Purifying Recombinant Protease Inhibitors from Yeast
Figure 1
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Mixed-mode chromatography was used successfully to purify several recombinant Kunitz-type protease inhibitor muteins from
yeast. Mutein I was expressed in Saccharomyces cerevisiae and secreted into the fermentation supernatant using a complex medium with a conductivity >20 mS/cm that contained a large
amount of colored impurities (i.e., melanoidins). The protease inhibitor (pI ~6.3) showed a reasonable solubility of ~13 mg/mL
at pH <3.2 that decreased significantly to <0.5 mg/mL when the pH value was increased only slightly (Figure 1). At a neutral
or slightly basic pH value, an acceptable solubility could be achieved only at high-salt conditions.
Figure 2
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The design of a suitable purification strategy focused on the following objectives: 1) identifying chromatography steps that
keep the protein solution at a pH value below 3 or at high salt concentration and 2) using the low solubility of mutein I
at neutral pH to find a suitable crystalline storage form for the bulk drug substance. Because the use of conventional ion
exchange (IEX) resins was not possible given the unusual solubility profile of mutein I and the high conductivity of the starting
material, the salt-tolerant properties of two mixed-mode resins (Capto MMC and HEA Hypercel), which are orthogonal with respect
to their binding and elution pH levels, proved to be particularly useful. The purification process is shown schematically
in Figure 2. The protein concentration of the feed stock was <0.5 mg/mL, and the clarified fermentation supernatant was applied
at >20 mS/cm directly onto Capto MMC at a pH value of 5.5. Elution was achieved at a pH value of 8.5 under high-salt conditions
(2 M NaCl), and, subsequently, the protein was applied directly onto HEA Hypercel without any conditioning steps. Elution
at a pH value of 2.7 made it possible to keep the protein in solution under low-salt conditions. Lastly, increasing the pH
value to more than 4 in a controlled mode of operation initiated crystallization, which led to a stable storage form of the
bulk drug substance.
Table 2. Experimental conditions for a typical Capto MMC capture run of the recombinant protease inhibitor mutein I
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Capturing mutein I with Capto MMC was the key chromatography step of the purification process (Tables 2 and 3). The clarified
fermentation supernatant was applied directly at relatively high conductivity (>20 mS/cm) without further dilution. The maximum
capacity of 17 mg/mL was only moderate but sufficient for an early-phase production process. Process- and product-derived
impurities were removed efficiently, and the purity of mutein I was >90% after this chromatography step, which indicates that
Capto MMC acts as a pseudo-affinity resin in this case. This was particularly possible by using stringent washing conditions
(Tables 2 and 3). High-salt washing conditions at a pH value of 5 and a low-salt washing step at a pH value of 8.5 led to
an almost complete removal of the colored impurities (melanoidin) from the fermentation medium, and the Capto MMC eluate was
colorless and clear. De-colorization by Capto MMC is mainly a pH-driven process. During the development of a production process
for another mutein of a protease inhibitor (mutein II, see Table 1), we were able to show that colored impurities from complex
yeast media bind to Capto MMC at pH values from 3 to 6, but can be completely removed at a pH value of more than 6.5. Thus,
the binding of the desired protein to the resin under these conditions is of particular importance for a successful de-colorization
strategy using Capto MMC.
Table 3. Purification data from a typical Capto MMC capture run of the recombinant protease inhibitor mutein I
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Several approaches for intermediate purifications were tested during process development. Reversed-phase chromatography under
strongly acidic conditions worked well for purification of research-grade material, but using high-pressure equipment was
not considered suitable for the large-scale manufacturing process. Because mutein I was eluted from Capto MMC at 2 M NaCl,
pH 8.5, we screened for a resin that allowed binding at high-salt conditions as well as elution at a low pH value under low-salt
conditions. The mixed-mode Hypercel-based resins from Pall meet these requirements because they allow salt-tolerant binding
at neutral or slightly basic pH value, and elution is usually achieved at a low pH value by electrostatic charge repulsion.
