Downstream Processing
Chromatographic Purification
in Downstream Processing
New Sorbents and Membranes for Process Chromatography
I
n many biopharmaceutical companies, chromatographic
purification in downstream processing is a key focus for
optimization studies. The objective is to streamline the
process, which may be achieved by the elimination of
intermediate unit operations. The main question, therefore, is
how to optimize the adjustment of individual chromatographic
process steps.
Currently there is a broad range of next-generation sorbents
and membranes available for process chromatography. These
new chromatography media are characterized by significantly
improved performance compared to their classical
predecessors: e.g., higher dynamic binding capacities, higher
operational flow rates, and specific and distinctive retention
mechanisms. Nevertheless, traditional media (based on agarose
or polymers) continue to be routinely used for new method
development due to their proven suitability for protein
purification in FDA- or EMA-approved applications. In these
cases, the potential of modern ion exchangers and novel
mixed-mode or multimode sorbents still remains poorly
exploited. As a result, significant additional costs in production
may be incurred. Practical experience shows that the
optimization of individual process steps (capture, purification,
and polishing) already offers important cost reductions. But it is
mainly through a combination of all sorbent- and membranebased unit operations deployed in downstream processing that
the door is opened for effective process cost savings.
Ion-Exchange Chromatography
Anion and cation exchangers have been among the most
important tools in protein purification. Ion exchange is well
known by regulatory authorities and is a user-friendly
technique. Recently, new sorbent manufacturing processes
have significantly improved their capacity and performance. An
example is Pall’s Q HyperCel™ and S HyperCel ion-exchange
sorbents, which were designed with differentiated selectivity
for the purification of proteins at laboratory, pilot, and full
manufacturing scales.
Q HyperCel and S HyperCel sorbents exhibit high dynamic
binding capacities at high flow rates (and low residence times
such as 1 or 2 minutes, as in Figure 1), and enable increased
throughput.
74 BioProcess International
Mixed-Mode Chromatography
New perspectives for biopurification are also provided by
sorbents that interact with the target protein by a multiple
retention mechanism. Mixed-mode MEP, HEA, and PPA
HyperCel sorbents’ binding mechanism is primarily a
combination of hydrophobic and/or pseudoaffinity interactions
with the target. Typically this is achieved without additional
modification of the feedstock. MEP HyperCel is uncharged
under typical loading conditions (pH ~7), whereas the majority
of proteins carry a net negative charge. At acidic pH, both MEP
ligand and bound proteins take on a net positive charge.
Elution is induced by electrostatic repulsion, by decreasing the
pH. HEA and PPA HyperCel sorbents carry different ligands
(aromatic and aliphatic) and operate under similar conditions,
but offer a different selectivity. Recently, process developers
have shown that the addition of 0.1–0.5 M arginine to MEP
HyperCel sorbent elution buffers allows for protein elution at
pH values around neutrality (Arakawa et al., 2009).
T wo-Step A ntibody P urification
Monoclonal antibody purification traditionally implies a protein
A capture step followed by two orthogonal steps. According to
the antibody, the sequence and type of steps after protein A
may vary. But generally, intermediate unit operations are
required such as diafiltration for buffer exchange or addition of
salt for hydrophobic interaction.
An alternative two-step purification process is based on the
use of MEP HyperCel sorbent for the primary capture and
S HyperCel sorbent for the second chromatographic step
(Ferreira et al., 2007). These two sorbents are complementary to
each other and assure both yield and purity of the target.
Requirements for intermediate unit operations are eliminated
for better process economics. Non-protein A purification
schemes, including MEP HyperCel at intermediate step
following Mab capture by cation exchange, have also been
recently reported (Conley et al., 2011).
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Figure 1: Dynamic binding capacities (DBC; 10% breakthrough) for Q HyperCel sorbent (a, left) and S HyperCel sorbent (b, right); upper graphs
document the influence of residence time on DBC. Samples tested were bovine serum albumin (BSA) in 50 mM Tris-HCl at pH 8.4 (a) and human IgG in
50 mM sodium acetate at pH 4.7 (b). For both, an LRC glass column (1.0 × 10 cm) was used. Plots below show DBC as a function of buffer conductivity
and pH, which differs significantly from other ion exchangers (particularly cation exchangers) that offer different selectivities.
S HyperCel TM
B
Q HyperCel TM
A
200
200
100
100
0
0
1
2
3
4
0
5
0
1
11
180
160
140
120
100
80
60
40
20
0
10
9
8
Conductivity
7
5
4
7.0
7.2
7.4
7.6
pH
7.8
8.0
8.2
3
4
8
8.4
Membrane Chromatography
Typically, the capture and intermediate chromatographic steps
are in positive mode (the target molecule is bound to the
sorbent). However, to ensure the remaining level of
contaminants (DNA, HCP, endotoxin, and virus) meet regulatory
approval, the final chromatographic step tends to be in negative
mode. In this mode, contaminants are bound, and the target is
in the flow through. Chromatography membrane adsorbers are
ideally suited for negative mode or polishing applications. A
significant advantage of membranes over classical sorbents is
their larger pore size, which allows enhanced accessibility for
macromolecules (e.g., DNA and viruses) and allows for faster
processing using much higher flow rates than packed-bed
resins. Membrane adsorbers are available in disposable capsule
formats that eliminate the need for column packing and
cleaning. The family of Mustang® XT membrane adsorbers with
scalable formats meets all requirements from laboratory-scale to
process-scale applications.
5
DBC (mg/mL)
180
160
140
120
100
80
60
40
20
0
7
6
5
6
3
DBC (mg/mL)
DBC (mg/mL)
Conductivity
DBC (mg/mL)
12
2
Residence Time (minutes)
Residence Time (minutes)
4
3
2
4.0
4.2
4.4
4.6
pH
4.8
5.0
5.2
5.4
Figure 2: Chromatography columns for use in laboratory, pilot, and
process scales — (a) PRC prepacked columns (1 and 5 mL); (b) LRC glass
columns (≤900 mL); (c) Resolute columns (≤300 L); and (d) Resolute
columns (≤1,500 L)
A
B
C
D
Process H ardware
A reproducible and robust manufacturing process under GMP
conditions requires high-performance columns (Figure 2) and
control skids to ensure the performance of the validated
process. Flexible and robust Resolute® columns and packing
stations in combination with PK systems for process-scale
chromatography and PKP systems for pilot-scale
chromatography offer the optimal hardware platform for all
types of sorbent or membrane-based operations. •
76 BioProcess International
Dr. Dirk Sievers is marketing manager at Pall GmbH Life Sciences,
Dreieich BioPharmaceuticals Central Europe; 49-6103-307-582, fax
49-6103-307-295; dirk.sievers@europe.pall.com. Dr. Sylvio Bengio is
marketing chromatography and scientific communications for Pall Life
Sciences, Cergy France; 33-1-3420-7823; sylvio.bengio@europe.pall.com;
www.pall.com/biopharm.
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