Peer-Review Research

Purification of Protein by
HIC: Mechanistic Modeling
for Improved Understanding
and Process Optimization
LUCRÈCE NICOUD, YOHANN LE GUENNEC, EDOUARD NICOUD,
ANTONIO CARDILLO, ELENA LIETTA, ELISA INNOCENTI, AND ALESSANDRO PIERI

ABSTRACT
Hydrophobic interaction chromatography (HIC) is a separation technique widely
used for the purification of therapeutic proteins. The main leverages identified
to operate HIC processes are solution pH and ionic strength. In this article, the
ArtemisDiana/Stock.Adobe.com

authors show that simplistic mathematical models derived from statistical analysis
cannot describe the impact of pH and ionic strength observed in HIC processes.
A new mechanistic model accounting for the protein charge in solution (varying
with pH) and the solution non-ideality (activity coefficient varying with the ionic
strength) is proposed here. This simple model is shown to accurately describe
experimental data and paves the way for numerical optimization of HIC processes.

Lucrèce Nicoud*, lucrece.
nicoud@ypso-facto.com, is
head of product; Yohann Le
Guennec is project manager;
and Edouard Nicoud is
head of customer success;
all at Ypso-Facto, France.
Antonio Cardillo, PhD, is
expert scientist & GSK fellow;
Elisa Innocenti is scientist;
and Alessandro Pieri, PhD,
is scientific leader & GSK
associate fellow; all at GSK,
Italy. Elena Lietta, PhD, is
from Politecnico di Torino,
Duca degli Abruzzi, Italy.
To whom all correspondence
*
H
ydrophobic interaction chro-
matography (HIC) is widely
used for the purification of
therapeutic proteins (1,2),
in particular for aggregate removal (3).
Designing a HIC process requires deter-
mining numerous operating parameters
such as buffer pH, buffer ionic strength,
loading volume, f low rate, and others.
Simulation can be used as a tool to investi-
gate different operating conditions digitally
and thus reduce the experimental burden. It
can also be used to perform process optimi-
zation and reduce the environmental impact,
in particular by decreasing raw materials
consumption and waste generation.
Existing HIC models are often inspired
by the protein solubility behavior observed
in solution (1). An interesting study treats
salt-induced protein precipitation as a liq-
uid–liquid phase separation with a pro-
be applied in a straightforward manner
to HIC. In another study, a HIC model
inspired by a complexation mechanism with
ligands has been proposed (5). This model
has the advantage of allowing the predic-
tion of the mean retention coefficient with a
rather limited number of model parameters,
but it does not provide a way to explic-
itly describe the impact of pH and ionic
strength on chromatograms.
In this article, the authors propose a sim-
ple chromatographic model capable of pre-
dicting the impact of pH and ionic strength
on HIC chromatograms. This mechanistic
model is based on the dependence of the
protein charge with pH and on an explicit
expression of the protein solution activity
coefficient with the ionic strength. The
model is first tested on literature data. Then
it is applied to a set of experimental data
generated by GSK. Finally, it is used to per-

should be addressed.
tein-poor supernatant and a protein-rich
precipitate (4). A theoretical model capa-
ble of accounting for both pH and ionic
PEER-REVIEWED strength effects was derived. However,
Submitted Sept. 15, 2022 this model counts many model parameters
Accepted: Dec. 8, 2022. that are difficult to estimate and cannot
form process optimization.
MATERIALS AND METHODS
Experimental part
Materials. A mmon iu m su l fate w a s
purchased from Carlo Erba Reagents

