Chetanya Jangra 2K20/EC/73

Current research breakthroughs in DSP of therapeutic

proteins
Overview:

Downstream processing techniques (DSP) currently account for most production costs of
pharmaceutically relevant proteins. Biopharmaceuticals and their production are constantly on
the rise as they are needed to treat and to prevent multiple diseases. Therefore, an urgent need
for process intensification in downstream processing (DSP) has been identified to produce
biopharmaceuticals more efficiently. We are going to discuss promising studies in the fields of
chromatography, aqueous two-phase systems, precipitation, crystallization, magnetic
separation, and filtration for the purification of pharmaceutically relevant proteins.

Introduction:

The growing demand for therapeutic proteins, particularly monoclonal antibodies (mAbs), poses
a challenge in efficiently producing large quantities of these products. While upstream
processing has been improved, downstream processing (DSP) now becomes the bottleneck in
production. DSP involves various steps such as clarification, capture, purification, and polishing
using chromatography and filtration techniques. DSP aims to enhance the purity and
concentration of the target molecules. Conventional chromatography, though effective, may
struggle to handle the increasing titers and volumes of upstream processing. To address this,
alternative technologies for process intensification are being researched, including continuous
integrated processes, single-use equipment, improved process control, and scale-down models
for efficient process development.

Fig: DSP strategies for process

intensification resulting aiming an
increased efficiency.

1
 Chetanya Jangra 2K20/EC/73

The goal of DSP is to improve the purity and increase the concentration of target molecules.
New and alternative technologies that allow for process intensification are increasingly being
investigated and a summary of innovative DSP strategies is highlighted in the following figure.

Fig: Overview of DSP intensification strategies

1.Precipitation

We will discuss the potential of precipitation as a technique for downstream processing (DSP) of
therapeutic proteins.
● The advantages of precipitation include fast and robust processing, scalability, high
yields, and low costs. Continuous processes using tubular reactor designs can be used
for process intensification.
● Several studies have shown that precipitation can be a valuable alternative to the current
protein-A chromatography for mAb capturing.
● Efficient precipitation processes using ZnCl2 and polyethylene glycol (PEG) have been
demonstrated. Dutra et al. developed a precipitation process based on ZnCl2 without
PEG, which reduces the viscosity of the processed fluid for improved harvesting and
washing of precipitates.

2
 Chetanya Jangra 2K20/EC/73

● Affinity precipitation using, for example, stimulus-responsive elastin-like polymers or

Ca2+-dependent calsequestrin(Calsequestrin is a calcium-binding protein that acts as a
calcium buffer within the sarcoplasmic reticulum) fused to affinity peptides, was recently
successfully employed for selective capture and purification of mAbs and other
therapeutic proteins
● Current research in process analytical technology (PAT) aims to speed up precipitation-
process development and ensure product quality, aligning with the "Quality by Design"
(QbD) concept introduced by the FDA.

2.Crystallization

● Protein crystallization, such as antibody crystallization, is a thermal process that

depends on the supersaturation of the protein. The crystallization of proteins is like the
crystallization of small molecules but needs mild condition changes such as salts or
polymers, as well as slow pH, ionic strength, and/or temperature changes (cooling
crystallization).
● However, a major obstacle to the crystallization process is the difficulty of its
implementation. The large size and complex configuration of proteins, especially of
mAbs with their flexible hinge region, hinder a simple crystallization process.
● Grob et al. recently demonstrated the enhancement of crystallizability of an alcohol
dehydrogenase by rational crystal-contact engineering, which could open a broader use
of crystallization as a purification technique.
● Nanoparticle-induced precipitation and crystallization are an upcoming trend for
pharmaceutical protein purification. Especially, iron oxide nanoparticles play a great role
in the precipitation and crystallization of proteins such as lysozyme or trypsine and
nanobodies.
● Protein crystals possess ordered protein configurations that generally have higher
physicochemical stability and purity than amorphous precipitates. Therefore,
crystallization can be used in intermediate and polishing steps in DSP. Another
advantage is the possible timely controlled release of therapeutic proteins from the
crystal lattice when used as drug formulation. These advancements in crystallization
techniques could have significant implications for the development of efficient and
effective DSP methods in the biopharmaceutical industry.

