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\begin{document}

\title{Purification of polyclonal Immunoglobulin G from human serum using
LigaGuardTM and LigaTrapTM adsorbents}



\author[1]{Wenning Chu}%
\author[1]{Hannah Reese}%
\author[2]{Dipendra Bhandari}%
\author[2]{Jae Sly}%
\author[2]{Michael Crapanzano}%
\author[1]{Stefano Menegatti}%
\affil[1]{North Carolina State University}%
\affil[2]{LigaTrap Technologies LLC}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
This study presents the chromatographic purification of immunoglobulin G
(IgG) from human plasma using a two-column process integrating the
peptide-based adsorbents LigaGuardTM, which captures non-Ig plasma
proteins in flow-through mode, and LigaTrapTM, which isolates IgG in
bind-and-elute. Buffer composition and column loading were optimized for
both adsorbents. Two process configurations were evaluated. In the first
design, plasma was fed to a LigaGuardTM column to capture plasma
proteins, the effluent was loaded on the LigaTrapTM column, and the
bound IgG was eluted with 63.8\% global recovery and 99.7\% purity; in
comparison, Protein G agarose afforded \textasciitilde{}67\% recovery
and 97.2\% purity. In the alternative design, the LigaGuardTM column was
utilized to polish the LigaTrapTM elution stream, affording 82.3\%
global recovery and 98.8\% purity. Collectively, these results
demonstrate the potential of a fully chromatographic process for
purifying polyclonal IgG from plasma feedstocks.%
\end{abstract}%



\sloppy


\textbf{Purification of polyclonal Immunoglobulin G from human serum
using LigaGuard\textsuperscript{TM} and
LigaTrap\textsuperscript{TM}adsorbents}

Wenning Chu\textsuperscript{1}, Hannah R. Reese\textsuperscript{1},
Dipendra Bhandari\textsuperscript{2}, Jae Sly\textsuperscript{2},
Michael Crapanzano\textsuperscript{2}, and Stefano
Menegatti\textsuperscript{1,2,3,*}

\textsuperscript{1} Department of Chemical and Biomolecular Engineering,
North Carolina State University, 911 Partners Way, Raleigh, NC 27606

\textsuperscript{2} LigaTrap Technologies LLC, Durham, NC 27701

\textsuperscript{4} Biomanufacturing Training and Education Center
(BTEC), North Carolina State University, 850 Oval Dr, Raleigh, NC 27606

\textsuperscript{*} corresponding author: smenega@ncsu.edu; (919) 515
6398.

\textbf{Abstract.} This study presents the chromatographic purification
of immunoglobulin G (IgG) from human plasma using a two-column process
integrating the peptide-based adsorbents LigaGuard\textsuperscript{TM},
which captures non-Ig plasma proteins in flow-through mode, and
LigaTrap\textsuperscript{TM}, which isolates IgG in bind-and-elute.
Buffer composition and column loading were optimized for both
adsorbents. Two process configurations were evaluated. In the first
design, plasma was fed to a LigaGuard\textsuperscript{TM} column to
capture plasma proteins, the effluent was loaded on the
LigaTrap\textsuperscript{TM} column, and the bound IgG was eluted with
63.8\% global recovery and 99.7\% purity; in comparison, Protein G
agarose afforded \textasciitilde{}67\% recovery and 97.2\% purity. In
the alternative design, the LigaGuard\textsuperscript{TM} column was
utilized to polish the LigaTrap\textsuperscript{TM} elution stream,
affording 82.3\% global recovery and 98.8\% purity. Collectively, these
results demonstrate the potential of a fully chromatographic process for
purifying polyclonal IgG from plasma feedstocks.

\textbf{Keywords:} Affinity ligands, protein chromatography,
LigaGuard\textsuperscript{TM}, LigaTrap\textsuperscript{TM}, polyclonal
antibodies.

\textbf{1. Introduction}

Polyclonal immunoglobulin G (pIgG), also known as Intravenous
immunoglobulins (IVIgs), is a therapeutic preparation containing
polyspecific IgGs extracted from the pooled plasma of healthy donors
{[}\emph{\textsuperscript{1}} {]}. Since 1980s, IVIg has been
extensively investigated in term of molecular structure and functions,
purification strategy, and clinical applications
{[}\emph{\textsuperscript{1}} {]}. A key component of IVIg formulations
are antibodies targeting microbial antigens, which, together with
autoantibodies and anti-idiotypic antibodies
{[}\emph{\textsuperscript{2, 3}} {]}, provide the biomolecular arsenal
forming the immune defense {[}\emph{\textsuperscript{4-6}} {]}.
Accordingly, IVIg has been widely utilized in treating immunodeficiency
as well as autoimmune and inflammatory disorders
{[}\emph{\textsuperscript{7, 8}} {]}. Furthermore, IVIgs from immunized
donors have also shown to aid patients stricken by infectious diseases
{[}\emph{\textsuperscript{9, 10}} {]}; in this context, the most recent
case includes clinical evidence of pooled plasma from SARS-CoV-2
convalescent donors successfully utilized to treat patients affected by
severe acute respiratory syndrome (SARS COVID19)
{[}\emph{\textsuperscript{11-13}} {]}.

With a steadily increasing annual consumption
{[}\emph{\textsuperscript{14}} {]}, large-scale IVIg production poses a
mounting pressure on companies operating in the plasma product
landscape. Currently, pIgG extraction from pooled plasma is based on the
Cohn-Oncley or the Kistler-Nitschmann processes, which fractionate
plasma proteins by precipitation under controlled conditions including
ethanol and caprylate concentration, conductivity, temperature, and pH
{[}\emph{\textsuperscript{15-17}} {]}. The immunoglobulin-rich fractions
undergo subsequent processing via ion exchange chromatography to isolate
pIgG {[}\emph{\textsuperscript{18,}} \emph{\textsuperscript{19, 20}}
{]}. These processes, however, are inherently batch operations,
characterized by high capital and operational costs and long processing
times. Furthermore, Protein A/G affinity chromatography employed in the
purification of therapeutic monoclonal antibodies (mAb) is unsuitable
for downstream processing of IVIgs: the enormous volumes of plasma to be
processed are in fact incompatible with the high cost of Protein
A/G-based resins, and the related risks of antibody aggregation and
ligand leakage during elution {[}\emph{\textsuperscript{21, 22}} {]}.

Fully chromatographic fractionation of plasma has the potential to
substantially accelerate the isolation of plasma proteins - especially
IVIgs - and significantly reducing process footprint, thus lowering the
cost of therapy {[}\emph{\textsuperscript{23, 24}} {]}. This, however,
requires the design of novel chromatographic tools and processes that
combine high binding capacity and selectivity with scalability,
affordability, and safety.

Our group has developed the concept of ``flow-through affinity
chromatography'', wherein all the non-target species in a complex
feedstock are captured by peptide ligands immobilized on a
chromatographic substrate (LigaGuard\textsuperscript{TM}), allowing the
target protein product to flow through unbound
{[}\emph{\textsuperscript{25}} {]}. A
LigaGuard\textsuperscript{TM}adsorbent tailored for the purification of
therapeutic mAbs from Chinese Hamster Ovary (CHO) cell culture harvests
has been demonstrated in prior work {[}\emph{\textsuperscript{26, 27}}
{]}. In this study, a LigaGuard\textsuperscript{TM} adsorbent tailored
for IVIg purification from human plasma has been developed, whose
peptide ligands bind all non-Ig plasma proteins. Our group has also
developed a Ig-targeting peptide- {[}\emph{\textsuperscript{28}} {]} and
peptoid-based {[}\emph{\textsuperscript{29-31}} {]} adsorbents
(LigaTrap\textsuperscript{TM}), which have been demonstrated by
purifying pIgG from different mammalian plasma as well as therapeutic
mAbs from industrial cell culture fluids.

In this study, we implemented the pair of
LigaGuard\textsuperscript{TM}and LigaTrap\textsuperscript{TM} adsorbents
into a two-step process of chromatographic purification of polyclonal
immunoglobulin G (IgG) from human plasma: the
LigaGuard\textsuperscript{TM} adsorbent operates in flow-through mode to
capture plasma proteins, while LigaTrap\textsuperscript{TM} isolates the
IgG product in bind-and-elute mode. The buffer composition and pH, the
column loading, and residence time were optimized for both adsorbents to
maximize product recovery and purity. Specifically, 20 mM Bis-Tris HCl
buffer at pH 5.5 (Buffer A) was selected as running buffer for
LigaGuard\textsuperscript{TM}, whereas 0.1 M phosphate buffer at pH 7.4
containing 0.5 M sodium chloride and 25 mM sodium caprylate (Buffer B)
was selected as binding and washing buffer for
LigaTrap\textsuperscript{TM}.

Two alternative processes were evaluated. The first design utilized a
LigaGuard\textsuperscript{TM} column for scrubbing plasma proteins prior
to the IgG purification-and-concentration step using a
LigaGuard\textsuperscript{TM} column; the plasma diluted in Buffer A was
fed to a LigaGuard\textsuperscript{TM} column, the effluent was
exchanged to Buffer B and loaded on a
LigaTrap\textsuperscript{TM}column, from which the pIgG produced was
eluted with 63.8\% global recovery and 99.7\% purity, corresponding to a
2-fold concentration and a 3.28 logarithmic removal value (LRV) of
plasma proteins. For comparison, Protein G agarose afforded a
\textasciitilde{}64\% recovery and \textasciitilde{}97\% purity alone,
and \textasciitilde{}67\% recovery and \textasciitilde{}97\% purity when
preceded by LigaGuard\textsuperscript{TM}. In the second design, the
adsorbents were switched: the LigaTrap\textsuperscript{TM} column was
initially utilized to capture and concentrate pIgG from plasma; the
eluted fraction was exchanged to Buffer A and fed to a
LigaGuard\textsuperscript{TM} column to polish the elution stream,
affording 82.3\% global recovery and 98.8\% purity (3.41 LRV).
Collectively, these results demonstrate the potential of a fully
chromatographic process for purifying polyclonal IgG from plasma
feedstocks.