22 BioPharm International  April 2023  www.biopharminternational.com

 2.2 Ammonium A and Buffersulfate B was set was7.0.purchased The
1−ε e flowrate was 1.16 mL/min, which corresponds to

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and model
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protein from the
that:
protein
model
of the
two [13]
(6)
For
fitting
may (i)
pulse
species
[13] (3)
contributions
theof
(2) the
pK
experim
adsorprotof
cient
we sed number
of
protein
of
The
the + − the
each
obtain: H4is
assuming
where defined
following
charge amino of
particles
in A positive
solution
vs ,
8.06 B
that:
acid,
reactions aspHare , thewas
Cand averaged
to and
(i)
UVin slope
are calculated
and
the
perform the
solution negative
3
saturation HMW.
two of together
pK the
contributions
were as
simulations. a of charges
parameters
was a
adsorption
taken in function
the C
observed k into .
amino were
The
were and of
isotherm
account:
To H at C
pH
then computed
activity
acid
IP k
improve is
around
OHP =
experimental
from Nthe λ side
charge.
summed H k at γ
coefficient
H the
ionic
Equilibrium
+ 2500
k 0
the C
low
2 P at
chains
k amino
to
O N 
1 kmAU. each
−
strength
1
description H
2
data,
concentration.
obtain + ε γ  + H k are acid pH
Hquantifies
+ 1
the + 2 as assumptions
+2.15 defined
net ofconditions on in pKaEquation
were
Kfitting a =as progressively
λretention
γk (Cof5. The →
− (8)0)k . tThe relaxed. Indeed, a
b9.23
+ In the literature, results are often presented in terms of 3k ,,OH which ,2is ×related to kK follows:
−14.00 a0
H SO hthe tain:
+
4 +
amino 6.82 H solution
acid, as k follows:
1.92
and k
was2.2the k
calculated
Modeling two contributions as
part a a func-
were shown then in flowrates
Equation
K(pH) summed O = 2− under b 7: + to P non
b+ obtain
,O a×
3− t binding
pH −+ εH −
+ the bnet 12.38 2 (van
2 Purification
Deemter plot).
protein by0)HIC
located in the pores of the particles 0b are ,pH 2averaged together (3) of
ε2accessible k in C activity co
4Vand 11
ng that the 3the k4−
acontributions from the species adsorbed on surface or
kinetics To as simulate follows: the experimental kmodel
pulse experiments, the lumped λdata Henry acquiredcoefficient by H GSK
can O
charge. tbe in H the
calculated + aframe bof
from the measured
14.00 time
pK where C iswas the concentration of species in solution 4 and
where K iscalculated the alumped aHenry coefficient εfrom a ,z(9)a− its
,ionic valence. are λparameters. →
− For
4 K =
were rm: HSO determined
+ +parameters,
impact from
its apparent protein
Conly the net pulse
pK charge: values experiments and
itquantifies has This
number
been performed
provided of
independent
reported e col that bfirstat 1of different
the curve
aspartic/glutamic the pK eof of the charge
strength,
b1amino acids
acids vs (ii)pH
and the
depend tohistidine perform
kspatial strongly kγ (C
kresidues simulations.
configuration kon Toth i
+ H 1.92 ,
umber protein of solution,
positive and while negative k the coefficientcharges were computed the at adsorption
each pH based ,strength 4b + on k with
− R
each
obtain: of(ii)
erformed
2.15 2− the parameters
amino
the
4 9.23protein pKspatial
K acid,
assuming
a= in
K Akand
λ where
kand
γ ,k (C
solution
where
configuration
= B λ ,the
that: γ →
− KCkare The
(C k0) is
were
two is (i)the
→
− the
material estimated
contributions
0) lumped
the ofconcentration the pK eluting asHenry
kby
protein
K of
K(pH) =comparing
were
from
theε coefficient
function
of H+ =species
does then
the
amino
(1
P b tK(I)
O − K(pH)
of
+ model
=
HIC
H not
ε bpH and
ksummed
) P= Hin
Calculations
acid
× OHP play
column
a 2pH =
Oa
simulations
solution  +0(9)
O side k + 02−
a aH 10the
a + 1toH
b + was
× + P role. 1obtain
chains
× amino
and
I2+
H
O × +
pH +
wereb2that
OH
collected pH with
0+ a z,2+ ak+ KbH ×
6.82+
acid
1its ,+experimental
the
first
are I 22 ×
valence.
inare
net pH
performed
2.154.7 fitting
14.00
mL model
data.
assuming
fractions parameters.(corresponding
(6) that: (3) (i) For (4)
the (2) to pK (3)the o
O +
ptions
les
xing 4 +
are
2− H
12.38
cell tion
were
where
averaged
14.00
model +as k of
progressively
were C 8.06
was
follows:
In
k pH
together the
pulse
consideredused from k
C k
literature, in
experiments, k
kto describe the
C relaxed.
kask . the amino
The adjustable concentrations
results activity
the the acid
Indeed, are a
hydrodynamics
lumped coefficient
often
parameters. the of the
presented
Henry ionic
non-ideality
i 2
Hydrodynamics
experimental
The γ 0R
H
inRegarding
speciesquantifies
k coefficient
4 K(I)
approach
This 2 N strength
SO
i
the
3 1 in H 
1 Qof
−
+
provided
4 k
column,
terms
= 4 0
data,  in
the√
Calculations
ε a can
usedthe
HSO 4Nthe + √
1encountered
protein
and of
the H
ε 2 2
a data
bewhile
a k
in
−
liquid +
× , +
kinetics
calculated
ε
assumptions
first 4
this H
which
I H solution,
the
+ 2
were and
curve a
article were is
× solid
first
related
I
offromextracted
1.92
To2 while phases,
9.23
were
can the simulate
performed
the
be tothe K from
measured
progressively
charge extended respectively.
coefficient
the
as
C vs assumingthe
=experimental
follows:
pH toλ literature
retention
relaxed.
toany
γ λ Cperform
k A that:
other[Eq.
quantifies time [14, 9]
data
Indeed, (i)
kind 15]
t
simulations the
acquire
the (2)
of to
the
a
pK
adsoper
ads io
H+ditions SO
ch −
c the medium.
amino + (van
Hacid, their
The Deemter
and
pulse location
8.06
parameter the experiments, two
where plot). [11, λ
iscontributions K 12] was is and
the estimated some
lumped
lumped were amino by then
Henry acid
comparingsummed coefficient residues model
to obtaincan may+ 0

