Solutions for Purification of Fc-fusion Proteins - When platform processes are applied to fusion molecules, innovation and flexibility are needed - BioPharm International


Solutions for Purification of Fc-fusion Proteins
When platform processes are applied to fusion molecules, innovation and flexibility are needed

BioPharm International Supplements

Figure 1. EA2 pH stability. Concentrated purified enzyme was incubated at 21 3 oC for various times at the indicated pH. Treatment was stopped by pH neutralization and samples were stored at 2–8 C prior to measuring enzymatic activity. A) low pH stability, B) high pH stability
The next step was to test alternate resins (i.e., Protein A mimetics) and alternate elution conditions.3–8 The Protein A mimetic tested did not exhibit adequate binding capacity. Chaotropic elution with potassium thiocyanate (KSCN) was physically successful, but destroyed the product's activity. However, elution with Tris at pH 11 has proven to be moderately successful and repeatable, and the product was shown to be stable at this pH (Figure 1b).

Figure 2. High pH Protein A elution screening
One downside to this procedure was that the elution peak tailed badly, and a modest amount of product did not elute until the low pH strip condition. Decreasing the elution buffer flow rate decreased the elution buffer usage and peak width. It seems that the product has a slow elution off rate. Given that Tris has essentially no buffer capacity at pH 11, these phenomena are perhaps not surprising. However, it seems that pH is not the sole driver of elution in this case. Glycine at pH 10 has a reasonable buffer capacity, but it failed to elute product when tested, doing even worse than Tris at pH 10 (Figure 2). Further investigation of this effect and optimization of the step are pending. We hypothesize that using a buffer with buffering capacity at pH 11 along with Tris would allow complete product elution.

Viral Inactivation

With the loss of low pH as a viral inactivation step, an alternative robust viral inactivation step was needed. Solvent/detergent (S/D) processes have a long history, and thus were the first to be examined. We decided that performing this unit operation just before the Protein A step was the best location in the overall process. At this point, the product was reasonably concentrated, minimizing the amount of S/D needed. Being early in the process, just before an affinity step, should afford good clearance of the S/D chemicals.

Figure 3. EA2 solvent/detergent stability. Final detergent concentration is 1.0% of Tween 80 or Triton X-100. At indicated time points, samples were taken and snap frozen, and stored frozen until enzymatic activity assay.
First, it was necessary to prove that the product was resistant to the chemicals. For this, partially purified product (Protein A, low pH elution with immediate neutralization) was incubated with two different detergents at two different Tri-n-butyl phosphate (TNBP) solvent concentrations for up to 24 hours (Figure 3). The detergents tested, Tween 80 and Triton X-100, were those most commonly cited in the literature and shown to be effective.

Although the results were somewhat noisy, they indicated that the product was reasonably stable to the treatment conditions. Also, the Tween-treated samples seemed more stable than the Triton samples over the long term, so Tween was the detergent of choice.

Figure 4. Small-scale Protein A purification of S/D-treated EA2. Load samples incubated in 1.0% Tween 80 plus indicated TNBP% for 60 minutes before loading onto column.
After this, we evaluated whether the S/D conditions would interfere with the subsequent Protein A chromatography. As Figure 4 shows, this proved not to be the case. As activity recovery with 1.0% TNBP and 0.3% TNBP seemed to be essentially equivalent (Figures 3 and 4), the 0.3% TNBP level was selected.

Polishing: Ion Exchange

The use of ion-exchange chromatography for polishing is very typical. Ion exchange is well known, well characterized, efficient, and economical. One typical modality is a flow-through anion exchange, more recently in a membrane format. This membrane format is disposable, has high throughput, and has good efficiency in removing negatively charged impurities such as nucleic acids, virus, and if needed, endotoxin, and some host cell proteins.

The pI of the product is variable, depending on the degree of sialylation. However, it is in the range of pH 5.2–6.5. Nonetheless, at pH 5.5, even with high salt, only 4% of the product did not bind to a Q membrane. This behavior was attributed to existence of a concentrated negatively charged patch built into the product molecule. Based on this, flowthrough anion exchange was not pursued.

