Computational and Structural Biotechnology Journal 14 (2016) 238–244

Contents lists available at ScienceDirectjournal homepage: www.elsevier.com/locate/csbj

Application of the Protein Maker as a platform puriﬁcation system fortherapeutic antibody research and development
Geneviève Hélie a, Marie Parat a, Frédéric Massé b, Cory J. Gerdts c, Thomas P. Loisel a, Allan Matte a,⁎abcProtein Puriﬁcation, Human Health Therapeutics, National Research Council Canada, 6100 Royalmount Ave. Montreal, QC H4P 2R2, Canada
Primary Assays, Human Health Therapeutics, National Research Council Canada, 6100 Royalmount Ave. Montreal, QC H4P 2R2, Canada
Protein BioSolutions Inc., Suite 280, 401 Professional Drive, Gaithersburg, MD, 20879, USAa r t i c l e

i n f oArticle history:
Received 29 March 2016
Received in revised form 8 June 2016
Accepted 13 June 2016
Available online 16 June 2016
Keywords:
Parallelized protein puriﬁcation
Antibody
Protein A
Protein G
Hybridoma
Process developmenta b s t r a c t
Within the research and development environment, higher throughput, parallelized protein puriﬁcation isrequired for numerous activities, from small scale puriﬁcation of monoclonal antibodies (mAbs) and antibody
fragments for in vitro and in vivo assays to process development and optimization for manufacturing. Here, we
describe speciﬁc applications and associated workﬂows of the Protein Maker liquid handling system utilized inboth of these contexts. To meet the requirements for various in vitro assays, for the identiﬁcation and validationof new therapeutic targets, small quantities of large numbers of puriﬁed antibodies or antibody fragments areoften required. Reducing host cell proteins (HCP) levels following capture with Protein A by evaluating various
wash buffers is an example of how parallelized protein puriﬁcation can be leveraged to improve a processdevelopment outcome. Stability testing under various conditions of in-process intermediates, as an example,
the mAb product from a clariﬁed harvest, requires parallelized protein puriﬁcation to generate concurrent samples for downstream assays. We have found that the Protein Maker can be successfully utilized for small-to-mid
scale platform puriﬁcation or for process development applications to generate the necessary puriﬁed protein
samples. The ability to purify and buffer exchange up to 24 samples in parallel offers a signiﬁcant reduction intime and cost per sample compared to serial puriﬁcation using a traditional FPLC system. By combining theProtein Maker puriﬁcation system with a TECAN Freedom EVO liquid handler for automated buffer exchange
we have created a new, integrated platform for a variety of protein puriﬁcation and process development
applications.
Crown Copyright © 2016 Published by Elsevier B.V. on behalf of Research Network of Computational and
Structural Biotechnology. This is an open access article under the CC BY license (http://creativecommons.org/
licenses/by/4.0/).

1. Introduction
Puriﬁcation of antibodies and antibody fragments are key activities
in the generation of critical reagents for various in vivo and in vitro
assays as part of biotherapeutic lead identiﬁcation and process development. Often, it is necessary to purify large numbers of antibodies withmilligram yield, relatively quickly and at minimal cost. Various strategies are available to achieve such puriﬁcation outcomes, and can involve
to various extents both automated and manual methods [1,2]. While
parallelized puriﬁcation methods yielding sub-milligram quantities ofpure proteins based on packed columns, 96-well plates containing
small quantities of chromatographic resins or ligands immobilized tothe surfaces of membranes have been developed, there are relatively

Abbreviations: CHO, Chinese Hamster Ovary; DPBS, Dulbecco's phosphate buffered saline;
HC, IgG heavy chain; HCP, host cell protein; IMAC, immobilized metal ion afﬁnity
chromatography; LC, IgG light chain; mAb, monoclonal antibody; OAA, one-armed antibody.
⁎ Corresponding author.
E-mail address: allan.matte@nrc-cnrc.gc.ca (A. Matte).

