Developing a MAb Aggregate Removal Step by High Throughput Process Development

ABSTRACT

This article presents a case study illustrating the benefits of using a high throughput process development (HTPD) approach for choice of media and optimization of the polishing step of a monoclonal antibody in a two-step purification process. The most commonly used format for HTPD is the 96-well plate to facilitate rapid screening of chromatographic parameters. The use of the 96-well plate format enabled the efficient screening of three different media and optimization of chromatographic parameters to maximize yield at the desired purity level.

Biopharmaceutical manufacturers are under increasing pressure to develop and produce biopharmaceuticals cost-effectively and within tight timelines. The US Food and Drug Administration is also nudging the industry to implement its Quality by Design (QbD) initiative in the manufacturing processes. QbD stipulates a better understanding of the influence of raw materials and intermediates, and control of process parameters. This initiative also is applicable to the development of downstream processes in the manufacture of monoclonal antibodies (MAbs). In this area, a new approach of high throughput process development (HTPD) is emerging as a useful tool for compliance with QbD. HTPD uses a screening format to facilitate the identification of optimal experimental conditions by directed, rapid screening of experimental parameters. A large experimental space can be investigated and characterized in a very short timeframe, enabling a better understanding of the effect of process conditions.

(GE HEALTHCARE)

Traditionally, MAbs are purified in a three-step process. First, the target is captured by chromatography with Protein A media, and two subsequent polishing steps are then performed according to a variety of protocols, often involving a combination of ion-exchange chromatography and hydrophobic interaction chromatography. For particular antibodies, it is possible to apply a more efficient two-step process that involves one capture step and a single polishing step. In the polishing step of a two-step process, a multimodal chromatography medium capable of several interactions (e.g., hydrophobic, ion-exchange interactions, hydrogen bonding) can be used, which allows the selective removal of impurities like antibody aggregates, host cell proteins (HCPs), and leaked ligands. The complexity of these media requires a more thorough process optimization study. The most commonly used format for HTPD is the 96-well plate to facilitate rapid screening of chromatographic parameters.

This case study illustrates the benefits of using the HTPD approach for choice of media and optimization of the polishing step in a two-step MAb purification process. The use of a 96-well plate format enabled efficient screening of three different media and optimization of chromatographic parameters to maximize yield at the desired purity level. The initial capture step was performed using a Protein A medium (MabSelect SuRe, GE Healthcare), which was then used diluted or undiluted for the specific experiments.¹ Three chromatography media have been investigated in this study (Table 1).

Table 1. Chromatography media tested in this study

INITIAL SCREENING OF CHROMATOGRAPHIC CONDITIONS

Initial experiments focused on finding the ideal incubation times and MAb concentrations for the remaining experiments (Table 2). These experiments were performed using 96-well plates (PreDictor, GE Healthcare) filled with the three chromatography media being studied, which eliminated the need for the more time-consuming column chromatography.

Table 2. Initial screening conditions for the three chromatography media tested

The chromatography media in the 96-well plate were first equilibrated at specific pH and salt concentrations, and the sample was then added at four different incubation times. The flow-through fraction was collected in a collection plate using centrifugation at 300g and was analyzed using size-exclusion chromatography (SEC) to determine monomer and aggregate contents.

Initial screening on the two anion-exchange media showed that antibody binding only occurred at pH 8.5 and 0 mM NaCl. The binding kinetics for all three media were fast for the monomer. After 10 min, the binding was complete, while the binding of the aggregates was approximately three-times slower—at least 30 min was required to achieve complete binding. The monomer content increased slightly at longer incubation times, possibly because of the displacement of monomer by aggregates. It was concluded that an incubation time of 1 h and a protein concentration of 5.3 g/L were the most appropriate conditions.

OPTIMIZATION OF FLOW-THROUGH PURIFICATION

A further screening in 96-well plate format was performed for all these media to identify the chromatographic conditions to reduce the aggregate levels to <1%. The starting material had very low levels of HCPs, so these were not considered in this particular study.

