr.

References
[1]* Mehta A, Zydney AL. Permeability and selectivity analysis for ultrafiltration membranes. J
Memb Sci 2005;249:245–9. https://doi.org/10.1016/j.memsci.2004.09.040.
This communication presents data on the hydraulic permeability and bovine serum albumin

19
selectivity for various ultrafiltration membranes from literature showing the inverse, or trade-off,
relationship, and describes this with a theoretical model.
[2]** Sadeghi I, Kaner P, Asatekin A. Controlling and expanding the selectivity of filtration
membranes. Chem Mater 2018;30:7328–54.
https://doi.org/10.1021/acs.chemmater.8b03334.
This perspective reviews and discusses recent advances in fabrication of liquid filtration membranes
for enhancing their selectivity based on size, charge and other molecular criteria.
[3] Qiu XY, Yu HZ, Karunakaran M, Pradeep N, Nunes SZ, Peinemann K V. Selective separation of
similarly sized proteins with tunable nanoporous block copolymer membranes. ACS Nano
2013;7:768–76. https://doi.org/10.1016/j.proeng.2012.08.450.
[4] Oss-Ronen L, Schmidt J, Abetz V, Radulescu A, Cohen Y, Talmon Y. Characterization of block
copolymer self-assembly: From solution to nanoporous membranes. Macromolecules
2012;45:9631–42. https://doi.org/10.1021/ma301611c.
[5] Peinemann KV, Abetz V, Simon PFW. Asymmetric superstructure formed in a block copolymer
via phase separation. Nat Mater 2007;6:992–6. https://doi.org/10.1038/nmat2038.
[6] Clodt JI, Bajer B, Buhr K, Hahn J, Filiz V, Abetz V. Performance study of isoporous membranes
with tailored pore sizes. J Memb Sci 2015;495:334–40.
https://doi.org/10.1016/j.memsci.2015.07.041.
[7] Yu H, Qiu X, Nunes SP, Peinemann KV. Self-assembled isoporous block copolymer membranes
with tuned pore sizes. Angew Chemie - Int Ed 2014;53:10072–6.
https://doi.org/10.1002/anie.201404491.
[8] Yu H, Qiu X, Moreno N, Ma Z, Calo VM, Nunes SP, et al. Self-assembled asymmetric block
copolymer membranes: Bridging the gap from ultra- to nanofiltration. Angew Chemie - Int Ed
2015;54:13937–41. https://doi.org/10.1002/anie.201505663.
[9] Radjabian M, Abetz V. Tailored pore sizes in integral asymmetric membranes formed by
blends of block copolymers. Adv Mater 2015;27:352–5.
https://doi.org/10.1002/adma.201404309.
[10] Shevate R, Kumar M, Cheng H, Hong PY, Behzad AR, Anjum D, et al. Rapid size-based protein
discrimination inside hybrid isoporous membranes. ACS Appl Mater Interfaces 2019;11:8507–
16. https://doi.org/10.1021/acsami.8b20802.
[11] Shevate R, Kumar M, Karunakaran M, Canlas C, Peinemann K V. Surprising transformation of a
block copolymer into a high performance polystyrene ultrafiltration membrane with a
hierarchically organized pore structure. J Mater Chem A 2018;6:4337–45.
https://doi.org/10.1039/c7ta09777h.
[12] Striemer CC, Gaborski TR, McGrath JL, Fauchet PM. Charge- and size-based separation of