fewer options available for generating puriﬁed quantitates of proteinin the intermediate (5–100) milligram scale. A few examples ofcustomized solutions to this problem exist, involving integration ofexisting puriﬁcation platforms such as the ÄKTA Puriﬁer with a CETAC
autosampler [3], ÄKTA Pure [4] or liquid handling robotics [5] havebeen reported. Other solutions include the design and fabrication ofcustomize robotics platform, including the Protein Expression and Puriﬁcation Platform [6]. While some commercial instruments for puriﬁcation of small quantities of protein have been developed, such as the
QIAcube for puriﬁcation of His-tagged proteins [7], there are few examples of commercial instruments that can be utilized for platform puriﬁcation at milligram scale.
In the context of process development applications, various
commercially available scale-down protein puriﬁcation products havebeen developed, including Predictor plates (GE) and Robo-columns(GE and Atoll Bio). While very useful for early-stage screening of various
chromatographic conditions, the maximum size of the columns possible
in these platforms (600 μL bed volume) results in a considerable gap inthe scale between screening and further optimization of process

http://dx.doi.org/10.1016/j.csbj.2016.06.001
2001-0370/Crown Copyright © 2016 Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology. This is an open access article under the CC
BY license (http://creativecommons.org/licenses/by/4.0/).

G. Hélie et al. / Computational and Structural Biotechnology Journal 14 (2016) 238–244

conditions. Some examples of higher throughput, automated solutions
to puriﬁcation process development have been reported [8,9]. While automated, sequential puriﬁcation of samples is possible using achromatography system connected to an auto-sampler, this cannot beparallelized using a single instrument, thereby reducing the possible
number of samples processed.
A speciﬁc instrument which has been designed around accomplishing
the task of parallelized, medium scale puriﬁcation is the Protein
Maker system, originated by Emerald BioStructures [10] and subsequently developed and marketed by Protein BioSolutions. The
Protein Maker is an automated protein puriﬁcation platform
designed for puriﬁcation of feed volumes of various sizes, from~ 10 mL to 1 L (~ 1 mg to 100 mg) or more utilizing up to 24 chromatography columns, each with an independent ﬂow path. The main
components of the system are (i) the syringe pumps with the associated 9-port valve, mixing syringe and sample lines, which together
form the initial portion of the ﬂow path, (ii) the column gantry,
columns and associated tubing from the syringe pumps, which
form the subsequent portion of the ﬂow path and (iii) the deck,
which contains up to 19 positions for SBS format plates and a dedicated waste position.
While puriﬁcation of a variety of proteins from any number ofsources is in principle possible with the instrument, the focus herein
are examples of puriﬁcation of antibodies and their fragments
generated from mammalian expression systems. We have utilized theProtein Maker as a key component of a platform puriﬁcation systemthat integrates automated buffer exchange implemented on a TECAN
Freedom EVO liquid handler. This protein puriﬁcation platform canbe used for both parallelized, small-medium scale puriﬁcation of
antibodies and their fragments, as well in various process development
applications.
2. Materials and methods
2.1. Antibody production
Murine IgG samples were produced in hybridoma culture in IMDM
supplemented with 10% heat-inactivated FBS and mouse IL-6 by aprocedure previously described [11]. For some antibodies, cultures
were performed transiently in Chinese Hamster Ovary (CHO) cells as
previously described [12]. Productions were harvested by centrifugation or ﬁltration (0.22 μm or 0.45 μm) and IgG containing supernatants
stored at 4 °C until puriﬁed.
2.2. Puriﬁcation of mAbs and Fabs
For development of Protein Maker puriﬁcation methods, protein
samples were puriﬁed using 1 mL HiTrap columns (GE Life Sciences),
including Protein G HP, MabSelect SuRe™ (Protein A) and Ni Sepharose
Excel™ mounted on an ÄKTA Puriﬁer 10/100 system. Chromatographic
proﬁles were monitored at 280 nm. Columns were equilibrated in
Dulbecco's Phosphate Buffered Saline Solution (DPBS, HyClone
Laboratories), the sample applied at the appropriate residence time
(1 min for Ni Sepharose Excel or 3 min for MabSelect SuRe or Protein
G HP) and a portion of the ﬂow-through fraction collected for subsequent non-reducing SDS-PAGE analysis. Columns were washed withDPBS and bound proteins eluted with two column volumes (CV) of
sodium-citrate buffer pH 3.6 (MabSelect SuRe), two CV of 100 mM
glycine-HCl pH 2.6 (Protein G HP) or one CV of DPBS with 500 mM
imidazole pH 8 (IMAC puriﬁcation). For proteins eluted from Protein A
or Protein G columns, samples were pH adjusted using 1 M solutionsof sodium HEPES or Tris–HCl buffer to a ﬁnal pH of 6–7.
Platform, parallelized puriﬁcation experiments were performed
using the Protein Maker running the Protein Maker v2.0 software(Protein BioSolutions). Puriﬁcation runs were performed using the
1 mL HiTrap columns and the chromatography conditions (residence

