Developing a MAb Aggregate Removal Step by High Throughput Process Development - High throughput process development allows rapid screening of chromatographic parameters. - BioPharm International

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Developing a MAb Aggregate Removal Step by High Throughput Process Development
High throughput process development allows rapid screening of chromatographic parameters.


BioPharm International
Volume 23, Issue 4

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.


Figure 5
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 6
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%.

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,

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: http://www.gelifesciences.com/


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