FUTURE CHALLENGES IN DOWNSTREAM PROCESSING
Because of the broad range of new molecules, hosts, and delivery methods being investigated, recent biopharmaceutical development
has resembled that from the early stages of the industry. A broad range of alternative BioTx modalities have obtained regulatory
approval and are currently in clinical trials (17–21) including antibody fragments, single-domain mAbs, Fc-fusions, vaccines,
antibody-drug conjugates, nonantibody protein scaffolds, PEGylated proteins and peptides, and viral vectors.
Some of the newer BioTx use nonmammalian expression systems such as yeast and E. coli if there is no requirement for post-translational modifications. These alternate expression systems may offer cost advantages.
However, other issues may arise, including undesired mannosylation in a transferrin-exendin-4 fusion molecule expressed in
yeast (22). This unwanted modification required an extra process step using hydrophobic interaction chromatography (HIC) to
separate these modified molecules from the desired product. E. coli expression often yields high levels of protein production, often in the form of inclusion bodies, which require extra steps
involving their isolation and the subsequent extraction and refolding to obtain the desired product (23).
For a difficult-to-refold protein such as neurotrophin-4, an additional step involving sulfonation of the cysteines was also
required to obtain a useful refold yield (24). The use of high throughput screening with Design of Experiments has been used
to accelerate the determination of optimal conditions for refolding difficult proteins (25).
Each of these BioTx modalities poses a unique challenge in downstream processing because of the lack of a platform process
resembling that used for mAbs. Examples of the purification challenges involved with two Fc-fusion proteins will be described
in the following sections.
Because each Fc-fusion protein has a different protein sequence fused to an antibody Fc region, the protein amino acid sequence,
charge, size, and hydrophobicity vary more than those of antibodies. In addition, they are often thermally, chemically, or
enzymatically unstable, which can lead to high levels of aggregates, clips, or inactive species.
Case study 1.
A purification process was developed for an acidic Fc-fusion protein that was unstable at ambient temperatures and low pH
and that quickly formed high molecular weight oligomers (HMW1) and dimers (HMW2).
To improve stability, the protein A chromatography step was performed at 2–8 o C and the low pH eluate was neutralized immediately. The capacity of the protein A resin for the molecule was less than 15
mg/mL which is typical for Fc-fusion proteins.
A combination of HTS methods and gradient chromatography was employed to identify resins and operating conditions for high
molecular weight (HMW) species removal. Anion exchange, cation exchange, and HIC resins were screened in 96 well plates in
an HTS format. Additionally, ceramic hydroxyapatite (CHA), immobilized metal affinity chromatography (IMAC), and HIC methods
were screened with elution gradients.
Due to the low pI of this Fc-fusion protein, a WPC–AEX step was not successful. Based on the resin screening, CHA chromatography
was selected for optimization. An initial test of CHA was performed using a phosphate gradient. This method allowed identification
of step elution conditions that reduced the HMW levels from 29% to 10.1% with 87% yield. The HMW1 flowed through the column
and the peak contained primarily dimer species (HMW2). The CHA step had a capacity of 20 g/L.
The HTS analyses also indicated that a HIC resin under certain loading and elution conditions could provide reduction of HMW.
An initial experiment using protein A peak pool and a salt gradient indicated the resin had a relatively low capacity for
the product but was effective at reducing both the levels HMW species and less active monomer variants.
Further development was performed using the protein A pool as a load to the HIC step to identify conditions where the product
would flow through the column while the HMW and less active monomer would bind. A successful method was developed but the
HMW1 species began to flow through the column at about 10 mg/mL of load.
Because the CHA step was successful at removing the HMW1, but not HMW2 species, the CHA pool was used as a load for the HIC
step. The HIC step was loaded with the CHA pool containing primarily HMW2 at 20 mg/ml. The HIC step provided a six-fold reduction
in HMW2 levels. In addition, the HIC step reduced the level of host cell proteins by 1.4 log10 . Based on these data, the final process used a Protein A capture step with elution at low pH followed by immediate neutralization.
This was followed by a bind-elute CHA step to remove HMW1 followed by a flow through HIC step to remove HMW2 (see Figure 3).
The first two chromatography steps were performed in the cold to maintain product stability.
Figure 3: Yield and percentages of HMW1 and HMW2 versus step in the Fc-fusion purification process described in case study
Case study 2.
During development of an early-phase purification process for an acidic Fc-fusion protein expressed in a CHO cell culture
system, it was determined that the protein displayed a range of 24–100% activity. Several types of chromatographic resins,
including cation exchange, anion exchange, HIC, CHA, and IMAC, were evaluated to remove the less active molecules along with
other product and process-related impurities. The process and conditions described above for Fc-fusion protein 1 were not
applicable to Fc fusion protein 2.
The CHA is a bimodal resin with two primary functional groups: phosphate and Ca2+ . The negatively charged phosphate groups serve as cation exchangers and the Ca2+ groups serve as chelators to proteins. For purification of basic and neutral proteins, the CHA column is usually equilibrated
with phosphate buffer at a neutral pH to utilize a cation exchange mechanism for the separation of protein monomers from HMW
The CHA column, when equilibrated with phosphate, resolved fully active protein from partially active protein. The product
was in the unbound fraction while the less active molecules were bound to the column. However, the load capacity was low and
removal of high molecular weight species was poor using the phosphate-charged CHA column in flow-through mode.
A calcium-charged CHA column operated in bind-elute mode successfully removed the less active monomer species, as well as
high molecular weight species, from the process stream. In this case, the CHA column was equilibrated with a low concentration
of CaCl2 in a neutral pH buffer, and then loaded with a protein mixture also containing CaCl2. After washing the column with a neutral pH buffer, the column was eluted with a phosphate buffer and stripped with a high
phosphate buffer. A high yield of the product was obtained in the elution peak. The less active protein and HMW species
were present in the strip fraction. These data demonstrate that although Fc-fusion proteins are more challenging to purify
than mAbs, other chromatography resins can be used to yield high-quality product.
These two examples illustrate the challenges posed by the different Fc-fusion molecules. In one case, calcium charging of
the CHA column was required for removal of a lower activity monomer species. In another case, a bind-elute CHA column followed
by a flow through HIC column was required for adequate HMW removal, resin capacity, and product yield. The sequence of the
polishing steps is also important for both purity and capacity.