239

time, column washing and sample elution) established using the ÄKTApuriﬁcation system. Sample and buffer lines were cleaned in placewith 0.5 M NaOH and equilibrated in DPBS or appropriate buffer
solutions. During puriﬁcation, a portion of the ﬂow-through fraction
was collected for subsequent non-reducing SDS-PAGE analysis. Protein
samples were eluted in three steps, consisting of a pre-elution volume,
elution volume and post-elution volume. Protein concentration measurements (A280 nm) on these fractions were used to establish theﬁnal pooled sample.
2.3. Process development for mouse IgG2a puriﬁcation
The Protein Maker system was used to purify in parallel ﬁve murine
IgG2a samples from mouse Hybridomas using MabSelect SuRe and Protein G HP 1 mL HiTrap columns. For each mouse IgG2a, 20–22 mL of supernatant (~ 0.5 to 2 mg of mouse IgG2a, depending on titer) was
puriﬁed on either column using the puriﬁcation method describedabove. For protein G puriﬁcations, elution was performed in two steps,
ﬁrst with 100 mM citrate buffer pH 3.6 and then with 100 mM
glycine-HCl buffer pH 2.6. Elution fractions were neutralized using
1 M Tris. The quantity of IgG2a contained in elution fractions were determined based on A280 nm. For protein G puriﬁcation, the quantity of
IgG2a obtained from the two elution steps was summed for calculating
the yield.
2.4. Development of a post-load wash step to improve HCP removal during
protein A puriﬁcation
Data were obtained with three different antibodies expressed in
CHO cells. The Protein Maker system was used to perform parallel
puriﬁcations using MabSelect SuRe 1 mL HiTrap columns. For each antibody, eight wash conditions were tested. For each tested condition,
10 mL of supernatant (16.3 to 17.5 mg of antibodies) were loaded at aresidence time of 3 min. HiTrap columns were then washed and antibodies were eluted using 2.5 CV of 100 mM citrate buffer pH 3.0. Elution
fractions were neutralized using 1 M HEPES. The quantity of antibody inelution fractions was determined by A280 nm. The quantity of HCP in
elution fractions was measured using a CHO HCP ELISA kit (Cygnus
Technologies).
2.5. Buffer exchange and aseptic ﬁltration
Buffer exchange into DPBS following afﬁnity puriﬁcation was
performed manually either using Zeba-spin columns (Thermo-Fisher
Scientiﬁc) by centrifugation or using PD-10 desalting columns (GEHealthcare) by gravity according to the manufacturer's instructions.
Alternatively, sample buffer exchange using PD Miditrap G-25 columns
was automated on a TECAN Freedom EVO150® liquid handler according to gravity protocols from GE Healthcare. The Freedom EVO150®
was equipped with a liquid displacement Liquid Handler (LiHa)
conﬁgured with 8 channels (4 disposable tips and 4 washable tips), a
Robotic Manipulator (RoMa), a Tecan Vacuum (TeVac), carriers for 11
microplates, shelf for 4 microplates, one reservoir position for elution
buffer, and tip carriers for hanging tips. All tubing and components of
the liquid displacement system were cleaned in-place with 0.5 M
NaOH for at least 15 min and rinsed with sterile water prior to operations. A script was developed with the ﬂexibility to process from 24 to
96 samples at once. Before starting, the storage solution from PD
MidiTrap G25 columns was removed manually, the columns placed ina 24 position custom holder and the rack positioned on the Freedom
EVO150® worktable. Puriﬁed protein samples from the Protein Maker
were stored in 24 deep well microplate (Seahorse Bioscience). The
system liquid was replaced with DPBS (HyClone Laboratories) and thescript started. First, the RoMa arm brought the column racks onto the
TeVac and columns were equilibrated with three bed volumes using
the Freedom EVO150® system liquid (DPBS). The equilibration buffer

