Concentrating Fc fusion protein by ultrafiltration with AcroPrep™ 24-well filter plate
Affinity chromatography with a protein A sorbent often forms the first step in capture of monoclonal antibodies and derivatives as Fc fusion proteins. Elution of these proteins from the protein A sorbent generally takes place at low pH. As prolonged exposure to low pH can induce aggregation, the protein A eluents are subsequently neutralized (NPE) prior to further processing (1).
Here we describe ultrafiltration with AcroPrep™ 24-well filter plates with 30K MWCO Omega™ ultrafiltration membrane as a method for concentration of NPEs prior to further processing during early discovery. If necessary, this procedure can be modified to include buffer exchange. The plates provide an option for processing of samples with low to medium volumes in an automated environment. Processing proved very reproducible and provided high protein recoveries (96% to 97%). No loss of protein to filtrate was observed.
Results
A CHO cell expressed Fc fusion protein with approximate Mr of 150kD was eluted from Protein A sorbent in 0.1 M sodium acetate buffer with pH 4. The samples were then neutralized with 1 M Trizmau base to pH 6.0, 6.5, 7.0, or 7.5, with resulting conductivities of 10 to11 mS/cm. These samples, with total volumes of 21 mL and a protein concentration of 2.35 g/L were loaded in the plate as 7 mL/well in three wells (Figure 1). The samples contained 2.2% soluble aggregates (high molecular weight species, HMW) as determined by size exclusion chromatography, and 47 ppm of host cell protein as determined by ELISA assay.
As indicated in Figure 1, samples (of indicated pH) were distributed over three wells each of an AcroPrep 24-well filter plate with 30K MWCO and processed on a multi-well plate manifold with a vacuum pressure of 29 inHg. The remaining 12 unused wells of the plate were covered with AlumaSealu II sealing film to minimize impact on vacuum processing of the samples. Filtration was carried out for a total of 35 h with filtrates being removed at intervals as shown in Table 1. At each time point, the protein concentrations of the filtrates were determined by measuring absorption at 280 nm. No appreciable amount of protein was detected in the filtrate (data not shown). Figure 2 shows that the filtration rate gradually decreased with time as the protein concentration of the retained sample increased. The recovered filtration volumes of the four samples were very similar, but the samples neutralized to pH 7.0 and pH 7.5 appeared to provide slightly higher filtrate volumes with higher protein concentrations of 9.2 g/L and 10.8 g/L, respectively, in the retentates (Table 2). However, protein recoveries were near identical and very high at 96% to 97% for all samples (Table 2).
Fig 1. Wells used in AcroPrep 24-well filter plate for ultrafiltration of samples with indicated pH. The unused wells were covered with AlumaSeal II sealing film during filtration.
Table 1. Filtrate volume collected at indicated filtration intervals
|
Filtration time (h) |
Filtrate volume collected (mL) |
||||
|
Cumulative |
Interval |
pH 6.0 |
pH 6.5 |
pH 7.0 |
pH 7.5 |
|
6 |
6 |
6.5 |
6.3 |
6.5 |
6.7 |
|
13 |
7 |
3.5 |
3.3 |
4.0 |
4.0 |
|
30 |
17 |
3.8 |
3.8 |
3.9 |
3.8 |
|
35 |
5 |
1.0 |
1.1 |
1.1 |
1.2 |
Fig 2. Retentate volume of Fc fusion protein samples filtered with 30K MWCO plate.
Table 2. Protein recovery of samples after ultrafiltration
|
|
Start |
End |
|
||||
|
pH |
Volume (mL) |
Concentration (g/L) |
Amount (mg) |
Volume (mL) |
Concentration (g/L) |
Amount (mg) |
Recovery (%) |
|
6.0 |
21.0 |
2.35 |
49.4 |
5.38 |
8.8 |
47.3 |
96 |
|
6.5 |
21.0 |
2.35 |
49.4 |
5.52 |
8.7 |
48.0 |
97 |
|
7.0 |
21.0 |
2.35 |
49.4 |
5.20 |
9.2 |
47.8 |
97 |
|
7.5 |
21.0 |
2.35 |
49.4 |
4.40 |
10.8 |
47.5 |
96 |
Conclusions
Concentrating Fc fusion protein with AcroPrep 24-well filter plates with 30K MWCO Omega™ ultrafiltration membrane proved very reproducible. Processing took place over a period of 35 h, which can possibly be shortened by using plates with 50K MWCO. The recovered filtration volumes of the four samples were very similar, with the samples neutralized to pH 7.5 providing the lowest retentate volume. The differences in filterability of the samples, however, do not result in differences in protein recoveries, which for all samples were very high at 96% to 97%.
References
- Chen Z, Huang C, Chennamsetty N, Xu X, Li ZJ. Insights in understanding aggregate formation and dissociation in cation exchange chromatography for a structurally unstable Fc-fusion protein. J Chromatogr A. 2016 Aug 19;1460:110-22. doi: 10.1016/j.chroma.2016.07.023.