The MAb was loaded on the chromatography media in a range of pH values and NaCl concentrations (Table 3). After 60 min of incubation with the sample (5.3 g/L), the flow-through fractions were collected and the concentration of monomer and aggregates in the fractions was determined by SEC.

Table 3. Screening conditions for flow-through purification

Using the monomer and aggregate concentration in the starting material (C_ini,m, Cini,a) and the concentration in the flow-through fraction (Cm,Ca), the binding capacity for monomer (Qm) and for the aggregates (Qa) can be calculated according to the following equation:

For the strong anion exchanger (Capto Q), the highest capacity for both monomer and aggregates was observed at high pH values and the lowest NaCl concentration (data not shown). For all conditions, the monomer capacity was higher than the aggregate capacity. The capacity for the weak anion exchanger (Capto DEAE) leveled off at higher pH levels (8.8–9.2, data not shown). This is thought to be because of the nature of the DEAE ligand, which loses its charge around this pH, while the quaternary ammonium ions remain charged at these conditions.

The calculations with the multimodal medium (Capto adhere) revealed the highest capacity for both monomer and aggregates at high pH values and low NaCl concentrations, as shown in Figure 1. For phosphate, the capacity decreased with increasing NaCl concentration, and the opposite trend was seen for citrate. Given that this was a flow-through step, the objective was to identify conditions when the aggregate capacity was much higher than the monomer capacity. For all conditions, the monomer capacity was higher than the aggregate capacity, which would mean a low monomer yield in a flow-through step.

Figure 1

The binding capacity data from the 96-well plate experiments were then used to predict yield and purity of the monomer in the flow-through fraction. This is possible using the assumption that the monomer plate binding capacity equals the dynamic binding capacity in a column, which is a good approximation for longer residence times. Purity and yield can then be calculated using the following equations:

in which Vload is volume of sample loaded onto a column, C is concentration, CV is column volume, and SBC is the binding capacity found in the plates.

A column prediction for the strong anion exchanger at a simulated load of 150 g/L is shown in Figure 2, which also shows the strong anion exchanger's predictions. The yield increased with decreased pH while the purity increased with increased pH. In this case, the purity was considered the most important factor for the flow-through step. The highest purity was identified by following the "ridge" from pH 8.0 without NaCl to pH 9.2 with approximately 50 mM NaCl.

Figure 2

Predictions for yield and purity at different sample loads also were performed for the multimodal medium in the 96-well plate format and the prediction at the most favorable conditions was compared with the column data. Based on the previous data, a column prediction of purity and yield at various sample loads could be made to find the best conditions for the flow-through step. Figure 3 shows the data for a load of 122 g/L. The yield increased with decreased pH while the purity increased with increased pH. In this case, the purity was considered to be the most important factor for the flow-through step, with the highest purity prediction highlighted with the red box.

Figure 3

These results were verified using a 1-mL prepacked column (Figure 4, HiTrap, GE Healthcare). The column was equilibrated with 25 mM sodium phosphate at pH 7.5. The sample (desalted MabSelect Sure eluate at approximately 7 mg/mL) was loaded at 10 min residence time. Experiments were performed with two sample loads, 130 and 260 g/L, and the corresponding prediction calculations were made for these two loads.

Figure 4

The predicted yield and purity at both sample loads (130 and 260 g/L) were compared to the experimental values obtained using both 96-well plate and 1-mL HiTrap columns. The results correlated well between columns and plates (Figure 4), demonstrating that the experimental conditions in 96-well plates can be successfully scaled up to the column format. For both formats the yield increased with increased load, but the purity showed the opposite trend. With purity being the most important factor, the lower sample load was of greater interest because it provided 98% (96-well) and 97% (column) purity. However, in both cases the yields were significantly below the target 85%.

CHOICE OF MEDIA

The results from the screening experiments were used to compare the performance of the strong anion exchanger (Capto Q) with the weak anion exchanger (Capto DEAE) and the multimodal medium (Capto adhere). The predicted performance was almost identical at similar loads for all media: at the best purity (100% monomer content), the yield was at most 60%.