\textbf{2. Experimental}

\emph{2.1. Materials.} The Fmoc-Cys-(Trt)-Rink polystyrene resin was
purchased from Anaspec (Fremont, CA, USA), Toyopearl AF-Amino-650M resin
was obtained from Tosoh Corporation (Tokyo, Japan), and WorkBeads 40 ACT
resin was from Bioworks (Uppsala, Sweden).
Fmoc-N-{[}3-(N-Pbf-guanidino)-propyl{]}-glycine was from PolyPeptide
(Torrance, CA, USA). Fluorenylmethoxycarbonyl- (Fmoc-) protected amino
acids Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,
Fmoc-Phe-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-His(Trt)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, and
Fmoc-Glu(OtBu)-OH, Hexafluorophosphate Azabenzotriazole Tetramethyl
Uronium (HATU), diisopropylethylamine (DIPEA), piperidine, and
trifluoroacetic acid (TFA) were obtained from ChemImpex International
(Wood Dale, IL, USA). Kaiser test kits, triisopropylsilane (TIPS), and
1,2-ethanedithiol (EDT) were obtained from Millipore Sigma (St. Louis,
MO, USA). N,N'-dimethylformamide (DMF), dichloromethane (DCM), methanol,
and N-methyl-2-pyrrolidone (NMP) were obtained from Fisher Chemical
(Hampton, NH, USA).

Human polyclonal Immunoglobulin G (IgG) in lyophilized form was
purchased from Athens Research \& Technology, Inc (Athens, GA, USA).
Ig-rich paste, and cryo-rich and cryo-poor human plasma were a gift of
CSL Behring (King of Prussia, PA, USA). Sodium phosphate monobasic
(NaH\textsubscript{2}PO\textsubscript{4}), sodium phosphate dibasic
(Na\textsubscript{2}HPO\textsubscript{4}), hydrochloric acid, sodium
hydroxide, Bis-Tris, ethanol, sodium chloride (NaCl), and sodium
caprylate (NaCapr) were purchased from Fisher Scientific (Hampton, NH,
USA). Phosphate buffered saline at pH 7.4 was purchased from Millipore
Sigma (St. Louis, MO, USA). Vici Jour PEEK chromatography columns (2.1
mm ID, 30 mm length, 0.1 mL volume), Alltech chromatography columns (3.6
mm ID, 50 mm length, 0.5 mL volume), and 10 \selectlanguage{greek}µ\selectlanguage{english}m polyethylene frits were
obtained from VWR International (Radnor, PA, USA). The Yarra 3 \selectlanguage{greek}µ\selectlanguage{english}m
SEC-2000 300 x 7.8 mm size exclusion chromatography column was obtained
from Phenomenex Inc. (Torrance, CA, USA). Protein G
Sepharose\textsuperscript{TM} Fast Flow resin was purchased from
Millipore Sigma (Burlington, MA, USA). The 10-20\% Tris-Glycine HCl
SDS-PAGE gels and Coomassie blue stain were purchased from Bio-Rad Life
Sciences (Carlsbad, CA, USA). A Pierce BCA Protein Assay Kit was
purchased from Fisher Scientific\textsuperscript{} (Pittsburgh, PA,
USA). All chromatographic experiments were performed using a Waters
Alliance 2690 separations module system equipped with a Waters 2487 dual
absorbance detector were purchased from Waters Corporation (Milford, MA,
USA).

2.2. Preparation of LigaTrap\textsuperscript{TM} Human IgG resin and
LigaGuard\textsuperscript{TM} resin. The peptoid ligand PL-16 was
synthesized and conjugated to WorkBeads 40 ACT resin as described in
prior work (\emph{note:} the conjugation strategy and the value of
peptoid density on WorkBeads resin are proprietary information of
LigaTrap Technologies LLC) {[}\emph{\textsuperscript{32, 33}} {]}. The
resulting LigaTrap\textsuperscript{TM} Human IgG resin was rinsed in
water and stored in 20\% v/v aqueous methanol for long-term storage. The
peptide-based LigaGuard\textsuperscript{TM} resin was produced by direct
peptide synthesis on Toyopearl AF-Amino-650M resin via Fmoc/tBu strategy
as described in prior work (\emph{note:} the values of peptide density
on Toyopearl resin are proprietary information of LigaTrap Technologies
LLC) {[}\emph{\textsuperscript{34}} {]}, and stored in 20\% v/v aqueous
methanol for long-term storage.

2.3. Dynamic binding capacity of pure IgG on
LigaTrap\textsuperscript{TM} Human IgG resin. The dynamic binding
capacity of LigaTrap\textsuperscript{TM} Human IgG resin at 10\%
breakthrough of IgG (\emph{DBC} \textsubscript{10\%}, mg/mL resin) was
measured as reported in prior studies {[}\emph{\textsuperscript{28,
35-37}} {]}. A volume of 0.1 mL of LigaTrap\textsuperscript{TM} Human
IgG resin was wet packed in a Vici Jour PEEK column, washed with 10
column volumes (CVs) of 20\% v/v ethanol, deionized water (3 CVs), and
finally equilibrated with 10 CVs of PBS buffer at pH 7.4. A volume of 2
mL of solution of human polyclonal IgG at either 5 mg/mL or 10 mg/mL in
PBS buffer was continuously loaded on the column at the flow rate of
either 0.05 mL/min (residence time, RT: 2 min) or 0.02 mL/min (RT: 5
min). Following load, the resin was washed with 10 CVs of PBS buffer at
the flow rate of 0.1 mL/min. IgG elution was then performed with 20 CVs
of 0.2 M acetate buffer at pH 4.0 at the flow rate of 0.2 mL/min. The
resin was regenerated with 10 CVs of 0.1 M glycine buffer at pH 2.5 at
the flow rate of 0.2 mL/min. The effluents were continuously monitored
by UV spectrometry at 280 nm and the resulting chromatograms were
utilized to calculate the \emph{DBC} \textsubscript{10\%} of IgG.

2.4. Purification of IgG from Ig-rich paste, cryo-poor, and cryo-rich
human plasma using LigaTrap\textsuperscript{TM} Human IgG resin in
bind-and-elute mode. A volume of 0.1 mL of LigaTrap\textsuperscript{TM}
Human IgG resin was wet packed in a Vici Jour PEEK column, washed with
20\% v/v ethanol (10 CVs), deionized water (3 CVs), and finally
equilibrated with binding buffer (10 CVs) at the flow rate of 0.2
mL/min. The following binding buffers were prepared:\emph{(i)} X M NaCl
in PBS at pH Y, wherein X is either 0, 0.15, 0.25, or 0.5, or Y is
either 6.5, 7.0, 7.4, or 8.0; and \emph{(ii)} 0.5 M NaCl and Z mM NaCapr
in PBS at pH Y, wherein Z is either 0, 25, 50, or 75, and Y is either
7.4 or 8.0. The Ig-rich paste was dissolved in the binding buffer to
achieve a total protein concentration of \textasciitilde{}10 mg/mL by
stirring the solution overnight at 4\selectlanguage{ngerman}°C; cryo-poor plasma was diluted in
binding buffer to achieve a total protein titer of 25.7 mg/mL and an IgG
titer of 7.4 mg/mL; similarly, cryo-rich plasma was diluted in binding
buffer to achieve a total protein titer of 30.0 mg/mL and an IgG titer
of 7.0 mg/mL; the feedstocks were filtered using 0.44 \selectlanguage{greek}μ\selectlanguage{english}m and 0.22 \selectlanguage{greek}μ\selectlanguage{english}m
Millex-GP syringe filters (MilliporeSigma, Burlington, MA). A volume of
either 0.2 mL of Ig-rich paste solution (corresponding to 1.5 mg of
IgG), 0.2 mL of cryo-poor plasma solution (1.5 mg of IgG), or 0.2 mL of
cryo-rich plasma solution (1.4 mg of IgG) were loaded on the column at
the flow rate of 0.02 mL/min (RT: 5 min). After washing the adsorbent
with 10 CVs of binding buffer at 0.1 mL/min, the bound IgG was eluted
with 20 CVs of 0.2 M acetate buffer at pH 4.0 at 0.2 mL/min and
neutralized upon collection using 3 M Tris buffer at pH 8.5. The
adsorbent was then regenerated with 10 CVs of 0.1 M glycine buffer at pH
2.5 at 0.2 mL/min, cleaned in place with 10 CVs of aqueous 0.1 M NaOH,
and finally equilibrated with binding buffer. The collected flow-through
and elution fractions were analyzed by Protein G
Sepharose\textsuperscript{TM} Fast Flow resin to obtain IgG yield, and
via size exclusion chromatography (SEC) and SDS-PAGE under reducing
condition to obtain IgG purity.

2.5. Capture of non-Ig plasma proteins in flow-through mode by first-
and second-generation LigaGuard\textsuperscript{TM}adsorbents. The
following mobile phases were prepared: 20 mM piperazine HCl buffer at pH
5.0 and 5.5; 20 mM Bis-Tris HCl buffer at pH 5.5, 6.0, 6.5, and 7.0; 20
mM citric acid and Na\textsubscript{2}HPO\textsubscript{4} at pH 6.0,
6.5, 7.0, and 7.4; 20 mM KH\textsubscript{2}PO\textsubscript{4} and
Na\textsubscript{2}HPO\textsubscript{4} at pH 6.0, 6.5, 7.0, and 7.4; 20
mM Tris HCl buffer at pH 7.0 and 7.4; and PBS buffer at pH 7.4. A volume
of 0.5 mL of either first- or second-generation
LigaGuard\textsuperscript{TM} resin was wet packed in a 0.5 mL Alltech
PEEK column, washed with 20\% v/v ethanol (10 CVs) and deionized water
(3 CVs), and finally equilibrated with binding buffer (10 CVs) at the
flow rate of 0.5 mL/min. Pure IgG solutions were prepared by dissolving
human polyclonal IgG in the above-listed buffers at 2.5 mg/mL. The
Ig-depleted plasma samples were prepared as the flow-through fractions
obtained by injecting 1.0 mL of cryo-rich plasma diluted 10-fold with
the corresponding buffer in the columns packed with 1.0 mL
HiTrap\textsuperscript{} Protein A HP and 1.0 mL of
HiTrap\textsuperscript{} Protein G HP. A volume of 7 mL volume of
either pure IgG solution or Ig-depleted diluted plasma (protein titer
\textasciitilde{}5.0 mg/mL) was continuously loaded on the
LigaGuard\textsuperscript{TM} column at the flow rate of 0.5 mL/min (RT:
1 min) and the flow-through fractions were collected at 0.5 mL
increments; after loading, the column was washed with 20 CVs of
equilibration buffer, and a pooled wash fraction was collected until the
280 nm absorbance decreased below 50 mAU. The resin was discarded after
one use (\emph{i.e.} , no elution or regeneration was performed). The
collected fractions were analyzed by Pierce BCA Protein Assay Kit,
Protein G Sepharose\textsuperscript{TM} Fast Flow resin, size exclusion
chromatography (SEC), and SDS-PAGE under reducing condition to obtain
the breakthrough ratio (\textbf{Equation 1} ), yield (\textbf{Equation
2} ), and binding (mg of protein per mL resin, \textbf{Equation 3} ).