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was allowed to enter the packed bed completely and the ﬂow-throughdiscarded in the TeVac waste. Samples were pipetted onto the columns
using disposable ﬁlter tips (Tecan) and time was allowed for them toenter the packed bed by gravity. Column racks were tapped 4× times
on the Te-Vac by the RoMa arm to remove any droplets and transported
on top of a 24 deep well block for elution. The LiHa pipetted 1.5 mL ofelution buffer (DPBS) to each MidiTrap, respectively, using disposabletips and the eluate containing the protein of interest was collected bygravity. Subsequent aseptic ﬁltration was performed by centrifugation
using either sterile Multiscreen 0.22 μm 96-well plates (Millipore) or
deep well 0.22 μm 96-well plates (Corning) stacked with a sterile receiver plate.
2.6. Analytical methods
Proteins were quantitated based on A280 nm values obtained usinga NanoDrop 2000 spectrophotometer (Thermo Scientiﬁc) and concentration values were corrected based on calculated extinction coefﬁcients
derived from the protein sequence. Non-reducing SDS-PAGE analysis
was performed using 4–12% Bis-Tris NuPage gels (Novex, ThermoFisher scientiﬁc) and stained with Sypro Ruby protein gel stain(Thermo-Fisher Scientiﬁc) as recommended by the manufacturer.
SDS-PAGE gels were imaged using a ChemiDoc MP imaging system(Bio-Rad Laboratories).

observed comparable results in terms of product yield and sample
purity when using the same puriﬁcation method on an ÄKTA puriﬁcation platform and the Protein Maker (results not shown).
A key factor in establishing the capture step is to determine theresidence time to achieve optimal capture of the product on the columnof interest. In the case of Protein G afﬁnity chromatography, using a
residence time of 3 min, no IgG was found in the ﬂow-through ofhybridoma-generated mAb samples as determined by non-reducing
SDS-PAGE with Sypro Ruby staining. For larger volumes of feeds containing small quantities of product, it is necessary to split samples
over two or more columns in order to reduce the total time required
for the binding step. Using such an approach, ~200–250 mL of product(~ 30 mg of protein in the case of Protein A) can be passed over 1 mL
columns in an overnight run.
We have found that using the approach of collecting elution
fractions in three steps (pre-elution (0.5 CV), main elution (0.5–
1.5 CV) and post-elution (1.5+ CV) volumes) allows for optimizationof the quantity of puriﬁed product in a minimum volume, allowing for
maximum product concentration. At the elution step, the practical outcome is to obtain ≥ 80% of the puriﬁed protein in a minimum volume
suitable for manual or automated buffer exchange as the second stepin the workﬂow shown in Fig. 2.

3. Results
3.1. Development and implementation of protein maker platform
puriﬁcation methods
Our strategy for the development and implementation of afﬁnity
puriﬁcation methods is summarized in Fig. 1. Key factors that inﬂuence
the puriﬁcation process, including residence time for product captureand elution volume are determined using the ÄKTA puriﬁcation runs.
The essential value of these runs is to provide the absorbance trace
during the puriﬁcation in order to quickly converge to appropriate
starting conditions for puriﬁcation. The essential features of thedifferent afﬁnity puriﬁcation methods are similar for Protein G, Protein
A and IMAC based puriﬁcation method development. Overall, we

Fig. 1. Protein Maker puriﬁcation development strategy.

Fig. 2. Protein G puriﬁcation workﬂow for puriﬁcation of mAbs from hybridoma
supernatants.

G. Hélie et al. / Computational and Structural Biotechnology Journal 14 (2016) 238–244

241

3.2. Platform puriﬁcation applications
3.2.1. Small scale puriﬁcation of mAbs from hybridoma supernatants
In order to generate puriﬁed mAbs for cell-based assays, we havedeveloped a Protein G based puriﬁcation workﬂow using Protein G HP
as the capture resin (Fig. 2). Each puriﬁcation cycle permits 23 mAb
samples along with one murine IgG control sample, to be puriﬁedusing Protein G, buffer exchanged into DPBS and aseptically ﬁlteredprior to aliquoting in bar coded tubes for storage. The purpose of thecontrol sample is to act as a sentinel for the puriﬁcation run via determination of the recovery of puriﬁed protein. In principle, this sample will
help to troubleshoot problems in executing the puriﬁcation method,
as for example, a change in column binding capacity resulting from excessive numbers of clean in place (CIP) cycles.
In most cases, approximately 0.1–1 mg of puriﬁed mAb wereobtained per hybridoma supernatant, mainly with a concentration
range of approximately 0.75 to 0.95 mg/mL. Non-reducing SDS-PAGE
followed by staining with Sypro Ruby, a high-sensitivity protein stain,
revealed no IgG in the ﬂow-through fraction (Fig. 3B). The purity of
the resulting puriﬁed mAbs was also veriﬁed by non-reducing SDSPAGE (Fig. 3A).
In early experiments, samples were buffer exchanged manuallyusing Zeba-spin centrifugal columns. While this approach minimizes
sample dilution, it is manually intensive, and sub-optimal within ahigh throughput protein puriﬁcation workﬂow (Fig. 2). Using PD
MidiTrap G-25 columns in gravity mode, we implemented an automated approach to buffer exchange samples following afﬁnity puriﬁcation,
reducing the manual labor, time and cost associated with this step. A
consequence of this change was increased dilution of the puriﬁed sample by ~1.5-fold compared to the Zeba-spin buffer exchange.