simulations
be be
the 3 4
calculated
1 poorly net e  with accessible
+ 2 from the because
measured “buried” retention in the
time t(3) R
ofmat
aHenry coefficient and a ε,H aanalysis ,3kpositions
a ,H bin ,+ b1adsorbed
,presence
barticle
2 are fitting inmodel
in HIC processes extremely high (several mol/L) and k parameters. For(and
,harge number 14.00 of positive and negative charges were computed at εit each is known pH based a,that the
on of salt kassignificantly
2.2.2 Mechanistic model 4 t
2
+ N H t SEC N H + 9.23 k LMW, kconfiguration
4i
eelution,
H 6.82ength,first4 the performed
2.15vs (ii) pH the K to kCspatial =In
assuming
perform λthe column
kkγ literature,
(C
configuration that:k
simulations. volume)
→
− (i)
0) results the and of pK
To are stored the often
ofthe
improve KHPat
the
protein presented
= -20°C
Oεamino 2−independent
εe4HSOthe
iVH + does before
The
A
(1
P bdescription
0O P acid
− in
zO −
O 2approach terms
3−
bnot ) side
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HP 0 play of of chains
O + 1k the
of2−
used 2 + which
ionic
× are
12.38
role. H (9)to
0pH 2is
strength,
+0determine
this related
6.82 to
(ii)
canthe K bethe
content extendedfollows:
spatial of to (6) any Monomer
Rother kind
arge this of
pproximation
article ++ article
performed of
1.92
can thesequence.
protein
lumped
while
9.23 be can the
core was
extended
assuming
where
protein be The in
approach
coefficient
of asTo solution
used
extended
the
inK = follows:
activitydo
to λ
isprotein,
solution to
that: any
γ so,
the k is
λ
where C kdescribe
towasthe
used,
quantifies
other k any
coefficient
lumped was (i)
thus K number
calculated meaning
kindisthe
other the Henry
not
calculated the theofof pKof
kinetics
adsorption
kind the
contributing
as
that
adsorption
lumped coefficient
athe
of
of
protein
as
function
of HenryK(pH)
adsorption
log(γ athe
Langmuir
tRεlog(γ
model,
contributions
strength
chromatographic
the internal present
isotherm,
function)inamino
and
to = of
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the−
=
coefficient
colHa
experimental
only
pH
mass
isotherm,
adsorption
of ,
−
Pwithk
study,
independent
like
apparent acid
a
+A
pH
4 kfrom
,
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k the
was √z1SO
transfer
a from
from
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− H
2−
blike
computed
+
pH
theInet
mixing
data, a
isotherm.ε, ethe
C P
0 +the b , of+amino
O4[6,
the
chains a
H
,
×
charge.
− bspecies
the
1 b
+
the ,The
2
I
amino 7].
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using
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parameter
model
ionic
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assumptions To ,were
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8.06 ,
2.15wasb
strength,
concentration
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reduce
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used
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model
2 were
k
the
the
fitting
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(ii)to
parameters. in
progressively
number
the
surface
describe
the model
estimated the original
spatial
model
of liquid or parameters.
the
fittingFor by
(7) [13]
relaxed.
articles
phase comparing
hydrodynamic
configuration C For
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H extracted 9.23 from as the k
follows:pulse literature k
experiments, k
[14, 15] the to lumpedaperform Henry preliminary )external
=
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can √ 4 2 + of C the 2× − +Inet + (7)
ofparameter
each impacts
amino acid, where the and values Vspatial the isof oftwo the pK contributions
column [8, 9,charges 10].
volume, Besides,
were then
the itkLangmuir is summed clear that porosity to the
obtain tbe kspatial andcalculated the Q configuration
the from
flowrate. the ofmeasured
the protein retention may time tR
1P  εside
0play 1first 4  k
of
+ s−To
herm. he 12.38
ionic
st
ters
.
a H
o,Hwere
the
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the are
strength,
Interactions
performed6.82
8.06The positive
progressively
number
located
charge
2.15
the
concentration λthe
number
Solution kvs
concentration
(ii)
assuming
ofwas
in and
2.2 positive
pH
in the the
with
experimental
of
in HIC negative
estimated
chemistry to
pulse
Modeling and
relaxed.
pores
mixing
the col the
perform
processes
in and
liquid
HMW.
that: experiments,
the cellssolid
charges
by configuration
negative
the
part phase
liquid data
comparing
Indeed, The
a(i)
J particles
simulations.
is phase
and the
were
extremely
C acquired
following
phase the
isthe pK
model are
indeed C
of
The
lumped ionic
experimental
characteristic
the
were
high by
of
averaged
reactions
acoefficient
is H
Tosimply
Linear K
kprotein
interactions
2
indeedthe
simulations
K(pH) GSK
whereSO
Therefore,
strength
computed
improve
Henry
(several
= The Q
4
ε HP 
This
amino C
together
replaced
in
Driving
H +
simply
ε in
1
net
data.
time
= kO +H
does
HSO
εP
coefficientwith
mol/L)
solution
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and
1
provided
O
with
B −only
encountered
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the
charge
+ of
+col− acid O

εby
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−
not
provided
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replaced
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adsorption
frame
C
ineach