2.6. Purification of IgG from cryo-rich and cryo-poor human plasma using
a 2-step chromatographic process comprising
LigaGuard\textsuperscript{TM} and LigaTrap\textsuperscript{TM} resins.
LigaGuard\textsuperscript{TM} and LigaTrap\textsuperscript{TM} resin
were wet packed in three 0.5 mL Alltech PEEK columns, and washed with
20\% v/v ethanol (10 CVs) and deionized water (3 CVs). The
LigaGuard\textsuperscript{TM} resin were equilibrated with 10 CVs of 20
mM Bis-Tris HCl buffer either at pH 6.0 or 5.5 (Buffer A), while the
LigaTrap\textsuperscript{TM} resin was equilibrated with 10 CVs of 0.1 M
phosphate buffer at pH 7.4 containing 0.5 M NaCl and 25 mM NaCapr
(Buffer B). Diluted plasma, prepared as described in \emph{Section 2.4}
, was loaded on the LigaGuard\textsuperscript{TM} column at the flow
rate of 0.5 mL/min (RT: 1 min), and the flow-through fractions were
collected at 0.5 mL increments; the loading was chased with 20 CVs of
0.2M acetate buffer at pH 5.0 (Buffer C). The IgG-rich effluent
collected during loading and buffer chasing was continuously mixed with
Buffer B and injected on the LigaTrap\textsuperscript{TM} column. Column
loading and washing, IgG elution, and column regeneration and cleaning
were performed as detailed in \emph{Section 2.5} . The collected
flow-through and elution fractions were analyzed by Protein G
Sepharose\textsuperscript{TM} Fast Flow resin to obtain IgG yield, and
via size exclusion chromatography (SEC) and SDS-PAGE under reducing
condition to obtain IgG purity.

2.7. Quantification of IgG yield by analytical Protein G chromatography.
The IgG concentration in the collected fractions were determined by
analytical Protein G chromatography using a Waters Alliance 2690
separations module system with a Waters 2487 dual absorbance detector
(Waters Corporation, Milford, MA, USA). Protein G
Sepharose\textsuperscript{TM} Fast Flow resin wet packed in a Vici Jour
PEEK 2.1 mm ID x 30 mm column (0.1 mL) was equilibrated with PBS, pH
7.4. A volume of 50 \selectlanguage{greek}µ\selectlanguage{english}L for each sample or standard was injected, and the
analytical method proceeded as outlined in \textbf{Table 1} . The
effluent was monitored by 280 nm absorbance (A280), and the
concentration was determined based on the peak area of the A280 elution
peak. Pure IgG at 0, 0.1, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, and 10.0 mg/mL
was utilized to construct the standard curve.

\emph{\textbf{Table 1.} HPLC method for IgG quantification by analytical
Protein G chromatography.}\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
\textbf{Time (min)}\strut
\end{minipage} & \begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
\textbf{Flowrate} \textbf{(mL/min)}\strut
\end{minipage} & \begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
\textbf{\% Buffer A}\strut
\end{minipage} & \begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
\textbf{\% Buffer B}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.5\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
100\%\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
2.00\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.5\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
100\%\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
2.01\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.5\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0\%\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
100\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
6.00\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.5\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0\%\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
100\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
6.01\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.5\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
100\%\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
10.00\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.5\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
100\%\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0\%\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

To assess the breakthrough ratio (C/C\textsubscript{0}), yield (\emph{Y}
, \%) and binding capacity (\emph{Q} , mg/mL resin) of IgG product, the
values of fractional breakthrough ratio, pooled yield, and protein
binding as a function of CV were calculated using\textbf{Equations 1} --
\textbf{3} , respectively.

\textbf{Equation 1}\({\mathbf{\ }C/C_{0}}_{fractional,\ \ x}=\frac{C_{IgG,x}}{C_{IgG,L}\ }\)

\textbf{Equation 2}\({Y\left(\%\right)}_{pooled,\ x}=\frac{\sum_{x=1}^{N}{C_{IgG,x}\ \times\ V_{x}}}{C_{IgG,L}\ \times\text{\ V}_{L}}\times 100\ \%\)

\textbf{Equation 3}\({Q(mg/mL\ resin)}_{pooled,\ x}=\frac{\sum_{x=1}^{N}{\left(C_{IgG,L}-C_{IgG,x}\right)\ \times\ V_{x}}}{V_{R}}\)

Wherein C/C\textsubscript{0}(\%)\textsubscript{fractional,x} is the
fractional IgG breakthrough ratio at fraction x,
Y(\%)\textsubscript{pooled,x} is the pooled IgG yield at fraction x,
Q(mg/mL resin)\textsubscript{pooled,x} is the pooled binding capacity in
at fraction x, C\textsubscript{IgG,x} is the IgG concentration in
fraction x, V\textsubscript{x} is the volume of fraction x,
C\textsubscript{IgG,L} is the IgG concentration in the load samples, and
V\textsubscript{L} is the cumulative feed volume loaded, and N is the
number of fractions generated by loading V\textsubscript{L}, and
V\textsubscript{R} is the volume of selected resin.

To assess the breakthrough performance of non-immunoglobulin proteins,
the corresponding values were calculated using \textbf{Equation 1 -- 3}
, respectively.

2.8. Quantification of IgG purity by size-exclusion chromatography
(SEC). The collected fractions were then analyzed by analytical SEC
using a Yarra 3 \selectlanguage{greek}µ\selectlanguage{english}m SEC-2000 300 mm x 7.8 mm column operated with a
40-min isocratic method using PBS at pH 7.4 as mobile phase. A volume of
50 \selectlanguage{greek}µ\selectlanguage{english}L of sample was injected and the effluent continuously monitored by
UV spectrometry at 280 nm absorbance (A280). The fractional purity of
IgG (P, \%) was calculated using\textbf{Equation 4} .

\textbf{Equation 4}\({P(\%)}_{fractional,x}=\frac{A_{IgG,x}}{A_{IgG,x}+A_{impurity,x}}\times 100\%\)

Wherein P(\%)\textsubscript{fractional,x} is the fractional IgG purity
at fraction x, A\textsubscript{IgG,x} is the IgG peak area in the x-th
fraction, A\textsubscript{impurity,x} is the impurity peak area in the
x-th fraction.

2.9. Quantification of IgG purity by sodium dodecyl polyacrylamide gel
electrophoresis (SDS-PAGE). The collected fractions were analyzed by
SDS-PAGE using 4-20\%
Mini-PROTEAN\textsuperscript{TM}TGX\textsuperscript{} Precast protein
gels with Tris/Glycine/SDS buffer as running buffer. The fractions were
diluted or concentrated to a total protein concentration of
\textasciitilde{}1 mg/mL and a volume of 10 \selectlanguage{greek}µ\selectlanguage{english}L of different samples were
loaded to the wells of SDS-PAGE gels. The sample stripes were
concentrated under 80 V for about 30 min and separated under 120 V for
about 1 h. Then the gels were stained by Coomassie Brilliant Blue R-250
Staining solution for about 25 min and then distained using 10\% glacial
acetic acid, 5\% ethanol dissolved in Milli-Q water. Finally, the
stained protein strips were imagined by the Gel Doc2000 imaging system
from Bio-Rad.

\textbf{3. Results and Discussion}

\textbf{3.1. IgG purification from human Ig-rich paste using
LigaTrap\textsuperscript{TM} resin}

LigaTrap\textsuperscript{TM} resin has been developed by our team for
purifying \selectlanguage{greek}γ-\selectlanguage{english}globulins from polyclonal and monoclonal sources. This
affinity adsorbent has been used by purifying \emph{(i)} pIgG from a
variety of mammalian sources, including the human, rodent (mouse, rat,
and rabbit), caprine (goat and sheep), equine (horse and donkey),
camelid (camel and llama); \emph{(ii)} human monoclonal IgG, IgA, and
IgM from Chinese Hamster Ovary (CHO) and Human Embryonic Kidney (HEK)
cell culture supernatants {[}\emph{\textsuperscript{33, 38}} {]};
and\emph{(iii)} avian IgY {[}\emph{\textsuperscript{39}} {]}. Compared
to Protein A/G-based adsorbents, the LigaTrap\textsuperscript{TM} resin
features comparable binding capacity and selectivity, improved lifetime
{[}\emph{\textsuperscript{32}} {]}, and a substantially lower cost,
making it ideal for the chromatographic extraction of pIgG from large
volumes of pooled plasma.

In this study, we initially evaluated the performance of
LigaTrap\textsuperscript{TM} resin against a human Ig-rich paste
obtained via cold ethanol precipitation of plasma. To this end, we
optimized the process conditions focusing on protein loading (mass of
IgG loaded per volume of resin), residence time (\textbf{Figure S1} ),
and composition (sodium chloride and sodium caprylate), concentration
(0, 0.15, 0.25 and 0.5 M of NaCl), and pH (6.5, 7.0, 7.4 and 8.0) of the
binding and washing buffers. The eluted fractions, obtained at pH 4.0,
were analyzed via analytical Protein G HPLC to determine pIgG adsorption
and yield (\textbf{Figured 1A} and \textbf{1B} ), and size exclusion
chromatography (SEC, \textbf{Figure 2C} ) and gel electrophoresis to
determine product purity in the eluted fractions (\textbf{Figure 2} ).