When binding mutein I on the Pall mixed-mode resins was tested, HEA Hypercel showed the highest capacity (27 mg/mL) compared
to PPA Hypercel (19 mg/mL) and MEP Hypercel (9 mg/mL). We decided, therefore, to use HEA Hypercel for intermediate purification.
The Capto MMC eluate was applied directly without prior buffer exchange, and elution at a pH value of 2.7 enhanced the solubility
of mutein I under low-salt conditions (Tables 4 and 5). Product purity was increased only marginally during this step; only
traces of product-derived impurities were separated. However, HEA Hypercel enabled us to remove large amounts of salt from
the Capto MMC eluate and to immediately lower the pH value, which was necessary to prepare the protein for the crystallization
step.
Table 4. Experimental conditions of a typical HEA Hypercel intermediate step of the recombinant protease inhibitor mutein
I
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All unit operations were successfully scaled up by a factor of 5 to 10 and manufacturing was performed using a 5-L Capto MMC
and a 2-L HEA Hypercel column, respectively. Crystallization was carried out in a 5-L glass reactor. The overall process yield
was 70%.
Table 5. Purification data from a typical HEA Hypercel intermediate step of the recombinant protease inhibitor mutein I
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In summary, chromatography resins with multimodal ligands enabled us to purify a recombinant mutein of a protease inhibitor
with an unusual solubility profile in a three-step process. Ligands combining ionic interactions with hydrogen bonding and
hydrophobic interactions showed a unique selectivity as well as salt-tolerant binding and allowed for efficient removal of
major product- and process-derived impurities under conditions where classical chromatography methods like IEX or hydrophobic
interaction chromatography (HIC) failed.
Purifying a Recombinant Cytokine Mutein from E. coli
Ceramic hydroxyapatite (CHT, Bio-Rad Laboratories) is a crystalline mineral with multimodal functionalities that are significantly
different from the mixed-mode ligands described above. The amino groups of proteins bind to the phosphate ions of the mineral
by a classical cation exchange mechanism, and elution is typically achieved by increasing salt or phosphate buffer concentration.
In contrast, carboxylic side chains of proteins bind to the Ca2+ ions of the resin by a combination of calcium metal affinity and anion exchange. The combination of different binding modes
leads to excellent selectivity, which allows for efficient removal of aggregates and multimers in production processes of
antibodies or antibody fragments.7,9 We used CHT type I (particle size: 80 µm) to separate a closely related, product-derived impurity with a post-translational
modification during clinical manufacturing of a recombinant cytokine mutein from E. coli.
Figure 3. Flow scheme of the cytokine mutein purification process
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The production process of the cytokine mutein is shown schematically in Figure 3. The protein was expressed in E. coli as an inclusion body and refolded. After pH adjustment and depth filtration, reversed-phase chromatography was performed
to reduce the volume of the protein solution after the refolding step and to decrease the salt concentration. CHT type I (80
µm) was used as intermediate step to remove an N-formylated product-derived impurity. Finally, polishing was performed by
reversed phase chromatography to separate several product-derived disulfide isoforms. The overall process yield was 50%.
Figure 4a-b. Separation of an N-formylated cytokine mutein species by CHT type I chromatography. The figure shows the elution
profile of the native cytokine mutein (blue) and the N-formylated impurity (red) before (a) and after (b) optimization by
Design of Experiments
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Analytical characterization of the cytokine mutein intermediate after refolding and reversed-phase chromatography revealed
an N-formylated product-derived impurity that had to be removed from the desired nonformylated isoform. A broad screening
of commercially available ion exchange resins showed that a separation of the formylated and nonformylated species could be
achieved only with a 15-µm Source 15S cation exchanger operating at a pressure of 1–2 MPa. To perform intermediate purification
at low pressure, several CHT and ceramic fluorapatite (CFT) resins were tested. Purification with CHT type I (80 µm) resulted
in a reasonable separation of the formylated and nonformylated species (Figure 4a) when a column length of 9.5 cm and a column
load of 17.5 mg/mL were used. To further improve the removal of the formylated impurity, an optimization program using Design
of Experiments (DoE) was performed. Standard conditions for binding were 5-mM K-phosphate at pH 6.5, and elution was achieved
using a linear 5–500 mM K-phosphate gradient. Five process variables were selected as factors: column length (5–14 cm), column
load (5–30 mg protein/mL resin), flow rate (10–60 column volumes/h), gradient steepness (1–2 % eluent B/column volume), and
pH value (6.0–7.0). A chromatogram-response function (CRF) was selected as a response variable to reflect successful separation
of the formylated and nonformylated species.10 Purity and yield of the cytokine mutein were defined as additional response variables. A linear d-optimal design with center
points was used that was later augmented to a full response surface design (i-optimal design). Column length and column load
showed the largest effects, and several two-factor interactions were identified. Calculating the optimal factor settings (column
length: 14 cm, column load: 5 mg/mL) resulted in a significant improvement of the separation of formylated and nonformylated
species (Figure 4b). The optimized unit operation was successfully scaled-up (to a 2.8-L column) and used to manufacture large
quantities of material. The overall yield of the process step was 85%, and the purity of the cytokine mutein increased from
60 to 75%.