4I
internal
) +
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the
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1 H
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I kpH
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pH t+
approximation
−
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it 1εthe
O of
2 The
takenby
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P
the−
be
k +
+a 2−
mass
is
abased O
kchains εH role.
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calculated
protein
known
first
b +
e
curve
isotherm.
concentrations
1.92
activity
×
+
into
H of Hare
on
+
curve
pH
of
were
212.38
The
in
that
account:
was
6.82
the
of
The2.15
parameters
fromapproach
coefficient
solution
Purification
the the
charge
described
used
concentration
of
the
presence the
chargeto
vs
γused
measured
was by
species
kdescribe
pH
described
quantifies
calculated
of
vs of in the
protein
salt
pH
6to thiskin perform
retention
the
to in the
in by
significantly the
article as
perform the
kinetics
(6)
liquid
HICa liquid simulation
paragraph
time
function
(3)
phase
of
simulat and
t inte o
nry trength,
o
srotein + so,
article +the
2.2.2
coefficient
Hfollowing pulse
(ii)
number
can2.15 as
parameters,
the follows:
experiments,
Mechanistic
be
is defined of
extended where
spatial positive only
as K kconfiguration
the model the
to is
and slope the
any pKlumped negative lumped
aof other values the k Henry Henry
ofof
charges
kind and
adsorption the γ
number
of C
coefficient
protein
were .
adsorption
isotherm of
computed can accessible and
does be 4
at k
isotherm, calculated
alow not
at , a eeach
aspartic/glutamic
concentration. , 4
play t,pHlike from
b a+ , basedb role.
the the
, b measured
on are acids fitting and retention
model
histidine parameters.time residues t For R
i
performed impact assuming its as apparent that:
follows: (i) net the charge: pK isof it the
has k amino
been t=
reported acid i = e 2 side
col 0 that i
chains −
1k 1H + are e 2−
2  K 2 (4)(3)
concentrations
were4 1.92 +
curve + reported
12.38
in of the
solution of
charge the
in
adsorption was the The
species vs
calculated original pH ionic
isotherm: to in strength
the
performas articles a liquid function Isimulations.
aand(and defined solid not
pH HSOas:
kphases,
the
from k
To
lumped
t
not K K(pH)− full=
improve
γ
R
the
the respectively.
 C εsummed
experimental aminochromatograms).
SOH .
iapproach +
hydrodynamic= (1
P
2− 4the

b O −
1 0 +
acid + ε1the
 b
descriptionA 1) −
is data,
+ × HPεpK−2
used, pH 4aO
/ K k
the
0 of
kinetic +
meaning
8.06 b 1amino
of + 
assumptions
× H 2pHacids
+
parameters.that 6.82 depend
thewere contributions strongly
progressively on R relaxed.
from (6)
(4) the Indee
species
c+K del
mptions
y, − aH
which was
of
strength, each
+ were computed
used
the
6.82were
as amino
where(ii)to
non-ideality
follows: Equilibrium
progressively
determined acid,
the at
describe
impacts
A each
,A as Band
spatial of follows:
,in from pH
the the the
Cperform based
relaxed.
values
are twohydrodynamics
configuration
protein
protein 3 the on
contributions
of
fitting the
solution,
pulse
pK Indeed, pK [8,
parameters of
experiments while
9, in
were
The
thethe
10].
The the
sequence. R then
the protein
lumped
ionic
keyand H
column,
k
4 performed
Besides, coefficient
model
HP2 SO
k
Istrength To
Henry
experimental
is OεImay
2Q does
ite44the
4 2−
Vcando
is√ 0while
parameters  4 HSO
clear toλ
so,
coefficient
at
ionic knot
P 1 obtain
encountered 1
O the
quantifies
the
different 4[Eq.
that− 3− t data, +
playε
15the
strength number
−+tare
H 7]
the
the 4 is the
εHeflowrate. a
+
the2 net
the
defined
spatialcan
role.
as of 1.92