\textbf{Hosted file}

\verb`image1.emf`  available at \url{https://authorea.com/users/425045/articles/529963-purification-of-polyclonal-immunoglobulin-g-from-human-serum-using-ligaguardtm-and-ligatraptm-adsorbents}

\par\null

Figure 1.\emph{Values of \textbf{(A)} pIgG adsorption (mg of pIgG per mL
of LigaTrap\textsuperscript{TM} resin),\textbf{(B)} elution yield (\%),
and \textbf{(C)} pIgG purity in the eluted fraction (\%) obtained by
purifying pIgG from anIg-rich paste using LigaTrap\textsuperscript{TM}
resinand binding buffers with different pH (6.5, 7.0, 7.4, and 8.0) and
NaCl concentrations (0, 0.15, 0.25, and 0.5 M).}

\textbf{Hosted file}

\verb`image2.emf`  available at \url{https://authorea.com/users/425045/articles/529963-purification-of-polyclonal-immunoglobulin-g-from-human-serum-using-ligaguardtm-and-ligatraptm-adsorbents}

\emph{\textbf{Figure 2.} SDS-PAGE analysis (reducing conditions,
Coomassie staining) of the chromatographic fractions obtained by
purifying pIgG from an Ig-rich paste using
LigaTrap\textsuperscript{TM}resin and binding buffers with different pH:
\textbf{(A)} 6.5,\textbf{(B)} 7.0, \textbf{(C)} 7.4, and \textbf{(D)}
8.0, and sodium chloride concentrations (0, 0.15, 0.25, and 0.5 M).
Labels: MW, molecular weight marker; hIgG, human polyclonal IgG
standard; F, feedstock obtained by dissolving the Ig-rich paste in
binding buffer; FT-X, flow-through fraction obtained by loading Ig-rich
paste diluted in a binding buffer with XM sodium chloride concentration;
E-X, elution fraction obtained by loading Ig-rich paste diluted in a
binding buffer with XM sodium chloride concentration; HSA, human serum
albumin; IgG HC, heavy chain of IgG; IgG LC, light chain of IgG.}

As shown in \textbf{Figure 1} , the value of pH in the binding buffer is
the major determinant of pIgG binding strength and selectivity on
LigaTrap\textsuperscript{TM} resin; conversely, the value of NaCl
concentration has a rather minor influence on product capture and
purity. At all values of salinity, in fact, pIgG binding and yield
continuously increased as the binding pH ranged from 6.5 to 8.0.
Specifically, pIgG binding and elution yield reached their maximum of
\textasciitilde{}11.4 mg/mL at pH 7.4 and \textasciitilde{}99.0\% at pH
8.0, respectively; conversely, within each value of binding pH, both
binding and yield remained constant as the NaCl concentration increased,
demonstrating that LigaTrap\textsuperscript{TM} resin features
salt-tolerant adsorption of pIgG. These results are coherent with the
chemical make-up of the LigaTrap ligand, which features a combination of
hydrophobic (indolyl and isopropyl), cationic (pyridinyl and guanidyl),
and hydrogen bond-forming (guanidyl and backbone amides) moieties
{[}\emph{\textsuperscript{32}} {]}. As the isoelectric point of pIgG
varies in the range 7.0 - 8.1 {[}\emph{\textsuperscript{40, 41}} {]}, it
stands to reason that binding is highest at 7.4, being driven mainly by
hydrophobic and hydrogen bond interactions, which provide for
salt-tolerant binding; as the pH is lowered to 4, elution is triggered
by the electrostatic repulsion between the induced cationic charges on
the pIgG and the ligand. While being a key driver of binding, the
hydrophobic interactions do not compromise the selectivity of pIgG:
ligand binding, which increases the purity of the target product 2-fold,
from \textasciitilde{}43 \% in the feedstock to \textasciitilde{}90\% in
the eluted fraction. When column loading is conducted at low salinity
(\emph{i.e.} , up to 0.15 M NaCl), the purity of eluted pIgG shows a
decreasing trend with pH. This is imputed to anionic plasma proteins,
such as \selectlanguage{greek}α\selectlanguage{english}\textsubscript{1}-antitrypsin (pI \textasciitilde{} 4.6),
albumin (pI \textasciitilde{} 4.7), fibrinogen (pI \textasciitilde{}
5.5), and transferrin (pI \textasciitilde{} 6)
{[}\emph{\textsuperscript{42}} {]}, which acquire a strong binding drive
towards the cationic moieties of the ligand as the pH increases. This,
however, can be effectively mitigated by increasing the conductivity of
the binding and washing buffers: as the NaCl concentration is increased
to 0.5 M, in fact, the pIgG purity in the eluted fraction is
consistently at 90\%. These results are visually concerned by the
electrophoretic analysis of the chromatographic fractions reported
in\textbf{Figure 2} : \emph{(i)} as the pH and the conductivity of the
binding buffer increase, the presence of pIgG in the flow-through
fraction decreases, while the amount of non-Ig plasma proteins
increases; \emph{(iii)} at pH 7.0, the purity of pIgG in the eluted
fraction is the highest across all values of binding conductivity.
Overall, binding at higher conductivity effectively prevents
electrostatic binding of non-Ig plasma proteins and promotes the
hydrophobic component of IgG adsorption, affording high product capture,
yield ([?] 85.0 \%), and purity (\textasciitilde{}90.0\%). Accordingly, we
adopted NaCl concentration of 0.5 M in both binding and washing buffer
for the remainder of this study.

To further improve the performance of the
LigaTrap\textsuperscript{TM}resin for its final implementation in the
proposed 2-step process, we evaluated the addition of sodium caprylate
to minimize the capture of the highly abundant non-Ig serum proteins,
chiefly albumin. Accordingly, new feedstock solution was prepared by
dissolving the Ig-rich paste in different buffers prepared by adjusting
a base aqueous solution of 0.1 M phosphate and 0.5 M NaCl at different
values of pH (7.4 or 8.0) and sodium caprylate concentration (0, 25, 50,
or 75 mM). The key chromatographic results -- namely, pIgG binding, and
elution yield and purity -- are summarized in\textbf{Figure 3} , while
the electrophoretic analysis of the collected fractions is reported in
\textbf{Figure 4} .

\textbf{Hosted file}

\verb`image3.emf`  available at \url{https://authorea.com/users/425045/articles/529963-purification-of-polyclonal-immunoglobulin-g-from-human-serum-using-ligaguardtm-and-ligatraptm-adsorbents}

\emph{\textbf{Figure 3.} Values of \textbf{(A)} pIgG adsorption (mg of
pIgG per mL of LigaTrap\textsuperscript{TM} resin), \textbf{(B)} elution
yield (\%), and \textbf{(C)} pIgG purity in the eluted fraction (\%)
obtained by purifying pIgG from an Ig-rich paste using
LigaTrap\textsuperscript{TM} resin and binding buffers with different pH
(7.4 and 8.0) and sodium caprylate concentrations (0, 25, 50 and 75 mM),
and constant NaCl concentration (0.5 M).}

\textbf{Hosted file}

\verb`image4.emf`  available at \url{https://authorea.com/users/425045/articles/529963-purification-of-polyclonal-immunoglobulin-g-from-human-serum-using-ligaguardtm-and-ligatraptm-adsorbents}

\emph{\textbf{Figure 4.} SDS-PAGE analysis (reducing conditions,
Coomassie staining) of the chromatographic fractions obtained by
purifying pIgG from an Ig-rich paste using
LigaTrap\textsuperscript{TM}resin and binding buffers with pH of either
\textbf{(A)} 7.4 or\textbf{(B)} 8.0, and sodium caprylate concentrations
(0, 25, 50 and 75 mM), and constant NaCl concentration (0.5 M). Labels:
MW, molecular weight marker; hIgG, human polyclonal IgG standard; F,
feedstock obtained by dissolving the Ig-rich paste in binding buffer;
FT-X, flow-through fraction obtained by loading Ig-rich paste diluted in
a binding buffer with XM sodium caprylate concentration; E-X, elution
fraction obtained by loading Ig-rich paste diluted in a binding buffer
with XM sodium caprylate concentration; HSA, human serum albumin; IgG
HC, heavy chain of IgG; IgG LC, light chain of IgG.}

Notably, the addition of caprylate to the binding and washing buffers
substantially reduced the amount of bound non-Ig plasma proteins,
bringing the purity of pIgG in the elution from \textasciitilde{}90\%
(no caprylate) to 94\% (75 mM caprylate and pH 8.0); the rejection of
albumin in particular is evident in the electrophoretic analysis of the
flow-through fractions collected at pH 8.0 (\textbf{Figure 4} ). At the
same time, however, the addition of caprylate increases the strength of
IgG adsorption, bringing the yield of pIgG in the elution from
\textasciitilde{}99\% (no caprylate) to 64\% (75 mM caprylate and pH
8.0). In this regard, we speculate that, as the concentration of sodium
caprylate in the binding buffer increases, the caprylate anions
progressively saturate the binding sites on albumin and other plasma
proteins, eventually adsorbing on IgG and increasing the effective
hydrophobicity of its surface; this trend has been observed in prior
work using the mixed-mode resin MEP HyperCel
{[}\emph{\textsuperscript{43}} {]}, where it was shown to increase the
purity of pIgG at the cost of yield. Overall, column loading at pH 7.4
afforded values of yield that are significantly and consistently higher
(\textasciitilde{} 95 -- 100\%) than those obtained by loading at pH 8.0
(\textasciitilde{} 63 -- 83\%). Considering the high yield
(\textasciitilde{} 95\%) and purity (\textasciitilde{} 91\%), 0.1 M
phosphate buffer at pH 7.4 added with 0.5 M NaCl and 25 mM sodium
caprylate was selected as binding and washing buffer for the remainder
of this study.

\par\null

\textbf{3.2. Purification of pIgG from cryo-rich and cryo-poor plasma in
flow-through mode using LigaGuard\textsuperscript{} adsorbents}

The LigaGuard\textsuperscript{TM} adsorbent has been originally
developed by our team as a combination of resins functionalized with
multi-modal peptides for purifying monoclonal antibodies (mAbs) in
flow-through mode from recombinant sources
{[}\emph{\textsuperscript{44-46}} {]}. The ensemble of ligands is
capable of capturing a broad spectrum of protein impurities while
allowing the antibody product to flow through unbound. The multi-modal
character of the peptides provides them with the ability to capture
hundreds of protein contaminants that encompass a broad range of amino
acid composition, post-translational modification, size, and
concentration.