20
 macromolecules using ultrathin silicon membranes. Nature 2007;445:749–53.
https://doi.org/10.1038/nature05532.
[13] Zhang W, Cheng P, Cheng J, Li N, Wang Y, Yang J, et al. Ultrathin nanoporous membrane
fabrication based on block copolymer micelles. J Memb Sci 2019;570:427–35.
https://doi.org/10.1016/j.memsci.2018.10.066.
[14] Zhou M, Kidd TJ, Noble RD, Gin DL. Supported lyotropic liquid-crystal polymer membranes:
Promising materials for molecular-size-selective aqueous nanofiltration. Adv Mater
2005;17:1850–3. https://doi.org/10.1002/adma.200500444.
[15] Ichikawa T, Yoshio M, Hamasaki A, Kagimoto J, Ohno H, Kato T. 3D interconnected ionic nano-
channels formed in polymer films: Self-organization and polymerization of thermotropic
bicontinuous cubic liquid crystals. J Am Chem Soc 2011;133:2163–9.
https://doi.org/10.1021/ja106707z.
[16] Schenning APHJ, Gonzalez-Lemus YC, Shishmanova IK, Broer DJ. Nanoporous membranes
based on liquid crystalline polymers. Liq Cryst 2011;38:1627–39.
https://doi.org/10.1080/02678292.2011.603504.
[17] Karausta A, Bukusoglu E. Liquid crystal-templated synthesis of mesoporous membranes with
predetermined pore alignment. ACS Appl Mater Interfaces 2018;10:33484–92.
https://doi.org/10.1021/acsami.8b14121.
[18] Nyström M, Aimar P, Luque S, Kulovaara M, Metsämuuronen S. Fractionation of model
proteins using their physiochemical properties. Colloids Surfaces A Physicochem Eng Asp
1998;138:185–205. https://doi.org/10.1016/S0927-7757(96)03892-7.
[19] Van Reis R, Brake JM, Charkoudian J, Burns DB, Zydney AL. High-performance tangential flow
filtration using charged membranes. J Memb Sci 1999;159:133–42.
https://doi.org/10.1016/S0376-7388(99)00048-4.
[20] Ebersold MF, Zydney AL. Separation of protein charge variants by ultrafiltration. Biotechnol
Prog 2004;20:543–9. https://doi.org/10.1021/bp034264b.
[21] Bhushan S, Etzel MR. Charged ultrafiltration membranes increase the selectivity of whey
protein separations. J Food Sci 2009;74. https://doi.org/10.1111/j.1750-3841.2009.01095.x.
[22] Saxena A, Shahi VK. Isoelectric separation of proteins using charged ultrafilter membranes
with different functionality under coupled driving forces. Ind Eng Chem Res 2010;49:780–9.
https://doi.org/10.1021/ie900258d.
[23] Lebreton B, Brown A, Van Reis R. Application of high-performance tangential flow filtration
(HPTFF) to the purification of a human pharmaceutical antibody fragment expressed in
Escherichia coli. Biotechnol Bioeng 2008;100:964–74. https://doi.org/10.1002/bit.21842.
[24] Cromwell MEM, Hilario E, Jacobson F. Protein aggregation and bioprocessing. AAPS J 2006;8.

21
 https://doi.org/10.1208/aapsj080366.
[25]** Roberts CJ. Protein aggregation and its impact on product quality. Curr Opin Biotechnol
2014;30:211–7. https://doi.org/10.1016/j.copbio.2014.08.001.
This article provides a concise summary protein aggregation phenomena, by pointing out the kinds of
aggregation and mechanisms involved, and suggesting approaches to minimize adverse effects on
product quality.
[26] Durbin SD, Feher G. Protein crystallization. Annu Rev Phys Chem 1996;47:171–204.
https://doi.org/10.1007/978-1-4939-7000-1_2.
[27] Scopes RK. Protein Purification: Principles and Practice. Third Edit. New York: Springer-Verlag;
1993.
[28] Lesins V, Ruckenstein E. Patch controlled attractive electrostatic interactions between
similarly charged proteins and adsorbents. Colloid Polym Sci 1988;266:1187–90.
https://doi.org/10.1007/BF01414409.
[29] Leckband DE, Schmitt FJ, Israelachvili JN, Knoll W. Direct force measurements of specific and
nonspecific protein interactions. Biochemistry 1994;33:4611–24.
https://doi.org/10.1021/bi00181a023.
[30] Jurrus E, Engel D, Star K, Monson K, Brandi J, Felberg LE, et al. Improvements to the APBS
biomolecular solvation software suite. Protein Sci 2018;27:112–28.
[31] Velev OD, Kaler EW, Lenhoff AM. Protein interactions in solution characterized by light and
neutron scattering: Comparison of lysozyme and chymotrypsinogen. Biophys J 1998;75:2682–
97. https://doi.org/10.1016/S0006-3495(98)77713-6.
[32] Tessier PM, Lenhoff AM, Sandler SI. Rapid measurement of protein osmotic second virial
coefficients by self-interaction chromatography. Biophys J 2002;82:1620–31.
https://doi.org/10.1016/S0006-3495(02)75513-6.
[33] George A, Wilson WW. Predicting protein crystallization from a dilute solution property. Acta
Crystallogr Sect D Biol Crystallogr 1994;50:361–5.
https://doi.org/10.1107/s0907444994001216.
[34] Dumetz AC, Snellinger-O’Brien AM, Kaler EW, Lenhoff AM. Patterns of protein-protein
interactions in salt solutions and implications for protein crystallization. Protein Sci
2007;16:1867–77. https://doi.org/10.1110/ps.072957907.
[35] Scherer TM, Liu J, Shire SJ, Minton AP. Intermolecular interactions of IgG1 monoclonal
antibodies at high concentrations characterized by light scattering. J Phys Chem B
2010;114:12948–57. https://doi.org/10.1021/jp1028646.
[36]* Neal BL, Asthagiri D, Velev OD, Lenhoff AM, Kaler EW. Why is the osmotic second virial
coefficient related to protein crystallization? J Cryst Growth 1999;196:377–87.