number
assumptionsbe
adsorption
positive
12.38
defined as extended
the
configurationofinmixing and
slopestrength
were
Equation tonegative
of any
of the
cells
progressively the with other
5. adsorption
J protein charges
and
The kind the were
may
relaxed. isotherm
character comp In
ε12col
pulse experiments, the lumped Henry coefficient e
dmber pK of each were amino V
considered kacid, is ,kthe and as column Ctheadjustable two volume, contributions parameters. εHenry the external
were then porosity
Q summed ,be
and to calculated
ε,role. Q4eobtain the from bnet the measured 6 retention time tFor
on strength, +8, H we 6.82
obtain:
(ii) 2.2.2 the Mechanistic
spatial B configuration model of athe aprotein does  not play 
m.
mic
Hn
re
8.06
he
is of
+
meaning
ve
+ the
first
defined
was of
The 1.92
assumptions
aof
parameters
Solution
the
charge
that
the
12.38
used
their
performed
concentration
positive
as
number the
charge
the
to
where vs
location
were
and
slope and
chemistry
chromatographic
contributions
described
describe vs
pH
assuming
colnegative
 pKof progressively
where kthe
pH
6to [11,
of + kK
adsorption
each
the
to
k,12]
the in
charges
The that:
medium.
from
is
perform
kthe and
amino
kinetics
are
liquid
the − relaxed.
following (i) some
were 3isotherm
simulations.
lumped
paragraphs acid,
fitting
species
the
The phase
of
amino
computed
simulations.
pK Indeed,
reactions
parameter
internal
eatparameters
adsorbed
aClow ofacid
above
The
k atTo
the
is
the located
in ε indeed
coefficient
each
concentration.
net
mass
improve
on
residues
solution
λ
Vnumber
To
amino
ionictwere
HSO
and
the
waspH
charge
improve= in IHP
transfer
surface
simply
strength
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−
based
=theacid
estimated,
were
estimated
and 
is
O
1
the
of e− a2−
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4poresSO
pK0 be
theonside
taken
the ε [6,
or aawe
description
 replacedionic
zpoorly
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2− 1k 2 P
protein
+ of C
byof chains
7]. +
descriptiona− O
into
keach2and the
H
3− strength
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account: are
by
,particles
aminoK εH
bpK 1eof
+
,of
solution as are
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28.06
of model
acid,
defined
because
12.38
are fitting
wasaveraged
and
in
“buried”
model
calculated
isotherm,
simulations
the
Equation
two together parameters. such
with
contributions
in
as 5.
the
a in as
The
function C
the .R
k (5) The
were(4) of thenacti
pH
nding + H + conditions 12.38 parameters H
The (van
as 2 Oimpact
where follows:
ionic
activity Deemter A H wherekV
its
strength , + B coefficient
kis
OH
apparent
, Kplot).C
the is I were the
is
column net
defined
of lumped
estimated
the 14.00
charge:
volume, protein Considering
as: Henry by it
transfer
εto in has coefficient
comparing
ethe kcol
solution been
R Equation
t
H t, SOA 4 =reported
respectively, was z
model
Q and  8, col
I
computed HSO a e a4 ,
simulations that a 1 obtain:
which
+ , 4athe
+ ε H
using, b+were ,withb
K the , 1.92 amino
determined
b are
experimental
Truesdell-Jones fitting acids from depend
model
data. protein
model parameters. strongly
[13]pulse on
experimen
For (4)
γexternal porosity and Q the flowrate.
→
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+
umptions en:particles
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+ solution
ionic
amino the are
charge
strength,
8.06acid, core were wasaveraged
and vs
parameters
of calculated
where the
(ii) pH
progressively
the pulse +two the V K
together
protein, toa
experiments, =
perform
kspatial
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is the ,akthus B
inγfunction
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C
,+ Cknot
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simulations. were The
were of 0)
contributing activity
pH estimated
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volume,then
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To
summed Ccoefficient
the
log(γ
Henry = improve
the
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the
protein
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)toλ comparing
=
external obtain
coefficient
non-ideality −
i γC
the
apparent2 R 1kH acid
kdoes+
strength k
quantifies
description
the
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porosity 2
k
can √ and
model
not
4net
net
kof  be 0 K
+the play
encountered
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charge.
and Ccalculated4of
simulations
1
protein× −
a4Q 2
eI+ 
role.
the (9)
0
To H To 1
+reduce
from with
flowrate.
solution, simulate
2
the
1.92 experimental
the measured
while thethe
number experimental of data.
coefficient (8)
retention 6experimental
fitting (4)were (7) data
time
λ tacquired
quantifies
were
snumber
parameters.
H
tein
sumptionsofcs
that ssumptions
+
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+were
protein H +first
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1.92
first
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1.92
and
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2.2.2
the
Solution
mixing
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solution were
performed
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2.2.2
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simulatenegative in H
progressively
6
theirHIC
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contributions
assuming
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experimental
from chemistry
the
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col Nthe
location
Mechanistic
processes
assuming
coefficient
charges
calculated Jionic H
experiments,
experimental and +that:
3literature
relaxed. were H λ
strengthdata
[11,the
as
were
is
that:
model
The
relaxed. 12]
extremely
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amino acid,λand the two contributionsk were then summed to obtain
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Considering
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All
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SO
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+
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H
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by
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1.92