Fig. 4. Non-reducing SDS-PAGE and Sypro Ruby staining of CHO-produced and IMAC
puriﬁed Fab samples using the Protein Maker. Lanes 1, 9, 17, molecular weight markers,
in kDa; lanes 2, 10, CHO supernatants of expressed Fabs; Lanes 3,4,11,12, ﬂow-throughfractions; lanes 5,6,13,14, wash fractions; lanes 7,8,15,16, elution fractions. The ~50 kDa
species in the F/T fraction represents light chain (LC) dimers.

G or Protein A. The purity of representative Fab samples as determinedby non-reducing SDS-PAGE is shown in Fig. 4.
3.2.2. Antibody fragments — puriﬁcation of Fabs
We applied the Protein Maker platform to the puriﬁcation of a seriesof his-tagged Fab constructs generated for in vitro studies. Other thanthe buffer composition for column washing and sample elution, theoverall strategy is similar to that applied to Protein G or Protein A
puriﬁcation methods. Using this approach, up to 20 mg of puriﬁed Fabfrom ~200 mL CHO culture has been obtained. Recoveries post-buffer
exchange are similar to that obtained for mAbs puriﬁed using Protein

Fig. 3. Non-reducing SDS-PAGE and Sypro Ruby staining of ten different hybridomaproduced antibodies puriﬁed using Protein G HP with the Protein Maker (A) puriﬁed
mAbs, (lanes 2–11)(B) ﬂow-through fractions (lanes 12–21), revealing no unboundmAb (arrow). The mAb species (2HC + 2LC) is indicated with an arrow, and minor
species include 2HC (~ 100 kDa), half-antibody (~ 75 kDa) and free LC (~ 25 kDa) are
visible for some samples. Molecular weight markers (lanes 1, 22) are shown in kDa.

3.3. Process development applications
3.3.1. Evaluation of protein A vs protein G resin for puriﬁcation of murine
IgG2a
Protein G resin is typically considered as the ﬁrst choice to purify
antibodies produced from mouse Hybridomas, especially when the
IgG subclass is unknown. Indeed, Protein G has strong afﬁnity for all
mouse IgG subclasses, whereas Protein A has strong afﬁnity for only
certain subclasses, speciﬁcally IgG2a and IgG2b. For puriﬁcation ofmouse IgG2a, the choice of one resin vs the other is not straightforward.
According to some manufacturers, mouse IgG2a has strong binding
afﬁnity for both Protein A and Protein G, however, protein A columns
have a higher binding capacity and could be a better choice especiallyfor larger scale puriﬁcation. To determine which of Protein A or Protein
G is the better choice to purify mouse IgG2a, we used the Protein Maker

Fig. 5. Quantities of mouse IgG2a (μg) puriﬁed from mouse hybridoma supernatants using
either Protein A or Protein G.