In this study, the LigaGuard\textsuperscript{TM} adsorbent was utilized
for the first time for IgG purification from a polyclonal source.
However, the higher complexity of plasma, where the ratio of
Ig\emph{vs.} non-Ig proteins is about 1:5 {[}\emph{\textsuperscript{47}}
{]}, compared to that of recombinant sources, where the same ratio
varies between 5:1 and 10:1 {[}\emph{\textsuperscript{48-50}} {]},
required an optimization of both the composition of the
LigaGuard\textsuperscript{TM} ligands and the chromatographic protocol.

First, the chromatographic process was optimized by evaluating the
effect of composition and pH of the running buffer on the recovery of
pIgG and the retention of non-Ig plasma proteins. To this end, 20 mM
piperazine HCl buffer at pH 5.0 and 5.5; 20 mM Bis-Tris HCl buffer at pH
5.5, 6.0, 6.5, and 7.0; 20 mM citric acid and
Na\textsubscript{2}HPO\textsubscript{4} at pH 6.0, 6.5, 7.0, and 7.4; 20
mM KH\textsubscript{2}PO\textsubscript{4} and
Na\textsubscript{2}HPO\textsubscript{4} at pH 6.0, 6.5, 7.0, and 7.4; 20
mM Tris HCl buffer at pH 7.0 and 7.4; and PBS buffer at pH 7.4 were
evaluated as buffers. Injections of either pure human pIgG
(\textasciitilde{} 3.0 mg/mL) or Ig-depleted plasma (\textasciitilde{}
5.0 mg/mL) were performed to select a value of protein load and a
binding condition that hold the highest promise towards pIgG
purification from plasma in flow-through mode. Following prior work with
LigaGuard\textsuperscript{TM} adsorbent, the residence time was set at 1
min to ensure fast processing of plasma and minimize processing time.

The resultant profiles of pIgG yield (\emph{Y\textsubscript{pIgG}}
)\emph{vs.} loading volume are collated in \textbf{Figure 5} ; the
corresponding values of breakthrough ratio, namely ratio of pIgG titer
in the effluent \emph{vs.} feedstock
(\emph{C\textsubscript{pIgG}/C\textsubscript{pIgG}*} ), and binding
(\emph{Q\textsubscript{pIgG}} ) are reported in \textbf{Figures S4A}
and\textbf{S4B} , respectively. The profiles of non-Ig plasma proteins
capture (\emph{Q\textsubscript{PP}} ) \emph{vs.} loading volume are
collated in \textbf{Figure 6} , while the corresponding values of
breakthrough ratio (\emph{C\textsubscript{PP}/C\textsubscript{PP}*} )
are reported in \textbf{Figure S4C} . By highlighting the role of buffer
composition and pH on the binding of plasma proteins, these results
corroborate the classification of the
LigaGuard\textsuperscript{TM}peptides as advanced multi-modal ligands.
As observed in prior work, the pH is the most critical parameter in
determining binding capacity and selectivity: decreasing pH, in fact,
consistently improved binding selectivity by concurrently increasing
\emph{Y\textsubscript{pIgG}} and\emph{Q\textsubscript{PP}} .
Specifically, at the loading volume of 5 mL -- corresponding to 10
column volumes (CVs) -- at which the LigaGuard\textsuperscript{TM}
adsorbent has reached saturation on non-Ig plasma proteins -- decreasing
the loading pH from 7.4 to 5.5 improved\emph{Y\textsubscript{pIgG}} from
\textasciitilde{}35.0 \% to \textasciitilde{}80.0 \%. These results can
be directly tied to the isoelectric point of both pIgG and the
LigaGuard\textsuperscript{TM}peptides: as the environment becomes more
acidic, both the ligands (pI = 7.8-12.5) {[}\emph{\textsuperscript{51}}
{]} and pIgG (pI = 6.1-8.5) {[}\emph{\textsuperscript{40, 41}} {]}
acquire a net positive charge, and undesired pIgG capture is prevented
by electrostatic repulsion.

\textbf{Hosted file}

\verb`image5.emf`  available at \url{https://authorea.com/users/425045/articles/529963-purification-of-polyclonal-immunoglobulin-g-from-human-serum-using-ligaguardtm-and-ligatraptm-adsorbents}



\emph{\textbf{Figure 5.} Profiles of pIgG flow-through yield
(Y\textsubscript{pIgG}) vs. loading volume obtained by injecting a
solution of pIgG at 3 mg/mL in different binding buffers: \textbf{(A)}20
mM piperazine HCl buffer at pH 5.0 and 5.5; 20 mM Bis-Tris HCl buffer at
pH5.5; \textbf{(B)} 20 mM citric acid and
Na\textsubscript{2}HPO\textsubscript{4,}KH\textsubscript{2}PO\textsubscript{4}
and Na\textsubscript{2}HPO\textsubscript{4}, and Bis-Tris HCl buffer at
pH 6.0; \textbf{(C)} 20 mM citric acid and
Na\textsubscript{2}HPO\textsubscript{4,}KH\textsubscript{2}PO\textsubscript{4}
and Na\textsubscript{2}HPO\textsubscript{4}, and Bis-Tris HCl buffer at
pH 6.5; \textbf{(D)} 20 mM citric acid and
Na\textsubscript{2}HPO\textsubscript{4,}KH\textsubscript{2}PO\textsubscript{4}
and Na\textsubscript{2}HPO\textsubscript{4}, Bis-Tris HCl, and Tris HCl
buffer at pH 7.0; and \textbf{(E)} 20 mM citric acid and
Na\textsubscript{2}HPO\textsubscript{4,}KH\textsubscript{2}PO\textsubscript{4}
and Na\textsubscript{2}HPO\textsubscript{4}, Tris HCl buffer at pH 7.4,
and PBS buffer at pH 7.4 - on first-generation
LigaGuard\textsuperscript{TM}resin.}

Compared to pH, buffer composition has a minor, yet still noticeable
effect on pIgG yield. In particular, as the pH decreases, differences in
ionic strength -- determined by both concentration and valency of the
ions -- result in different profiles of Y\textsubscript{pIgG}
\emph{vs.}loading volume. At pH 7.4, in fact, the values of
Y\textsubscript{pIgG}obtained with different binding buffers -- namely,
20 mM citric acid-Na\textsubscript{2}HPO\textsubscript{4}, 20 mM
KH\textsubscript{2}PO\textsubscript{4}-Na\textsubscript{2}HPO\textsubscript{4},
20 mM Tris HCl, and PBS -- are virtually indistinguishable; at pH 6.5 -
7.0, a 10\% difference in Y\textsubscript{pIgG} across the entire range
of loading volume is observed between the monovalent Bis-Tris HCl buffer
and the trivalent citric
acid-Na\textsubscript{2}HPO\textsubscript{4}buffer; as the pH decreases
further to 5.0 - 6.0, the difference in\emph{Y\textsubscript{pIgG}}
among the various buffers grows to 20\%, with piperazine HCl buffer at
pH 5.0 affording the highest product yield in flow-through mode, namely
87\% at the cut-off loading volume of 5 mL (10 CVs). The capture of pIgG
during the initial stages of binding -- namely, up to a loading volume
of 1.5 mL (3 CVs) -- is due to the makeup of the load: in the absence of
non-Ig proteins competing with pIgG for the peptide ligands, a small
product loss due to non-specific adsorption is inevitable. In this
context, it is also important to notice that Y\textsubscript{pIgG} can
be increased by ``chasing'' the loading with buffer C; for example,
Y\textsubscript{pIgG} rose from 75\% to 87\% when the loading was
followed by flowing 20 mM Bis-Tris HCl buffer at pH 6.0, and rose from
77\% to 87\% when the followed by flowing 20 mM piperazine HCl buffer at
pH 5.5. While operating binding under acidic conditions reduced the loss
of pIgG, we resolved not to explore any buffer with pH \textless{} 5.0;
operating at lower pH, in fact, would likely result in a significant
flow-through of albumin (pI \textasciitilde{}
4.7\emph{\textsuperscript{52-54}} ) and increase the risk of pIgG
denaturation and aggregation \emph{\textsuperscript{55, 56}} .
Accordingly, we adopted 20 mM Bis-Tris HCl buffer at pH 6.0 and 5.5 as
well as 20 mM piperazine HCl buffer at pH 5.5 and 5.0 as buffers for
pIgG purification from human plasma in flow-through mode in the
remainder of this study.

Next, the capture of non-Ig plasma proteins by the LigaGuard adsorbent
was evaluated. A feedstock comprising of filtered and Ig-depleted plasma
with a protein titer of \textasciitilde{}6.0 mg/mL was injected at the
residence time of 1 min; the protein titer in the effluent fractions,
collected at 0.5 mL increments, was measured to determine the profiles
of non-Ig plasma proteins capture (\emph{Q\textsubscript{PP}}
)\emph{vs.} loading volume (\textbf{Figure 6} ).