3.Extraction (aqueous two-phase system)

3
 Chetanya Jangra 2K20/EC/73

● Aqueous two-phase systems (ATPS) are extensively studied for protein extraction due to
their favorable aqueous nature. A spontaneous formation of an ATPS can be observed,
when two hydrophilic phase-forming components remixed above a critical concentration.
● Several advantages for biopharmaceutical DSP applications include high
biocompatibility, high recovery yields and selectivity, low cost, fast equilibrium
adjustment, and scalability. However, the widespread use of ATPS in DSP is hindered
by several factors. Firstly, the underlying mechanisms of ATPS on biomolecule partition
are not fully understood, and there is a lack of large-scale studies and continuous
application process designs.
● Nonetheless, researchers are working towards filling these knowledge gaps through
investigating partition coefficient predictions.
● Different reactor systems, such as the use of coiled flow inverters for extraction
processes, are tested.
● Understanding and mathematical modeling are supported by the development of
(continuous) microfluidic screening.
● New phase-forming components, such as ionic liquids and the improvement of the
process strategy (e.g., via multistage extraction or phase/component recycling), are
currently being studied.
● Moreover, with reactive ATPS and magnetic-assisted ATPS, there are further
approaches to process intensification.
● It is crucial to gain a systematic understanding of ATPS through small-scale screening
for future process development and scale-up.

4.Adsorption (magnetic separation)

● The use of batch-adsorption systems based on magnetic separation in downstream

processing (DSP) and antibody purification is a current trend in the industry. These
systems utilize ferromagnetic, ferrimagnetic, or superparamagnetic carriers with specific
binding sites, such as protein-A or protein-G domains.
● The advantage of magnetic-based processes is the easy separation of the bound and
nonbound phases, making it suitable for protein-capture steps. Recent developments
include new adsorbents and binding strategies to magnetic particles, as well as
improvements in high-gradient magnetic separation processes.
● Protein-A-based magnetic beads can be used for purification processes and capture of
mAbs in analogy to protein-A chromatography.
● Brechmann et al. showed that magnetic beads can be used efficiently at very high cell
densities for antibody capture.

4
 Chetanya Jangra 2K20/EC/73

● These magnetic separation processes offer a sustainable alternative to protein-A

chromatography in DSP, as they provide energy-efficient separation without the need for
harvest filtration and centrifugation steps required in packed-bed systems. Current
research and advancements focus on optimizing separator and process design, as well
as integrating magnetic separation processes into the DSP workflow.

5.Filtration

● The filtration step in downstream processing (DSP) of therapeutic proteins is essential

for separating proteins based on their size, as well as for concentration and buffer
exchanges. Various filtration techniques, including microfiltration, ultrafiltration (UF),
nanofiltration, and reverse osmosis, are employed in the purification of pharmaceutically
relevant proteins. Recent trends in filtration focus on process intensification and the
optimization of processing strategies.
● High-performance countercurrent membrane purification has been introduced for protein
purification using bovine serum albumin as a model protein.
● The combination of separation driven by electric charge and filtration is an ongoing trend
in protein purification. A new model for electrostatic effects has been developed by
Briskot et al., which allows for better pH control for UF and diafiltration (DF) processes.

6.Chromatography

● Chromatography is conventionally used in multiple operational units for protein

separations. Chromatography describes the process of dynamic separation of mixtures.
The versatility of this technology is dependent on different separation mechanisms such
as specific interactions with a solid phase or different diffusivities. Chromatography is a
dynamic process that involves the separation of mixtures based on specific interactions
with a solid phase or different diffusivities.
● We focus on unconventional promising alternatives to packed-bed chromatography such
as membrane and monolith chromatography.
● Membrane chromatography offers higher flow rates compared to conventional
chromatography, which increases productivity.
● Cost-effective manufacture of membrane adsorbents offers the possibility of single-use,
reducing time-consuming and costly cleaning and validation procedures of packed-bed
columns in biopharmaceutical applications.

5
 Chetanya Jangra 2K20/EC/73

● Roshankhah et al. developed a cation exchange z2 laterally fed membrane

chromatography (z2LFMC) process with three times higher productivity compared with
conventional protein-A chromatography for antibody purification.
● Monolith chromatography is also characterized by high mass transfer efficiency. Simon
et al. recently demonstrated the cheap, robust, and customized fabrication of monolith
columns by 3D printing.
● The great potential of monoliths was confirmed, for example, by Wilke et al. who
demonstrated purification of IgG from human plasma with high productivity using
sintered glass monoliths with immobilized protein A.
● Despite advancements in novel approaches, chromatography remains the primary
method for purifying pharmaceutically relevant proteins.

Conclusion

Certain approaches discussed in the reviewed studies have the potential to enhance the
production of pharmaceutical proteins, making it more efficient and sustainable. However, one
significant challenge that needs to be addressed is convincing regulatory authorities to accept
new purification methods as alternatives to traditional chromatography methods. These findings
contribute to the ongoing efforts in improving DSP efficiency in the biopharmaceutical industry.