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to purify ﬁve mouse IgG2a samples in parallel on these columns. The
total quantity of mouse IgG2a obtained with either resin following
puriﬁcation is summarized in Fig. 5. For all tested mouse IgG2a, thequantity of puriﬁed protein obtained from Protein A was at least 50%
higher than those obtained from Protein G puriﬁcation.
3.3.2. Development of a post-load wash step to improve HCP removal
during protein A puriﬁcation
Removing impurities post-Protein A chromatography still
represents a signiﬁcant challenge to puriﬁcation process development
in order to achieve the required drug substance speciﬁcations suitablefor patient administration (HCP b 100 ppm). Using a post-load wash
step is a key means to achieve HCP clearance, since it has been demonstrated that HCP associates with antibodies and co-elute during theelution step. Basic pH and wash additives such as arginine seem toimprove HCP removal during the Protein A chromatography wash
step by disrupting interactions between the antibody and the HCPs
[13]. Here we evaluated both Tris and phosphate-based arginine wash
buffers at different pH values (7 to 9) in comparison to conventional
wash buffer (DPBS or citrate pH 5.0) using 1 mL HiTrap MabSelect
SuRe columns. These various wash conditions (Table 1) were tested inparallel with 3 different antibodies (CHO supernatants) using theProtein Maker. Yield and HCP levels obtained for each wash condition
are presented in Fig. 6. The use of basic wash buffers containing arginine
showed improved HCP removal (1.7 to 2.4-fold) compared to theconventional DPBS wash for the three antibodies tested, with detrimental effects on puriﬁcation yields only for one antibody (mAb3).
3.3.3. Stability hold of clariﬁed harvest for a mAb
In a series of experiments, a clariﬁed mAb harvest was held at either
2–8 °C or 19–23 °C and sampled at various time points corresponding to
t = 0, 1, 2, 3, 5 and 7 days before puriﬁcation using MabSelect SuRe
followed by buffer exchange to DPBS in order to evaluate changes in
the product using an analytical assay panel. The puriﬁcation performance at time points, t = 0 and t = 7 days, is shown in Table 2.
Puriﬁcation recoveries based on the measured product titer were atleast 85%. Analysis of charge variants and glycosylation proﬁles at eachtime point at either temperature revealed a signiﬁcant decrease in acidic
charge variants upon storage at 19–23 °C, corresponding to a drop in thesialic acid content of the mAb (results not shown). This assessment
made it possible to deﬁne the manufacturing hold time durationnecessary for process control during GMP manufacturing.
4. Discussion
During early-stage therapeutic antibody R&D projects, it is often
necessary to purify large numbers of samples that will be used asreagents for in vivo or in vitro screening assays. The nature of the
puriﬁcation strategy employed is determined by the quantity and ﬁnal
concentration of puriﬁed protein required; the number of samples
that need to be puriﬁed per unit time (throughput), the supernatantvolume and initial product titer, as well as the availability, if any, of
automated liquid handling instrumentation. While clearly there ismore than one possible solution to achieve the puriﬁcation objectives,

Fig. 6. Effect of wash buffers on (A) puriﬁcation yields and (B) HCP levels. Wash
conditions — A: PBS; B: 100 mM Citrate, 50 mM NaCl pH 5.0; C: 50 mM Tris, 100 mM
Arginine, 50 mM NaCl pH 7.0; D: 50 mM Tris, 100 mM Arginine, 50 mM NaCl pH 8.0; E:
50 mM Tris, 100 mM Arginine, 50 mM NaCl pH 9.0; F: 10 mM Phosphate, 100 mM
Arginine, 50 mM NaCl pH 7.0; G: 10 mM Phosphate, 100 mM Arginine, 50 mM NaCl
pH 8.0; H: 10 mM Phosphate, 100 mM Arginine, 50 mM NaCl pH 9.0. *: Yields below
80% — #: HCP reduction vs PBS greater than 1.7-fold.

we selected the Protein Maker as a platform using HiTrap columns,
including Protein G (Protein G HP), Protein A (MabSelect SuRe) andIMAC (Ni Sepharose Excel), and have applied these to feeds from either
hybridoma or CHO supernatants ranging from 10 mL to 200 mL volumes. A key outcome has been for puriﬁcation of small quantities(b1 mg) of puriﬁed mAbs using the Protein G method described here,with greater than 1000 mAbs puriﬁed and utilized as reagents forin vitro screening assays.
For the development of platform-based puriﬁcation methods on theProtein Maker, several parameters that inﬂuence the overall puriﬁcation outcome required evaluation. One of the most critical parametersis the residence time used in the binding step, as most of the overall
time required to execute the method involves binding of the productto the packed bed. Through several puriﬁcation campaigns utilizing
Protein G HP with murine IgG's, we have established that a threeminute residence time ensures capture of the product, as revealed by
non-reducing SDS-PAGE analysis. Another critical parameter is toestablish the elution volume range for the product. The elution volume
will depend on the column volume and the quantity of product boundto the packed bed, with larger elution volumes being required as one
approaches the dynamic binding capacity limit of the column. The optimal elution volume is a balance between the desired product concentration, yield and recovery of the puriﬁed product. One rule of thumb

Table 1
Wash conditions tested for Protein A post-load HCP removal.