\textbf{Hosted file}

\verb`image6.emf`  available at \url{https://authorea.com/users/425045/articles/529963-purification-of-polyclonal-immunoglobulin-g-from-human-serum-using-ligaguardtm-and-ligatraptm-adsorbents}

\emph{\textbf{Figure 6.} Profiles of non-Ig plasma protein capture
(Q\textsubscript{PP}) vs. loading volume obtained by injecting diluted
Ig-depleted plasma at 6 mg/mL in different binding buffers -- namely, 20
mM piperazine HCl buffer at pH 5.0 and 5.5; 20 mM Bis-Tris HCl buffer at
pH 5.5 and 6.0; and PBS buffer at pH 7.4 - on
LigaGuard\textsuperscript{TM} resin.}

When operating at pH 7.4, substantial flow-through of non-Ig plasma
proteins was observed: as soon as the loading a volume of
\textasciitilde{} 1 - 1.5 mL (2 - 3 CVs) was reached, until which
notable capture was observed,
the\emph{C\textsubscript{PP}/C\textsubscript{PP}*} rapidly rose to
\textasciitilde{} 0.8; combined with product loss discussed above, these
results disqualified PBS as a mobile phase for flow-through purification
of pIgG using the LigaGuard. To some surprise, loading in 20 mM
piperazine-HCl buffers at pH 5.0 and 5.5 failed to afford appreciable
capture of plasma proteins; for example, at the loading volume of 2.0 mL
(4 CVs) and pH 5.5, the breakthrough curves of both pIgG and plasma
proteins overlapped, resulting in a \emph{Y\textsubscript{pIgG}} of 59\%
and a \emph{Q\textsubscript{PP}} of 10.7 mg per mL of resin.
Accordingly, piperazine-HCl was excluded as a buffer for LigaGuard
flow-through operation. Conversely, loading in Bis-Tris buffers at pH
5.5 or 6.0 led to a notable increase in the capture of non-Ig plasma
proteins: at the loading volume of 1 mL (2 CVs),
the\emph{Y\textsubscript{pIgG}} reached 50\% while only 4\% of the
loaded non-Ig plasma proteins are found in the effluent, corresponding
to a\emph{Q\textsubscript{PP}} of 8.9 mg/mL; at the cut-off value of
loading (10 CVs), the \emph{Y\textsubscript{pIgG}} reached 80\% and
approximately 53.1\% of the non-Ig plasma proteins has been captured,
corresponding to a \emph{Q\textsubscript{PP}} of \textasciitilde{} 23.7
mg/mL. The profile of \emph{Q\textsubscript{PP}} reached a plateau value
of 25 mg/mL at a loaded volume between 4 - 5 mL (8 - 10 CVs), which is
coherent with what observed in prior work on the flow-through capture of
CHO HCPs using the LigaGuard adsorbent. As no appreciable differences
in \emph{Y\textsubscript{pIgG}} and \emph{Q\textsubscript{PP}} were
found between the effluents obtained with Bis-Tris buffers at pH 5.5 and
pH 6.0, the latter was adopted as binding buffer for subsequent studies
of flow-through purification of pIgG from whole human plasma.

Capitalizing on these results, we evaluated the LigaGuard adsorbent for
the flow-through purification of pIgG from cryo-rich plasma. The latter
features a total protein titer of \textasciitilde{}70 mg/mL, including
an Ig titer of 9.7 mg/mL and an albumin titer of \textasciitilde{}60
mg/mL (\emph{note:} small variations in protein titer are regularly
observed among batches of pooled plasma depending on donors and initial
processing) {[}\emph{\textsuperscript{47}} {]}. The plasma was diluted
by either 10- or 20-fold with the selected binding buffer prior to
injection on the column to overcome the innate buffering activity of
plasma and achieve the ionic strength and pH needed to ensure effective
capture of non-Ig plasma proteins by the LigaGuard peptides. Further,
the proposed dilution, by lowering the pIgG titer, reduces the
likelihood of non-specific loss of product, while still maintaining a
level non-Ig plasma proteins sufficient to ensure capture. The diluted
plasma feedstock was loaded on the
LigaGuard\textsuperscript{TM}adsorbent at the residence time of 1 min,
and the resultant profiles of\emph{Y\textsubscript{pIgG}} and
\emph{Q\textsubscript{PP}} \emph{vs.}loading volume are collated in
\textbf{Figure 7A} and \textbf{Figure 7B} ; the corresponding profiles
of\emph{C\textsubscript{pIgG}/C\textsubscript{pIgG}*}
and\emph{C\textsubscript{PP}/C\textsubscript{PP}*} are reported
in\textbf{Figure 7C} and \textbf{Figure 7D} .

\textbf{Hosted file}

\verb`image7.emf`  available at \url{https://authorea.com/users/425045/articles/529963-purification-of-polyclonal-immunoglobulin-g-from-human-serum-using-ligaguardtm-and-ligatraptm-adsorbents}

\emph{\textbf{Figure 7.} Profiles of \textbf{(A)} pIgG flow-through
yield (Y\textsubscript{pIgG}), \textbf{(B)} non-Ig plasma protein
capture (Q\textsubscript{PP}), \textbf{(C)} ratio of pIgG titer in the
effluent vs. feedstock (C\textsubscript{pIgG}/C\textsubscript{pIgG}*),
and \textbf{(D)} ratio of non-Ig plasma protein titer in the effluent
vs. feedstock (C\textsubscript{PP}/C\textsubscript{PP}*) vs. loading
volume obtained by injecting plasma diluted using 20 mM Bis-Tris HCl
buffer at pH 6.0 to a total protein titer of either 10 mg/mL (10-fold
dilution, D10X) or 5.1 mg/mL (20-fold dilution, D20X) on
first-generation LigaGuard\textsuperscript{TM} resin.}

Most notably, the amount of pIgG captured by the resin
(\emph{Q\textsubscript{pIgG}} ) was not affected by the dilution factor
and plateaued at \textasciitilde{} 2.5 mg per mL of resin; conversely,
the value \emph{Q\textsubscript{PP}} at the cut-off loading volume
doubled from 25 mg/mL to 50 mg/mL by decreasing dilution of the
feedstock from 20-fold to 10-fold. This translated in a
global\emph{Y\textsubscript{pIgG}} at the load cut-off of
\textasciitilde{}76.4\% and a 1.7-fold reduction of non-Ig plasma
proteins from 10-fold diluted plasma, corresponding to a 0.59-fold
product enrichment in the effluent (\textbf{Table 2} ). The improvement
in the cut-off value of product yield obtained from loading the
LigaGuard\textsuperscript{TM} adsorbent with plasma in lieu of pure pIgG
solution (\emph{Y\textsubscript{pIgG}} \textasciitilde{} 70\%) results
from the presence of non-Ig plasma proteins outcompeting pIgG for
binding the peptide ligands. Furthermore, the adsorbed pIgG was stripped
from the column by chasing the loading with 2 CVs binding buffer, which
further increased \emph{Y\textsubscript{pIgG}} up to 78.8\% without
compromising the global purity of the effluent. As shown by the
electrophoretic analysis of the combined effluents in \textbf{Figure 8}
, product purity decreased from 46\% to only 44\% by adding a buffer
chasing after loading the 10-fold diluted plasma, and the corresponding
pIgG enrichment factor decreased slightly from 0.76 to 0.63
(\textbf{Table 2} ).

\emph{\textbf{Table 2.} Values of Y\textsubscript{pIgG}, reduction of
non-Ig plasma proteins, and pIgG enrichment factor in the effluent
compared to feedstock obtained by loading 5 mL (10 CVs) of diluted
plasma onto LigaGuard\textsuperscript{TM} adsorbent at 1 min residence
time.}\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
\begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{Diluted plasma} \textbf{feedstock}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Y\textsubscript{pIgG}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Y\textsubscript{pIgG}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{Reduction of non-Ig} \textbf{plasma proteins}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{Reduction of non-Ig} \textbf{plasma proteins}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{pIgG enrichment} \textbf{factor}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{pIgG enrichment} \textbf{factor}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Loading only\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Loading + chasing\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Loading only\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Loading + chasing\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Loading only\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Loading + chasing\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
10-fold\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
77\%\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
80\%\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
4.7\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
4.9\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
1.3\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
1.0\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
20-fold\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
58\%\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
63\%\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
5.9\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
5.0\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
1.0\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
1.2\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Hosted file}

\verb`image8.emf`  available at \url{https://authorea.com/users/425045/articles/529963-purification-of-polyclonal-immunoglobulin-g-from-human-serum-using-ligaguardtm-and-ligatraptm-adsorbents}

\emph{\textbf{Figure 8.} SDS-PAGE analysis (reducing conditions,
Coomassie staining) of the chromatographic fractions obtained by
purifying pIgG from diluted cryo-rich plasma using
LigaGuard\textsuperscript{TM} resin and 20 mM Bis-Tris HCl buffer at pH
6.0 as binding buffer. Labels: Feed D10X, 10-fold diluted plasma; FT
D10X 2 CV, flow-through fraction obtained by loading 1 mL of 10-fold
diluted plasma; FT D10X 6 CV, flow-through fraction obtained by loading
3 mL of 10-fold diluted plasma; FT D10X 10 CV, flow-through fraction
obtained by loading 5 mL of 10-fold diluted plasma (i.e., cut-off value
of loading volume); FT D10X Chased, flow-through fraction obtained by
chasing the loading of 10-fold diluted plasma with the corresponding
binding buffer; FT D20X 2 CV, flow-through fraction obtained by loading
1 mL of 20-fold diluted plasma; FT D20X 6 CV, flow-through fraction
obtained by loading 3 mL of 20-fold diluted plasma; FT D20X 10 CV,
flow-through fraction obtained by loading 5 mL of 20-fold diluted plasma
(i.e., cut-off value of loading volume); FT D20X Chased, flow-through
fraction obtained by chasing the loading of 20-fold diluted plasma with
the corresponding binding buffer.}

With the aim of increasing both \emph{Y\textsubscript{pIgG}} and pIgG
enrichment factor in the effluent, we modified the LigaGuard resin by
improving and expanding its multi-modal binding character: specifically,
the anion exchange component was strengthened by quaternizing the amine
groups displayed on the cationic residues (\emph{i.e.} , lysine,
arginine, and histidine); further binding modalities were introduced by
integrating polar and thiophilic moieties. We anticipated that the
combination of amine quaternization and pH 6.0 would increase the
capture of non-Ig plasma proteins, which are for the most part anionic,
while further reducing the capture of pIgG.

To evaluate this second-generation LigaGuard adsorbent, we repeated the
flow-through studies with pure IgG and Ig-depleted plasma, while
maintaining 20 mM Bis-Tris HCl buffer at pH 6.0 as the mobile phase and
1 minute residence time. The resultant profiles
of\emph{Y\textsubscript{pIgG}} and \emph{Q\textsubscript{PP} vs.}
loading volume are collated in \textbf{Figure 9} , while the
corresponding values of
\emph{C\textsubscript{pIgG}/C\textsubscript{pIgG}*}
and\emph{Q\textsubscript{pIgG}} ,
and\emph{C\textsubscript{PP}/C\textsubscript{PP}*}
and\emph{Q\textsubscript{PP}} are reported in \textbf{Figures S5A}
and\textbf{S5B} , respectively.