experiments performed at different
f low rates under non-binding condi-
tions (van Deemter plot).
Rega rding the data that were
extracted from the literature (14,15)
to perform preliminary tests of the
model, only the peak positions were
reported in the origina l articles
(and not the full chromatograms).
Therefore, only the thermodynamic
parameters described in the earlier
paragraphs were estimated, and not
the hydrodynamic/kinetic parameters.
Simulations. All simulations pre-
sented in this article were performed
with the specific software (Ypso-Ionic).
RESULTS AND DISCUSSION
Preliminary results
based on literature data
Prel imina r y invest igat ions were
performed based on experimental
literature data obtained with chymo-
trypsinogen (14) and α–amyloglucosi-
dase (15). To do so, the protein Henry
coefficient measured at different val-
ues of the ionic strength (14) and solu-
tion pH (15) were considered. Two
approaches were used: a statistical
approach based on a quadratic expres-
sion and a mechanistic approach
based on physical phenomena. The
expressions used for the statistical
approach are given in Equations 2
and 3. The expressions used for the
mechanistic approach are given in
Equations 7 and 9.
T he resu lts a re presented in
Figure 1, where the lumped Henry
coefficient is plotted as a function of
the ionic strength (left) and solution
ALL FIGURES ARE COURTESY OF THE AUTHORS.
pH (right). The dots represent the
experimental data and the lines the
calculations. In examining the exper-
imental results, it is observed that the
lumped Henry coefficient goes through
a minimum as a function of the ionic
strength. At low ionic strength, the
retention is found to decrease with the
salt concentration. This phenomenon
is referred to as a “salting-in” effect. At
high ionic strength, the retention is
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Figure 1. Impact of pH and ionic strength on the protein retention quantified
in terms of the lumped Henry coefficient. Experimental results are represented
with symbols and calculations with lines. The experimental data were extracted
from Heinitz et al. (14) for the ionic strength impact (left) and from Xia et al. (15)
for the pH impact (right). Results were obtained with two different proteins in
isocratic conditions. For the ionic strength impact, experiments were performed
with chymotrypsinogen. A 20 mM phosphate buffer was used with varying
concentrations of sodium chloride (NaCl) and sodium sulfate (Na2SO4). For the
pH impact, experiments were performed with a high isoelectric point (pI) α–
amyloglucosidase using a 1 M Na2SO4 solution. Two calculation methods were
used: statistical (top) and mechanistic (bottom).
found to increase with the salt concen- chromatograms (instead of retention
tration. This phenomenon is referred times only as in this section).
to as a “salting-out” effect. In addition,
a maximum of retention is observed as Reference conditions
a function of pH, the retention being The UV and conductivity prof iles
maximal around the isoelectric point of obtained for the reference HIC exper-
the protein (around eight for the con- iment are shown in Figure 2a. The
sidered protein). Considering panels (a) steps are also indicated: first, crude
and (b) of Figure 1, it is observed that loading at a high ionic strength; sec-
the statistical model based on a quad- ond, wash at the same ionic strength;
ratic expression is not able to represent third elution at an intermediate ionic
the experimental trends. Indeed, some strength; fourth, regeneration at a
negative values of the lumped Henry low ionic strength; and fifth, strip-
coefficient are calculated, which does ping with water. Three peaks are
not make sense physically. observed in the UV profile, the first
Considering panels (c) and (d) of t wo during the elution phase and
Figure 1, it is observed that the sim- the last one during the regeneration
ple mechanistic model proposed in this phase. The perturbations of the UV
article represents well the experimental and conductivity signals at the begin-
trends of the lumped Henry coefficient ning of the experiment were caused
with the ionic strength and solution pH. by a temporary malfunction of the
In the rest of the article, the mechanis- system (probably due to the pres-
tic model is applied to novel experimen- ence of an air bubble) and were not
tal data. It is challenged against the full observed in subsequent runs.
April 2023 BioPharm International 25
Peer-Review Research
Figure 2. Results of the reference experiment (a) experimental ultraviolet (UV)
and conductivity profiles together with the percentages of low molecular
weight (LMW), monomer (Mono), high molecular weight (HMW) species
determined by size exclusion chromatography (b) simulation results showing
the species taken into account.
For each of the three peaks, frac-
tions were collected and analyzed
by SEC to qu a nt i f y t he Mono,
L M W, a nd H M W species. T he
analyzed results are presented in
Figure 2a. Interestingly, Peak 1 and
Peak 2 have similar compositions,
with around 90% of Mono. Peak 3
contains around 65% of impurities
(LMW+HMW) and 35% of Mono.
These results show that the three
species identif ied by SEC (LMW,
Mono, HMW) co-elute in the three
peaks observed in HIC. It is thus not
possible to match the peaks observed
in HIC with those observed in SEC
(e.g., associate Peak 1 with LMW,
Peak 2 with Mono, and Peak 3 with
HMW). This can be explained con-
sidering that HIC and SEC separate
26 BioPharm International  April 2023 
species based on different physical
properties, namely charge/hydropho-
bicity and size. As a consequence,
representing the crude as a mixture
of Mono, LMW, and HMW is not
suff icient. It is necessary to intro-
duce additional species, which are
likely to be protein charge variants.
Accordingly, the authors considered
LM W1, LM W2, and LM W3 for
the LMW species eluting in Peak
1, Peak 2, and Peak 3, respectively.
Similarly, they considered HMW1,
HMW2, and HMW3 for the HMW
species eluting in Peak 1, Peak 2,
and Peak 3, respectively. For the
Mono, two charge variants in Peak
2 were considered in an attempt to
describe peak broadness. The com-
position of the crude in terms of
charge variants was guessed based
on the SEC analysis of the chro-
matogram of the reference exper-
iment. T he fol low ing resu lts
were obta ined: L M W1 (0.66%),
LM W2 (2.56%), LM W3 (4.88%),
Mono1 (10.61%), Mono2 (43.90%),
Mono2bis (14.63%), Mono3 (8.46%),
HMW1 (0.26%), HMW2 (3.11%),
and HMW3 (10.93%).
The simulation of the reference
experiment (Run 1) with all the
considered spec ies is show n in
Figure 2b. The procedure used to
deter m ine model pa ra meters is
described in the materials and meth-
ods section. Briefly, the main param-
eters to be determined for each species
k are the Truesdell-Jones coefficients
Ak, Bk, Ck and the coefficient λk of the
adsorption isotherm. To reduce the
number of fitting parameters, it was
assumed that all species eluting in a
given peak have the same Truesdell-
Jones co-eff icients (e.g., LM W1,
Mono1, and HMW1 have the same
coefficients). In addition, it was con-
sidered that λ k = 1 for all species,
except Mono2bis, for which the λk was
estimated to be 1.2. The results of the
fitting for the five runs are presented
in terms of lumped Henry coefficient
as a function of the ionic strength in
Figure 3a. The results of the charge
as a function of pH are presented in
Figure 3b. As described in the mate-
rials and methods section, the pKa val-
ues and number of accessible aspartic/
glutamic acids and histidine residues
were adjusted compared to their theo-
retical values to allow a better descrip-
tion of experimental results.
The total protein concentration
(experimental and simulated) for
the reference experiment is shown
in Figure 4 (Run 1). It is seen that
the model allows correctly predict-
ing the overall shape of the chroma-
togram, with three peaks, the last
one being much narrower than the
first two. The retention times of the
peaks are accurately described, while
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 Peer-Review Research