Other factors must be considered to choose between the different media. One such factor could be the pH of the process step. At pH values >8, deamidation is a major problem, and the optimum conditions for Capto Q and Capto DEAE are at pH values of ≥8. Also, viral reduction is very effective with Capto adhere in a wider range of conductivities than for the other anion exchangers. Therefore, it is a better choice to use Capto adhere as the polishing step in the MAb purification process.

SELECTIVE ELUTION OF THE MONOMERIC ANTIBODY

To increase the yield of the monomer, a selective elution study in the 96-well plate format was performed using the multimodal medium. The objective was to selectively elute the bound monomer after the flow-through step while retaining the aggregates on the column.

The following parameters were studied after loading the plate at the optimal conditions for purity (Figure 3, red box):

• pH from 4.0 to 7.0

• pH 4.0 to 6.0 with 50 mM sodium citrate

• pH 6.0 to 7.0 with 50 mM sodium phosphate

• NaCl concentration from 0 to 550 mM NaCl.

Thorough analysis of the resulting monomer and aggregate content (determined using SEC) for the different elution conditions led to the conclusions that the monomer content in the elution pool was higher when using phosphate buffers than when using citrate buffers and that the differences were because of buffer type and not pH value. Therefore, the remaining results are focused on elution conditions for the data for the phosphate buffer.

To better visualize the relation between monomer and aggregates in the elution pool, the raw data were presented as an objective function (purity*yield) for all elution conditions (Figure 5). The highest values for the objective function were seen around pH 6 and NaCl concentrations around 250 mM.

Figure 5

To verify the results from the plate experiment, a single-column experiment was performed. The MAb was bound using the same conditions as in the selective elution experiment. The elution condition was selected from within the optimal area highlighted in Figure 5. When all three fractions (flow-through, wash, and elution) were pooled, both the yield and the purity reached the preset criteria (Figure 6). The aggregate level was reduced to 0.5% with a yield of 87%.

Figure 6

SUMMARY

Traditional downstream purification processes require two chromatographic polishing steps following the initial capture step with a Protein A medium. However, we have highlighted how a switch to an optimized two-step process consisting of a capture step and a single polishing step can achieve the desired levels of purity and yield of MAb. The use of the HTPD screening format facilitated identification of the optimal experimental conditions by directed, rapid screening of experimental parameters. This screening approach is ideal to support the QbD initiative implementation in the manufacturing process. A large experimental space can be investigated in a very short time-frame, enabling a much better understanding of the effect of process conditions. The following are the advantages of this approach for downstream processes development.

• In a relatively small time frame, many chromatography media and conditions can be tested, enabling a good understanding of the purification process and the effect of several factors on yield and purity using 96-well plates.

• Yield and purity can be predicted for several sample loads for various chromatographic conditions.

• A much larger experimental space can be investigated, enabling the adoption of QbD.

• Predictions with data from the 96-well plate experiments correlate well with those using column data which will support easier scale up of production processes.

When considering media selection, HTPD is a useful tool because it enables a broad range of chromatographic conditions to be explored. For example, in this study the availability of experimental data highlighted the impact of pH on the suitability of a weak anion exchanger such as Capto DEAE. At pH values >8.5, the ligand loses its charge, which limits the useful pH-range of this medium.

The purity and yield obtained using the three media were comparable. However, the multimodal medium was selected because it can be used at a more neutral pH. This minimizes the risk of deamidation of the MAb. For the multimodal medium, a selective elution HTPD study was then performed, which improved the yield to the required level of over 85% with a corresponding level of less than 1% of aggregates.

In conclusion, the availability of the right comparative experimental data for all the media enabled identification of the multimodal medium as a better choice for the polishing step in this two-step MAb purification process.

Gustav Rodrigo is a senior scientist and Kristina Nilsson-Välimaa is a research engineer, both at GE Healthcare, Uppsala, Sweden, +46 18 612 0000, gustav.rodrigo@ge.com

REFERENCES

1. High-throughput screening and optimization of a protein A capture step in a monoclonal antibody purification process. GE Healthcare; 2009:28–9468–58. Available from: www.gelifesciences.com