1
2
3
4
5
6
7
8

Wash 1 (5 CV)

Wash 2 (5 CV)

Wash 3 (5 CV)

PBS
PBS
PBS
PBS
PBS
PBS
PBS
PBS

PBS
100 mM Citrate, 50 mM NaCl pH 5.0
50 mM Tris, 100 mM Arginine, 50 mM NaCl pH 7.0
50 mM Tris, 100 mM Arginine, 50 mM NaCl pH 8.0
50 mM Tris, 100 mM Arginine, 50 mM NaCl pH 9.0
10 mM Phosphate, 100 mM Arginine, 50 mM NaCl pH 7.0
10 mM Phosphate, 100 mM Arginine, 50 mM NaCl pH 8.0
10 mM Phosphate, 100 mM Arginine, 50 mM NaCl pH 9.0

PBS
PBS
PBS
PBS
PBS
PBS
PBS
PBS

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243

Table 2Chromatographic Performance Table — MabSelect SuRe puriﬁcation, clariﬁed harvest stability hold study for t = 0 and t = day 7. The global process puriﬁcation yield is calculated as theratio of the ﬁnal yield over the initial load on the column.
PD-10 buffer exchange andaeptic ﬁltration

HiTrap Mab Select SuRe

Load

Final

Storage temperature(°C)

Time point(Day)

Volume(mL)

Load
Quantity(mg)

Quantity(mg)

Elution
Step yield(%)

quantity(mg)

quantity(mg)

Step yield(%)

Global process
yield (%)

N/A
2–8 °C
19–23 °C

0
7
7

50
50
50

16.6
16.5
16.6

14.8
14.0
15.3

89
85
92

13.6
12.8
14.0

13.6
12.6
13.7

99.9
98.7
97.5

82.0
76.6
82.6

would be to establish the elution volume such that ≥80% of the puriﬁedprotein is captured in one fraction.
In many antibody afﬁnity puriﬁcation work-ﬂows, puriﬁcation isfollowed by buffer exchange into a formulation buffer, including PBS,
in order to minimize protein aggregation as a result of unfavorable
conditions of pH or ionic strength. While it is possible to perform thebuffer exchange step in high-throughput mode using 96-well plates[14], this only applies to samples having a small volume, typically lessthan 130 μL. Initially, we performed this step in a more manuallyintensive manner using Zeba-spin buffer exchange columns. In order
to increase the throughput and operational efﬁciency of the buffer exchange step, a more automated approach was developed using PD
MidiTrap G-25 columns on a TECAN Freedom EVO 150 liquid handlingsystem. The integration of an automated buffer exchange step in a 24sample format, the same format as the Protein Maker, increases theoverall sample throughput, decreases manual manipulations andenhances the consistency of results.
In addition to platform-based puriﬁcation of full-size IgG's, there isoften a requirement to purify IgG fragments, including Fabs and scFv's,
as well as to evaluate the effectiveness of different afﬁnity puriﬁcation
resins for puriﬁcation of the same product. For puriﬁcation of murine
IgG2a, the choice of Protein A vs Protein G puriﬁcation requires experimental veriﬁcation prior to establishing the ﬁnal process to be used. By
performing all of the puriﬁcation experiments in parallel using the Protein Maker, a few hours were sufﬁcient to determine that protein A isthe better choice for puriﬁcation of murine IgG2a samples. Indeed, thequantity of puriﬁed protein obtained from Protein A was at least 50%
higher than those obtained from Protein G puriﬁcation, although inthe absence of additional data we cannot offer a deﬁnitive explanation
for this result. Executing the same experiment using one ÄKTA puriﬁcation system would require approximately ﬁve-fold more time thanperforming the puriﬁcation experiments in parallel with the Protein
Maker. It is noteworthy that these 5 antibodies were subsequently
puriﬁed at larger (2 L) scale using protein A resin with greater than
80% recovery for four out of the ﬁve antibodies processed. In the

Table 3
Possible platform afﬁnity puriﬁcation modes for antibodies and antibody fragments using
the Protein Maker.

FormatIsotype

Species

Capture
mode

Resin

mAb, bispeciﬁc
mAb, bispeciﬁc
mAb, bispeciﬁc
mAb, bispeciﬁc
OAA
scFv
HybridHybridHybridFabFabFabIgG1, IgG2, IgG4
IgG2
IgG1, IgG2, IgG3, IgG4
IgG1, IgG2, IgG3
N/A

Human
MurineHuman
Murine
Human
Human
Human
Human
Human
Human
Human
Human

Fc
Fc
Fc
Fc
Fc
LC (VL)Fc
HC (CH1)
LC (κ, CL)
HC (CH1)
LC (κ, CL)
LC (λ, CL)