Figure 3. (a) Experimental and simulated values of the lumped Henry coefficient as a function of the ionic strength at
pH 6.0, 7.0, and 8.0 for the species eluting in peak 2. The lumped Henry coefficient is equal to the activity coefficient
(Truesdell-Jones equation) because λ is equal to 1.0. (b) Calculated protein charge as a function of pH. The dots correspond
to the protein charge fitted to describe the experimental values of the lumped Henry coefficients.

Figure 4. Impact of the ionic strength (modulated by the % B) and pH: (a) experimental chromatograms at different ionic
strength values; (b) simulated chromatograms at different ionic strength values; (c) experimental chromatograms at different
pH values; (d) simulated chromatograms at different pH values.

their broadness could be improved.
The separation between Peak 1 and
Peak 2 is indeed underestimated. A
reduction in the characteristic time
for mass transfer of Mono2 was con-
sidered to improve peak resolution.
However, the broadness of Peak 2
was then signif icantly underesti-
mated. A plausible explanation is
that more than two charge variants
need to be considered to describe the
observed peak broadness. The impact
of multicomponent mixtures on peak
broadness has been studied experi-
mentally and theoretically in the lit-
erature (16); it is regarded as out of
the scope of the present study.
Impact of key
operating parameters
Ionic s trength. The left-hand
side of Figure 4 shows the impact
of the ionic strength on the chro-
matograms: experimental (top) and
simulated (bottom). It is observed
that the higher the %B (and thus the
lower the ionic strength), the more
retained are the species. This is due to
an increase in the activity coefficient
at high ionic strength, which is pre-
dicted thanks to the Truesdell-Jones
equation (Figure 3a). This behavior
is referred to as a “salting out” effect
(in contrast to the “salting in” effect
observed at low ionic strength).
Solution pH. The right-hand side
of Figure 4 shows the impact of pH
on the chromatograms: experimental
(top) and simulated (bottom). In the
investigated pH range, the higher the
pH, the more retained are the species.
This is related to the variation of the
charge of the protein with solution
pH (Figure 3b). Although the curve
seems flat in the pH range of interest,

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Peer-Review Research
Figure 5. Prediction of the impact of the ionic strength (%B) and solution pH. Conditions used to fit the model are
represented in black, while conditions fully predicted by the model are in red.
a slight decrease of the protein charge
with pH is actually obtained: z = 5.11
at pH 6.0, z = 4.94 at pH 7.0, and
z = 4.32 at pH 8.0. This is sufficient
to describe the increase in retention
observed in HIC experiments.
Overall, the peak positions and
shapes of the five performed experi-
ments are well represented by model
simulations. The proposed model is
thus capable of describing the impact
of t wo key operating parameters,
namely ionic strength and solution
pH. The model was also shown to be
capable of describing the impact of
loading volume and f low rate (data
not shown). A more ref ined ver-
sion of the model would consist in
improving the description of peak
broadness, potentially by including
more charge variants.
Illustration of
process optimization
Once model parameters are deter-
mined, the model can be used to pre-
dict unexplored operating conditions.
This predictive capability is illustrated
in Figure 5, where conditions used to
28 BioPharm International  April 2023 
fit the model are represented in black,
while conditions fully predicted by the
model are in red.
The choice of “optimal” process
conditions must be done consider-
ing given targets and constraints.
I n t he c a s e of u nder i nve st ig a-
tion, one can consider two options:
the collection of Peak 2 only, lead-
ing to a product of Grade 1 purity,
or the collection of Peak 1 and 2
simultaneously, leading to a product
of Grade 2 purity.
To obtain Grade 1 purity, pH 7.0
and 18 %B seem the most appro-
priate conditions to ensure a good
separation bet ween the f irst t wo
peaks. This is highlighted as option
(a) in Figure 5. To obtain Grade 2
purity, pH 6.0 and 28 %B seem the
most appropriate conditions to allow
a gain in productivity and decrease
in solvent consumption. Indeed,
the elution time can be sig nif i-
cantly reduced while still obtaining a
good separation between the f irst
two peaks and the third peak. This is
highlighted as option (c) in Figure 5.
Option (b) corresponds to the inter-
mediate situation obtained with pH
6.0 and 28 %B without reducing the
elution time. This is considered for
comparison purposes but has no real
practical interest.
Process performances (recover y,
p r o du c t i v it y, s ol v e nt c on s u mp -
tion) are quantif ied in Figure 5
for the three options. When mov-
ing from option (a) to option (c),
t he product iv it y is inc reased
by a fac tor of t wo a nd t he sol-
vent consu mpt ion reduced by a
factor of two.
This study illustrates how simu-
lation can help to perform process
optimization, increasing productivity
and reducing solvent consumption.
CONCLUSION
A simple mechanistic model of HIC
has been developed based on the
curve of the protein apparent charge
as a function of pH and the use of an
activity coefficient in solution.
It was show n that the model
allows predicting the impact of ionic
strength (%B) and solution pH on the
position and shape of chromatograms.
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 Peer-Review Research