22
 https://doi.org/10.1016/S0022-0248(98)00855-0.
This article emphasizes the theoretical definition of B22 based on molecular interactions, compares
experimentally measured and computationally obtained values of this parameter, and points out
how a few, local interactions can dominate overall behavior.
[37]* Okur HI, Hladílková J, Rembert KB, Cho Y, Heyda J, Dzubiella J, et al. Beyond the Hofmeister
series: Ion-specific effects on proteins and their biological functions. J Phys Chem B
2017;121:1997–2014. https://doi.org/10.1021/acs.jpcb.6b10797.
This article presents a contemporary discussion of salt-protein interactions based on state-of-the-art
computational and analyical methods, with emphasis on interpreting and extending the basic insight
on the Hofmeister series effects.
[38] Kamerzell TJ, Esfandiary R, Joshi SB, Middaugh CR, Volkin DB. Protein-excipient interactions:
Mechanisms and biophysical characterization applied to protein formulation development.
Adv Drug Deliv Rev 2011;63:1118–59. https://doi.org/10.1016/j.addr.2011.07.006.
[39] Bam NB, Cleland JL, Yang J, Manning MC, Carpenter JF, Kelley RF, et al. Tween protects
recombinant human growth hormone against agitation-induced damage via hydrophobie
interactions. J Pharm Sci 1998;87:1554–9. https://doi.org/10.1021/js980175v.
[40] Dumetz AC, Lewus RA, Lenhoff AM, Kaler EW. Effects of ammonium sulfate and sodium
chloride concentration on PEG/protein liquid-liquid phase separation. Langmuir
2008;24:10345–51. https://doi.org/10.1021/la801180n.
[41] Teske CA, Blanch HW, Prausnitz JM. Chromatographic measurement of interactions between
unlike proteins. Fluid Phase Equilib 2004;219:139–48.
https://doi.org/10.1016/j.fluid.2004.01.025.
[42] Tessier PM, Sandler SI, Lenhoff AM. Direct measurement of protein osmotic second virial
cross coefficients by cross-interaction chromatography. Protein Sci 2004;13:1379–90.
https://doi.org/10.1110/ps.03419204.
[43] Cheng YC, Bianco CL, Sandler SI, Lenhoff AM. Salting-out of lysozyme and ovalbumin from
mixtures: Predicting precipitation performance from protein-protein interactions. Ind Eng
Chem Res 2008;47:5203–13. https://doi.org/10.1021/ie071462p.
[44] Levy NE, Valente KN, Lee KH, Lenhoff AM. Host cell protein impurities in chromatographic
polishing steps for monoclonal antibody purification. Biotechnol Bioeng 2016;113:1260–72.
https://doi.org/10.1002/bit.25882.
[45] Aboulaich N, Chung WK, Thompson JH, Larkin C, Robbins D, Zhu M. A novel approach to
monitor clearance of host cell proteins associated with monoclonal antibodies. Biotechnol
Prog 2014;30:1114–24. https://doi.org/10.1002/btpr.1948.
[46] Filipe CDM, Ghosh R. Effects of protein-protein interaction in ultrafiltration based