\textbf{Hosted file}

\verb`image9.emf`  available at \url{https://authorea.com/users/425045/articles/529963-purification-of-polyclonal-immunoglobulin-g-from-human-serum-using-ligaguardtm-and-ligatraptm-adsorbents}

\emph{\textbf{Figure 9.} Profiles of \textbf{(A)} pIgG flow-through
yield (Y\textsubscript{pIgG}) and \textbf{(B)} non-Ig plasma protein
capture (Q\textsubscript{PP}) vs. loading volume obtained by
respectively injecting a solution of pIgG at 3.0 mg/mL and Ig-depleted
plasma at 5.6 mg/mL in 20 mM Bis-Tris HCl buffer at pH 6.0 on
second-generation LigaGuard\textsuperscript{TM} resin; \textbf{(C)} pIgG
flow-through yield (Y\textsubscript{pIgG}) and \textbf{(D)} purity
(P\textsubscript{pIgG}) vs. loading volume, and
\textbf{(E)}Q\textsubscript{PP} vs. loading volume obtained by injecting
cryo-rich plasma diluted using 20 mM Bis-Tris HCl buffer at pH 6.0 to a
total protein titer of either 15 mg/mL (5-fold dilution, D5X), 7.0 mg/mL
(10-fold dilution, D10X), or 3.9 mg/mL (20-fold dilution, D20X) on
second-generation LigaGuard\textsuperscript{TM} resin, \textbf{(F)} pIgG
flow-through yield (Y\textsubscript{pIgG}) and \textbf{(G)} purity
(P\textsubscript{pIgG}) vs. loading volume by injecting cryo-poor plasma
diluted using 20 mM Bis-Tris HCl buffer at pH 6.0 to a total protein
titer of 6.4 mg/mL (10-fold dilution, D10X).}

The second-generation LigaGuard adsorbent provided a remarkable
improvement in both product yield and capture of non-Ig plasma proteins.
Notably, the \emph{Q\textsubscript{PP}} at the cut-off value of loading
(10 CVs) grew from 24.1 mg per mL of the original LigaGuard resin to
37.6 mg/mL of second-generation LigaGuard resin; conversely,
the\emph{Q\textsubscript{pIgG}} at saturation remained unaltered at 3.8
mg/mL, translating in a \emph{Y\textsubscript{pIgG}} of 87.4\% at the
cut-off loading.

Following on these results, we proceeded with the flow-through
purification of pIgG from cryo-rich plasma using the second-generation
LigaGuard adsorbent. The plasma was diluted by either 5-, 10-, or
20-fold with 20 mM Bis-Tris HCl buffer at pH 6.0 and loaded on the
LigaGuard\textsuperscript{TM} adsorbent at the residence time of 1 min.
The resulting profiles of \emph{Y\textsubscript{pIgG}}
and\emph{Q\textsubscript{PP}} \emph{vs.} loading volume are reported
in\textbf{Figure 9C} and \textbf{Figure 9E} ; the corresponding profiles
of\emph{C\textsubscript{pIgG}/C\textsubscript{pIgG}*} are reported
in\textbf{Figures S6} . The loading of 20-fold diluted plasma, where the
titer of pIgG (0.6 mg/mL) and non-Ig plasma proteins (3.9 mg/mL) are
relatively low, resulted in efficient flow-through purification: the
adsorbent became progressively saturated with plasma proteins throughout
feedstock loading, while allowing pIgG to flow-through unbound.
Accordingly, by the cut-off loading volume (10 CVs), the cumulative pIgG
purity in the effluent reached 98.1\%, as shown by the electrophoretic
analysis in \textbf{Figure 10A} . The global yield, on the other hand,
reached 25.4\% only. This was imputed to the protein concentration in
the feedstock being inadequate to match the higher binding capacity of
the second-generation LigaGuard adsorbent: specifically, the titer of
non-Ig plasma proteins was insufficient to prevent undesired capture of
IgG or displace the bound pIgG molecules via the weak partitioning
mechanism described in prior work {[}\emph{\textsuperscript{26, 27}}
{]}. We therefore adjusted loading conditions by reducing the dilution
of the feedstock to 10-fold and 5-fold, which increased the titer of
pIgG to \textasciitilde{} 1.0 and 2.0 mg/mL, and non-Ig plasma proteins
to \textasciitilde{} 7.2 and 14.5 mg/mL, respectively.

The loading of 10-fold diluted plasma resulted in a significant increase
in pIgG yield with high purity; specifically, at the loading volume of
4.0 mL (8 CVs), \emph{Y\textsubscript{pIgG}}
and\emph{Q\textsubscript{PP}} respectively reached \textasciitilde{}71\%
and 27 mg per mL resin, corresponding to a cumulative product purity of
\textasciitilde{}80\%; beyond this point, however, a significant amount
of non-Ig proteins flow through the column, lowering product purity to
62\% at the loading volume of 5.5 mL. This is mirrored by the
electrophoretic analysis of the effluent in \textbf{Figure 10B} , which
shows that product purity is very high in the initial flow-through
fractions (\textgreater{} 85\% up to 5 CVs), decreases slightly to 81\%
during the injection of the CVs 6 and 7, during which albumin bands
appear, and finally drops to 72\% (CVs 8 and 9), when more plasma
proteins break through. Finally, the loading of 5-fold diluted plasma
showed a significant loss of product yield and purity. Upon loading 5
CVs, in fact, non-Ig plasma proteins began breaking through
(\emph{C\textsubscript{PP}/C\textsubscript{PP}*} \textasciitilde{} 5\%)
while the \emph{Y\textsubscript{pIgG}} was still relatively low
(\textasciitilde{} 61\%); yield increased appreciably through the
subsequent loading phase, although product purity dropped significantly,
reaching 76\% at 10 CVs. The rather early breakthrough of non-Ig plasma
proteins, visible in \textbf{Figure 9E} , indicate the rapid saturation
of the binding sites due to the high protein concentration.
Collectively, these results indicate that the 10-fold dilution ratio is
ideal for stripping non-Ig plasma proteins using the LigaGuard
adsorbent prior to, or in lieu of, affinity-based capture to improve the
performance of subsequent chromatographic steps and maximize product
yield and purity.

The optimized loading conditions (10-fold dilution; 20 mM Bis-Tris HCl
buffer, pH 6.0; RT: 1 min) were implemented to purify pIgG from
cryo-poor plasma. The resulting profiles of product yield and
purity\emph{vs.} loading volume reported in \textbf{Figure 9F}
and\textbf{Figure 9G} (the profiles
of\emph{C\textsubscript{pIgG}/C\textsubscript{pIgG}*} are
in\textbf{Figures S6} ) indicate a purification performance similar to
that obtained with 20-fold and 10-fold diluted cryo-rich plasma. At the
loading volume of 8 CVs, for example, \emph{Y\textsubscript{pIgG}}
\textasciitilde{} 50\% and \emph{P\textsubscript{pIgG}} \textgreater{}
93\% (\emph{Q\textsubscript{PP}} \textgreater{} 25.4 mg per mL of resin)
are obtained. Interestingly, the \emph{Y\textsubscript{pIgG}} profile
features two slopes, namely \textasciitilde{}7\%/CV and
\textasciitilde{}14\%/CV, respectively before and after the loading
volume of 7 CVs; this value also demarcates between the collection of
high-purity effluent and the breakthrough of non-Ig plasma proteins,
which lower the cumulative \emph{P\textsubscript{pIgG}} from 99\% to
82\% at 10 CVs (\textbf{Figure 10D} ). The profile
of\emph{Q\textsubscript{PP}} , in fact, shows an inflection above 10
CVs, indicating the saturation of the LigaGuard adsorbent at 30 mg/mL,
coherently with prior measurements.

\textbf{Hosted file}

\verb`image10.emf`  available at \url{https://authorea.com/users/425045/articles/529963-purification-of-polyclonal-immunoglobulin-g-from-human-serum-using-ligaguardtm-and-ligatraptm-adsorbents}

\emph{\textbf{Figure 10.} SDS-PAGE analysis (native conditions,
Coomassie staining) of the chromatographic fractions obtained by
purifying pIgG from cryo-rich plasma diluted using 20 mM Bis-Tris HCl
buffer at pH 6.0 to a total protein titer of \textbf{(A)} 15 mg/mL
(5-fold dilution, D5X), \textbf{(B)} 7.0 mg/mL (10-fold dilution,
D10X),\textbf{(C)} 3.9 mg/mL (20-fold dilution, D20X), or
\textbf{(D)}cryo-poor plasma diluted using 20 mM Bis-Tris HCl buffer at
pH 6.0 to a total protein titer of 6.04 mg/mL (10-fold dilution, D10X).
Labels: MW, molecular weight latter; hIgG, human polyclonal IgG
standard; F, diluted plasma feedstock; and FTi, i-th flow-through
fraction.}

\textbf{3.3. Purification of pIgG from cryo-rich plasma using a two-step
process: Guard-Capture \emph{vs.} Capture-Polish}

In prior work, we have demonstrated the use of
LigaGuard\textsuperscript{TM} resin as a scrubber of process-related
impurities prior to the Protein A-based affinity capture step in a
process of antibody purification from CHO cell culture fluids
{[}\emph{\textsuperscript{26, 27}} {]}. In analogy to that study, we
attempted the purification of pIgG from plasma using a two-column
process comprising a LigaGuard adsorbent that captures the non-Ig
plasma proteins in flow-through followed by a
LigaTrap\textsuperscript{TM} adsorbent that operates in bind-and-elute
mode to isolate and concentrate pIgG. The loading of diluted cryo-rich
plasma was ``chased'' with 20 CVs of buffer C, and the pooled effluent
(pIgG titer \textasciitilde{} 0.2 mg/mL) was adjusted to pH 7.4 and
loaded onto LigaTrap\textsuperscript{TM} adsorbent. The
``guard-capture'' process conditions detailing loading, buffer
composition, and residence time, along with the resulting values
of\emph{Y\textsubscript{pIgG}} and \emph{P\textsubscript{pIgG}} in the
fractions across the two-column process are listed in \textbf{Figure
11A} , while the electrophoretic analysis is shown in \textbf{Figure
11B} . Notably, the depletion of non-Ig plasma protein by the LigaGuard
adsorbent enabled a remarkable increase in the purity of pIgG eluted
from the LigaTrap\textsuperscript{TM} adsorbent
(\emph{P\textsubscript{pIgG}} \textasciitilde{} 99.9\%). Most
importantly, the capture of albumin, the major contaminant in the
affinity capture step, greatly facilitated the clearance of the residual
impurities by LigaTrap\textsuperscript{TM} adsorbent, thus improving its
effective binding capacity and selectivity. This process design,
however, suffers from limited throughput. The high concentration of
non-Ig plasma protein in the feed (6.0 mg/mL) and the binding capacity
of the LigaGuard adsorbent (32 mg per mL of resin), in fact, pose a
limit to the volume of feedstock that can be fed into the two-column
process.