Protein A
Protein A
Protein G
Protein G
Protein A, G
Protein L
Protein A, G
CH1
Kappa-SelectCH1
Kappa-SelectLambda-Fab Selectpuriﬁcation scheme presented here, Fabs are puriﬁed via his-tags
using IMAC, although Fab puriﬁcation can be achieved in some instances using protein A and more commonly using the CH1 domain of
the Heavy Chain (HC) or the conserved domain of the Light Chain (LC)as capture modes. Other therapeutic antibody formats, including onearmed antibodies, hybrids, bi-speciﬁcs and various Fc-fusion molecules
can also be puriﬁed in platform mode with the appropriate afﬁnity
resin. Examples of readily available afﬁnity chromatography resins
and how they can be applied to various antibody puriﬁcation requirements is summarized in Table 3.
Both the post-load wash step for optimization of HCP removal fromProtein A as well as the clariﬁed harvest hold stability study offer
examples of how the Protein Maker can be effectively utilized in thecontext of protein puriﬁcation process development. Minimizing hostcell protein levels at the Protein A capture step through modiﬁcationof wash buffer composition can improve the puriﬁcation process [13,15,16]. One cycle of parallelized puriﬁcation using the Protein Maker,
requiring approximately 3 h, was sufﬁcient to purify 24 samples(eight different wash conditions for three different antibodies). Executing the same experiment using one ÄKTA puriﬁcation system (i.e. 24
sequential puriﬁcations on an ÄKTA Puriﬁer) would have required approximately 37.5 h. In this experiment, the Protein Maker not only increased puriﬁcation throughput by 12.5-fold but also permitted
puriﬁcation of samples in an unbiased manner by having the capacity
to purify all of the samples at the same time. Indeed, under conditions
where a mAb is unstable in the clariﬁed harvest, the ability to purify
multiple samples in parallel offers a distinct advantage over serial
puriﬁcation using conventional puriﬁcation equipment, minimizing
misinterpretation of data due to sample degradation. As the effectiveness of optimized wash buffers and their effect on puriﬁcation yields
seem to vary depending on the antibody, the same wash conditions
cannot be used for all samples. Rather, post-load wash conditions
should be optimized for each antibody as part of puriﬁcation process development. Due to its ability to process multiple samples in parallel, theProtein Maker represents a preferred instrument for performing wash
buffer screening. Moreover, as multiple wash conditions can be evaluated in parallel, it is possible to minimize variability in sample handlingthat could bias data interpretation.
Therapeutic antibody products often pose various challenges
during the development of upstream and downstream processes,
including degradation, modiﬁcation of Fc or Fd glycans, reduction of intramolecular disulﬁdes, deamidation of Asn and Gln residues, as well as
aggregation [17]. In order to de-risk the overall process, an importantcomponent of the transition between upstream and downstream processing is to understand the stability of the product post-clariﬁcation
but prior to the initial capture puriﬁcation step. The removal of sialic
acid from mAbs by the action of extracellular CHO sialidase is an
example of the kinds of post-production, pre-puriﬁcation modiﬁcations
that are possible [18]. The utility of the Protein Maker in this application
is its ability to purify several samples in parallel, for example, if the clariﬁed harvest is to be held at various temperatures or at different pHvalues, or if multiple harvests are to be tested in parallel and puriﬁed
under the same controlled conditions.

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G. Hélie et al. / Computational and Structural Biotechnology Journal 14 (2016) 238–244

5. Conclusions
We have found the Protein Maker to be a versatile tool for a numberof puriﬁcation problems, ranging from small scale mAb and Fab puriﬁcation to various applications in process development. We have developed platform puriﬁcations methods using Protein G, Protein A andIMAC and have applied these to several projects. Use of the Protein
Maker and TECAN Freedom EVO150 together has resulted in reducing
both the required personnel time and creation of an integrated
workﬂow that includes both protein puriﬁcation and automated buffer
exchange in a 24-sample format. Future implementation of the UV
monitoring capability will add an additional dimension to the capabilities of this instrument, permitting 24-channel monitoring of the protein
absorbance signal, thereby expediting utilization of the instrument forvarious process development applications.
Acknowledgements
We wish to thank our colleagues within the Protein Puriﬁcation
Team who have provided valuable feedback on the various methods
that have been developed. We also wish to thank our colleagues invarious projects who have provided us with materials with which wehave developed the various methods described. In particular we thank
Maria Jaramillo and Anne Marcil for their encouragement. This research
was supported by the National Research Council Canada.
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