23
 fractionation processes. Biotechnol Bioeng 2005;91:678–87.
https://doi.org/10.1002/bit.20568.
[47] van Reis R, Zydney A. Bioprocess membrane technology. J Memb Sci 2007;297:16–50.
https://doi.org/10.1016/j.memsci.2007.02.045.
[48] Susanto H, Ulbricht M. Photografted thin polymer hydrogel layers on PES ultrafiltration
membranes: characterization, stability, and influence on separation performance. Langmuir
2007;23:7818–30.
[49] Li Q, Bi Q-Y, Zhou B, Wang X-L. Zwitterionic sulfobetaine-grafted poly(vinylidene fluoride)
membrane surface with stably anti-protein-fouling performance via a two-step surface
polymerization. Appl Surf Sci 2012;258:4707–17.
[50] Bengani-Lutz P, Converse E, Cebe P, Asatekin A. Self-Assembling Zwitterionic Copolymers as
Membrane Selective Layers with Excellent Fouling Resistance: Effect of Zwitterion Chemistry.
ACS Appl Mater Interfaces 2017;9:20859–72.
[51] Schönemann E, Laschewsky A, Rosenhahn A. Exploring the long-term hydrolytic behavior of
zwitterionic polymethacrylates and polymethacrylamides. Polymers (Basel) 2018;10:639.

24
[1]* Mehta A, Zydney AL. Permeability and selectivity analysis for ultrafiltration membranes. J Memb
Sci 2005;249:245–9. https://doi.org/10.1016/j.memsci.2004.09.040.
This communication presents data on the hydraulic permeability and bovine serum albumin selectivity
for various ultrafiltration membranes from literature showing the inverse, or trade-off, relationship, and
describes this with a theoretical model.

[2]** Sadeghi I, Kaner P, Asatekin A. Controlling and expanding the selectivity of filtration membranes.
Chem Mater 2018;30:7328–54. https://doi.org/10.1021/acs.chemmater.8b03334.
This perspective reviews and discusses recent advances in fabrication of liquid filtration membranes for
enhancing their selectivity based on size, charge and other molecular criteria.

[25]** Roberts CJ. Protein aggregation and its impact on product quality. Curr Opin Biotechnol
2014;30:211–7. https://doi.org/10.1016/j.copbio.2014.08.001.
This article provides a concise summary protein aggregation phenomena, by pointing out the kinds of
aggregation and mechanisms involved, and suggesting approaches to minimize adverse effects on
product quality.

[36]* Neal BL, Asthagiri D, Velev OD, Lenhoff AM, Kaler EW. Why is the osmotic second virial coefficient
related to protein crystallization? J Cryst Growth 1999;196:377–87.
This article emphasizes the theoretical definition of B22 based on molecular interactions, compares
experimentally measured and computationally obtained values of this parameter, and points out how a
few, local interactions can dominate overall behavior.

[37]* Okur HI, Hladílková J, Rembert KB, Cho Y, Heyda J, Dzubiella J, et al. Beyond the Hofmeister series:
Ion-specific effects on proteins and their biological functions. J Phys Chem B 2017;121:1997–
2014.
This article presents a contemporary discussion of salt-protein interactions based on state-of-the-art
computational and analyical methods, with emphasis on interpreting and extending the basic insight on
the Hofmeister series effects.
Conflict of Interest and Authorship Conformation Form

Please check the following as appropriate:

All authors have participated in (a) conception and design, or analysis and
interpretation of the data; (b) drafting the article or revising it critically for
important intellectual content; and (c) approval of the final version.

This manuscript has not been submitted to, nor is under review at, another
journal or other publishing venue.

The authors have no affiliation with any organization with a direct or indirect
financial interest in the subject matter discussed in the manuscript

The following authors have affiliations with organizations with direct or

indirect financial interest in the subject matter discussed in the manuscript:

Author’s name Affiliation

Emre Bukusoglu Middle East Technical University

Harun Koku Middle East Technical University
P. Zeynep Culfaz-Emecen Middle East Technical University