\textbf{Hosted file}

\verb`image11.emf`  available at \url{https://authorea.com/users/425045/articles/529963-purification-of-polyclonal-immunoglobulin-g-from-human-serum-using-ligaguardtm-and-ligatraptm-adsorbents}

Figure 11.\emph{\textbf{(A)} Two-column process comprising the
second-generation LigaGuard adsorbent operated in flow-through mode
followed by a LigaTrap\textsuperscript{TM}adsorbent operated in
bind-and-elute mode, and corresponding process (i.e., loading,
composition of the buffers, and residence time) and performance
(Y\textsubscript{pIgG} and P\textsubscript{pIgG}) parameters;
\textbf{(B)} SDS-PAGE analysis (reducing conditions, Coomassie staining)
of the chromatographic fractions obtained by purifying pIgG from
cryo-rich plasma diluted 10-fold using 20 mM Bis-Tris HCl buffer at pH
6.0 via the two-column process; labels: MW, molecular weight latter;
hIgG, human polyclonal IgG standard; F, diluted plasma feedstock; FT-LG,
flow-through fraction from the LigaGuard adsorbent; FT-LT, flow-through
fraction from the LigaTrap adsorbent; and E-LT, elution fraction from
the LigaTrap adsorbent.}

The global yield of the two-column process is evidently limited by the
first step (\emph{Y\textsubscript{pIgG}} \textasciitilde{} 49.9\%). In
response to this challenge, we lowered the pH of feedstock from 6.0 to
5.5 to enhance the electrostatic repulsion between IgG and the
second-generation LigaGuard adsorbent. All other process parameters
were kept unaltered; the values of \emph{Y\textsubscript{pIgG}}
and\emph{P\textsubscript{pIgG}} resulting from the analysis of the
process fractions are shown in \textbf{Figure S7} . This process
adjustment raised the global yield to 63.8\%, without altering final
product purity (99.7\%). For reference, the values of global pIgG yield
obtained by processing Ig-rich Cohn fractions via ion exchange
chromatography range between 40 - 70\% {[}\emph{\textsuperscript{16, 19,
57, 58}} {]}.

In the attempt to further pIgG recovery and productivity, we proposed an
alternative two-column process comprising an affinity-based capture step
using LigaTrap\textsuperscript{TM} adsorbent in bind-and-elute mode
followed by a polishing step using the LigaGuard adsorbent operating in
flow-through mode. The ``capture-polish'' process diagram, conditions,
the resulting values of \emph{Y\textsubscript{pIgG}}
and\emph{P\textsubscript{pIgG}} , and electrophoretic analysis of the
process fractions are reported in \textbf{Figure 12} .

\textbf{Hosted file}

\verb`image12.emf`  available at \url{https://authorea.com/users/425045/articles/529963-purification-of-polyclonal-immunoglobulin-g-from-human-serum-using-ligaguardtm-and-ligatraptm-adsorbents}

\emph{\textbf{Figure 12.} \textbf{(A)} Two-column process comprising the
second-generation LigaTrap adsorbent operated in bind-and-elute mode
followed by a LigaGuard\textsuperscript{TM} adsorbent operated in
flow-through mode, and corresponding process (i.e., loading, composition
of the buffers, and residence time) and performance
(Y\textsubscript{pIgG} and P\textsubscript{pIgG})
parameters;\textbf{(B)} SDS-PAGE analysis (reducing conditions,
Coomassie staining) of the chromatographic fractions obtained by
purifying pIgG from cryo-rich plasma diluted 10-fold using 20 mM
Bis-Tris HCl buffer at pH 6.0 via the two-column process; labels: MW,
molecular weight latter; hIgG, human polyclonal IgG standard; HSA, human
6serum albumin; F, diluted plasma feedstock; FT+W-LT, flow-through and
wash fraction from the LigaTrap adsorbent; FT-E, elution fraction from
the LigaTrap adsorbent; FT-CIP, clean in place fraction from the
LigaTrap adsorbent; and FT n-LG (n= 1,2 and 3), flow-through fraction n
from the LigaGuard adsorbent.}

Notably, preponing product capture improved substantially the global
recovery (\emph{Y\textsubscript{pIgG}} \textasciitilde{} 82.3\%), while
polishing still secured a high final product purity
(\emph{P\textsubscript{pIgG}} \textasciitilde{} 98.8\%). Furthermore,
the binding capacity of the LigaTrap adsorbent (10 mg per mL of resin)
enabled a 1.7-fold increase in the volume of plasma processed by the
``capture-polish'' compared to that enabled by the ``guard-capture''
using identical column volumes. On the other hand, the
``capture-polish'' imposes an intermediate step of buffer adjustment of
the elution stream from the LigaTrap adsorbent prior to loading into
the LigaGuard adsorbent, which lengthens the process and makes it less
streamlined; furthermore, conducting the polishing in flow-through mode
reduces the product concentration, thereby imposing a subsequent
ultrafiltration step.

The performance of the two alternative process designs is summarized
in\textbf{Table 3} , which reports, together with product yield and
purity, the corresponding values of enrichment of pIgG and the clearance
of non-Ig plasma proteins in the product stream. The remarkable levels
of purification achieved with the first process configuration
demonstrate the potential of the proposed technology for the
purification of plasma-derived therapeutics.

\emph{\textbf{Table 3.} Values of Y\textsubscript{pIgG},
P\textsubscript{pIgG,} reduction of non-Ig plasma proteins, and pIgG
enrichment factor in the effluent compared to feedstock obtained by
loading 1 mL cryo-rich plasma onto LigaGuard\textsuperscript{TM} and
LigaTrap\textsuperscript{TM} adsorbents at different resin sequence and
loading pH.}\selectlanguage{english}
\begin{longtable}[]{@{}lllllllll@{}}
\toprule
\begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{Two-column process}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
Y\textsubscript{pIgG}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
Y\textsubscript{pIgG}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
P\textsubscript{pIgG}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
P\textsubscript{pIgG}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{Reduction of} \textbf{non-Ig} \textbf{plasma proteins}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{Reduction of} \textbf{non-Ig} \textbf{plasma proteins}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{pIgG enrichment} \textbf{factor}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{pIgG enrichment} \textbf{factor}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
First column\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Global\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
First column\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Global\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
First column\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Global\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
First column\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Global\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
LG+LT (pH 5.5)\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
66.6\%\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
63.8\%\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
97.2\%\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
99.7\%\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
22\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
241\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
76\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
870\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
LG+LT (pH 6.0)\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
\textasciitilde{}50\%\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
\textasciitilde{}50\%\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
99.8\%\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
\textgreater{}99.9\%\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
37\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
366\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
134\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
1362\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
LT+LG (pH 6.0)\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
96.5\%\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
82.3\%\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
92.4\%\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
98.8\%\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
15\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
59\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
56\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
230\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{4. Conclusions}

The biomanufacturing of IVIG via isolation of pIgG from pooled plasma
has been performed for over half a century via precipitation-based
fractionation with minimal alterations since its initial design by Edwin
Cohn and Lawrence Oncley in the mid 40s. While greatly indebted to the
Cohn-Oncley process, the community of scientists and engineers operating
in the space of blood-derived proteins have defined the need to turn
towards more modern forms of biomanufacturing. In this context, protein
chromatography using affinity adsorbents functionalized with synthetic
ligands has gained momentum owing to a series of recent studies
demonstrating its potential for IVIG isolation from crude plasma. Our
group has invested significantly in this field by developing peptide-
and peptoid-based ligands for pIgG purification from animal -- human and
other mammals -- sources.

The two-step chromatographic process for pIgG purification from plasma
proposed in this study demonstrates significant advantages compared to
Cohn-derived processes in terms of process design, throughput, and
product yield and quality. Current IVIG manufacturing, which relies on
plasma fractionation via cold ethanol precipitation followed by
ion-exchange purification, is in fact a disconnected process that
suffers from the large footprint and extended times of precipitation
processes as well as the low recovery and product dilution typical of
ion-exchange chromatography. Conversely, the proposed two-step process
is fully integrated, and provides product yield and purity comparable -
or superior - to the current standard; furthermore, by operating 50\% in
flow-through mode, it provides a route towards process intensification
by enabling smaller footprint and continuous operation under reduced
residence time. Finally, when arranged in the ``capture-polish''
sequence it enables high productivity, while if arranged in the
``guard-capture'' sequence it provides for high product concentration.

The design conditions and the results presented in this study are by no
means definitive. Further adjusting the design of both adsorbents and
process is much needed. Specifically, we envision improving the binding
capacity and selectivity of the LigaGuard adsorbent by further
adjusting ligand composition and surface density as well as pore and
particle size; advancing the process by exploring in more detail the
effects of composition, concentration, and pH of the buffer as well as
loading rates and residence times; and introducing intermediate steps -
\emph{e.g.} , a tangential flow filtration - for continuous adjustment
of buffer composition and protein concentration between the LigaGuard
and LigaTrap adsorbents. Nonetheless, the technology proposed in this
study is the core of a new technology that shows tremendous potential
for a leap forward in the plasma fractionation technology for
large-scale IVIG manufacturing.

\textbf{Acknowledgements.} The authors wish to acknowledge CSL Behring
for funding this work; the authors specifically acknowledge Amy
Carruthers, David Boerema, Nathan Brinkman, and Laura Keigher at CSL
Behring for discussing our results and providing directions in
completing this work.

\textbf{Conflict of interest.} The authors Michael Crapanzano (CEO,
Ligatrap) and Jae Sly (CBO, LigaTrap) declare conflict of interest.

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