The use of anionic Q resin in a binding mode was also examined. At first, this seemed modestly successful. The high salt elution peak tailed significantly, and recovery was only modest, in the 80–90% range. However, analysis of the product determined that the material that was lost was concentrated in the highly sialylated product, which was the most desirable form. Thus, this modality was also ruled out.

Given the high pH stability of the product, we tried the weak anion exchanger DEAE, eluting with high pH. It was thought that the combination of the different ligand (DEAE versus Q), and different elution modality (high pH versus high salt) could impact the product recovery. However, even at pH 11, elution recovery was poor.

Finally, cation exchange chromatography was examined. Given the behavior of the product on anion exchange, it was not anticipated that cation binding would be possible at a pH high enough to be stable. Surprisingly, at pH 6 with low salt, only partial flow through was achieved. The bound product was eluted at pH 6.8. The recovery and the sialylation of the product was good. However, there was no clearance of host cell proteins, so this modality was also shelved.

Polishing: Pseudo-Affinity

As mentioned earlier, the product has two pseudo-affinity sites other than the Protein A binding Fc domain. Resins to take advantage of these sites are less well known and are more expensive than ion-exchange resins. However, they have the potential for very powerful purification, so with the failure of the ion exchangers, these were investigated as potential post Protein A polishing steps.

Table 2. Hydroxyapatite (HAP) recovery
One of these sites targets hydroxyapatite. The product bound quite well to commercially available chromatographic supports based on ceramic hydroxyapatite, Types I and II (Bio-Rad). However, neither high salt nor high phosphate could effectively elute the product (Table 2).

The other potential site is an enzyme, alkaline phosphatase, whose active site is known to be a binding target of dye ligands. However, the ligand recommended for this enzyme only binds to some variants of the enzyme.9 This proved to be a case where binding of the enzyme to the ligand was of inadequate strength to be suitable for chromatography.

Polishing: HIC and Mixed Mode

Given the high and persistent charge on the protein, in order for the product to bind to an HIC resin, a strongly hydrophobic resin and a high salt concentration were needed. A butyl resin was successfully tested, using slightly over 1 M ammonium sulfate to affect binding.

Figure 5. Hydrophobic interaction chromatography (HIC) recovery and host cell protein (HCP) clearance
Activity recovery off the column was generally good. A280 recovery was low because of the recovery of an unidentified colored material which co-eluted with the product off the preceding Protein A column (Figure 5).

Figure 6. Capto adhere chromatograms. Gradients from 0 to 100% B. A) pH 6 run; B) pH 8 run
The HIC was also effective in removing biological impurities (Figure 5). However, a third chromatography step was desired to further reduce impurity levels. Given the success of the HIC, and the near success of the Q binding step, a mixed-mode resin with hydrophobic and anion exchange characteristics was tested (Capto adhere, GE Healthcare). The choice eventually proved successful, but only after some unexpected results.

Figure 6. Capto adhere chromatograms. Gradients from 0 to 100% B. A) pH 6 run; B) pH 8 run
The manufacturer recommends loading conditions between pH 6 and 8. Typically, the manufacturer notes that pH 6 gives better recovery, but that pH 8 gives better impurity clearance. Initial gradient elution experiments at both pH 6 and pH 8 confirmed the recovery observation, with 93% recovery at pH 6 and 86% at pH 8 (Figure 6).

Figure 7. Capto adhere recovery and HCP clearance
Because impurity removal was the primary target of this step, development work first targeted pH 7 to 8. It became clear that in this range, the binding affinity for some of the product was low, and thus significant losses would occur if operated in a binding mode. On the other hand, a salt concentration of nearly 1 M would be necessary to operate this resin as a flowthrough column, and it seemed unlikely that this would enhance impurity clearance. Thus, we operated this column as a binding column at pH 6, as was done in the initial experiments, where complete binding was seen under the conditions used. Recovery was generally good, and impurity clearance was consistent (Figure 7).

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