The model was then used to illus- 86 (12), 1277–1283. DOI: 10.1002/ Selectivity. J. of Chromatogr. A 1988,
trate how numerical process optimi- prot.25608 443, 173–182.
13. Truesdell, A. H.; Jones, B. F. Wateq, a 15. Xia, F.; Nagrath, D.; Cramer, S. M.
zation can be performed with a view Computer Program for Calculating Effect of pH Changes on Water
to improving process performances Chemical Equilibria of Natural Waters. Release Values in Hydrophobic
such as recovery, productivity, and J. Res. US Geol. Surv 1974, 2 (2), Interaction Chromatographic Systems.
233–248. J. of Chromatogr. A 2005, 1079 (1–2),
eluent consumption. 14. Heinitz, M. L.; Kennedy, L.; 229–235.
Kopaciewicz, W.; Regnier, F. E. 16. Pfister, D.; Morbidelli, M.; Nicoud,
REFERENCES Chromatography of Proteins on R. M. A Continuum Theory for
1. Carta, G.; Jungbauer, A. Hydrophobic Interaction and Ion- Multicomponent Chromatography
Protein Chromatography: Process Exchange Chromatographic Matrices: Modeling. J. of Chromatogr. A 2016,
Development and Scale-Up, Mobile Phase Contributions to 1446, 50–58. ◆
2nd ed.; John Wiley & Sons,
Hoboken , NJ, 2020.
2. Curling, J. The Development of
Antibody Purification Technologies.
In Process Scale Purification of
Antibodies, Gottschalk, U., Ed.; John
Wiley & Sons, Hoboken , NJ, 2009; pp.
23–54. DOI:10.1002/9780470444894
3. Shekhawat, L. K. ; Rathore, A. S.
Mechanistic Modeling-Based Process
Analytical Technology Implementation
for Pooling in Hydrophobic Interaction
Chromatography. Biotechnol. Prog.
2019, 35 (2), e2758.
4. Kuehner, D. E.; Blanch, H. W.;
Prausnitz, J. M. Salt-Induced Protein
Precipitation: Phase Equilibria from an
Equation of State. Fluid Phase Equilib.
1996, 116 (1–2), 140–147.
5. Mollerup, J. M. Applied
Thermodynamics: A New Frontier for
Biotechnology. Fluid Phase Equilib.
2006, 241 (1–2), 205–215.
6. Nicoud, R.M. Chromatographic
Processes: Modeling, Simulation and
Design; Cambridge University Press,
Cambridge, UK, 2015.
7. Pfister D.; Nicoud, L.; Morbidelli,
M. Continuous Biopharmaceutical
Processes: Chromatography,
Bioconjugation, and Protein Stability;
Cambridge University Press,
Cambridge, UK, 2018.
8. Abe, Y.; Ueda, T.; Iwashita, H.; et al.
Effect of Salt Concentration on the
pKa of Acidic Residues in Lysozyme. J
Biochem. 1995, 118 (5) 946–952. DOI:
10.1093/jb/118.5.946
9. Lee, K. K.; Fitch, C. A.; Lecomte, J. T.;
García-Moreno E, B. Electrostatic
Effects in Highly Charged Proteins:
Salt Sensitivity of pKa Values of
Histidines in Staphylococcal Nuclease.
Biochemistry 2002, 41 (17), 5656–5667.
10. Kao, Y. H.; Fitch, C. A.; Bhattacharya,
S.; et al., Salt Effects on Ionization
Equilibria of Histidines in
Myoglobin. Biophys.J.
2000, 79 (3), 1637–1654.
11. Li, A. H.; Robertson, D.; Jensen, J.H.
Very Fast Empirical Prediction and
Rationalization of Protein pKa Values.
Proteins. 2005, 61 (4), 704–721. DOI:
10.1002/prot.20660
12. Pahari, S.; Sun, L.; Basu, S.; Alexov,
E. Delphipka: Including Salt in the
Calculations and Enabling Polar
Residues to Titrate. Proteins. 2018,

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