A Generic Strategy for Screening and Optimizing Mixed-Mode Resins
Figure 5. Flow scheme of a generic approach for screening and optimization of mixed-mode chromatography steps
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The interaction of mixed-mode ligands with the desired protein is often complex, and extensive optimization is required during
process development. Many of the mixed-mode chromatography steps described in Table 1 were optimized successfully without
using DoE. However, DoE offers considerable advantages for developing complex separation steps because it enables the identification
of main effects as well as two-factor interactions and critical/noncritical parameters. Furthermore, the model makes it possible
to calculate the impact of selected factors for each point in the design space, which offers an enormous flexibility for process
optimization in both early and late stages of development. We decided, therefore, to develop a generic strategy for optimizing
mixed-mode purification steps (Figure 5). This approach includes screening various resins (Capto MMC, Capto adhere, MEP Hypercel,
PPA Hypercel, and HEA Hypercel) using batch binding conditions, which allows for a selection of suitable resins based on binding
and elution of the desired protein at different pH values and salt concentrations. Then, resins are selected and further
optimized by DoE using small column chromatography (2 mL). We usually conduct a linear full-factorial design using four factors
(loading pH value, elution pH value, salt concentration in the elution buffer, and column load), a design that can be augmented
later to a full response surface design, if necessary. This approach was used successfully to optimize mixed-mode steps of
an antibody fragment expressed in Pichia pastoris and a monoclonal antibody expressed in mammalian cell culture. The complete experimental program takes approximately four
to five weeks and the total amount of protein needed is usually 1 to 2 g, which is acceptable even in early stages of process
development.
Conclusion
Salt-tolerant adsorption and unique selectivity are the major advantages of mixed-mode materials over single-mode resins.
These features enabled us to meet a variety of challenges in downstream processing, such as capturing proteins from complex
feedstocks, de-colorizing feed streams, purifying difficult proteins, and removing special product- and process-derived impurities.
Moreover, the overall number of unit operations was significantly lower in these cases when mixed-mode steps were implemented
in the downstream process. Thus, mixed-mode materials offer considerable flexibility for designing efficient downstream processes,
in particular if no generic affinity ligand such as Protein A is readily available. The complexity of interactions between
the ligand and the desired protein might be considered as a limiting factor. This can, however, be overcome easily by using
a combination of resin screening and statistical design of experiments, which makes it possible to optimize complex separation
steps in a generic and systematic fashion.
Acknowledgements
The contributions of Torsten Minuth and many other colleagues from Bayer Schering Pharma Biotech Development are gratefully
acknowledged. Solubility experiments were performed by Dirk Havekost, Bayer Technology Services. We thank Hans-Dietrich Hoerlein
for his support and many fruitful discussions.
FELIX OEHME, PhD, is a senior scientist in downstream process development and JOERG PETERS, PhD, is the pilot plant manager for biotechnology, both in Biotech Development, Global Biologics, at Bayer Schering Pharma AG,
Wuppertal, Germany, +49 202 36 7743, felix.oehme@bayerhealthcare.com
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