Process Development and Screening Strategies for High-Throughput Platform

With the rapid development of biopharmaceutical industry, the development of efficient and stable purification processes has become increasingly important. Through parallel experiments and automated means, high-throughput purification technology enables large-scale rapid screening and optimization of chromatography resin, buffer conditions and process parameters, significantly improving the efficiency and reliability of process development. This article will systematically introduce the core objectives and screening strategies of the high-throughput purification platform, and illustrate its application process based on actual cases.

Core Objectives of High-Throughput Purification Platform Development

•  Shorten Development Cycle: Accelerate the optimization of process parameters and resin screening through parallel experiments and automation technology.

• Reduce Development Costs: Reduce the consumption of samples and resins, optimize resource utilization, and alleviate the economic burden of process development.

• Improve Process Robustness: Clarify the correlation between critical process parameters (CPP) and critical quality attributes (CQA) through design of experiments (DOE) and data analysis to improve the repeatability and controllability of the process.

• Wide Application Adaptability: Support the purification process requirements of new biological drugs such as antibodies, antibody-drug conjugates (ADCs), viral vectors, and recombinant proteins.

High-Throughput Screening Strategy

Resin Screening

Affinity Chromatography Screening Strategy

• Target Product Characterization Analysis: fully evaluate the structure and affinity sites of the target product, such as the tag type of recombinant protein, the structure of therapeutic antibody, etc.

• Resin Primary Selection: Select the type of affinity resin according to the characteristics of the target product, such as affinity resin containing nickel ion for capturing recombinant protein containing histidine tag, and protein A or protein G affinity resin for capturing antibodies containing complete antibody structure.

• Preliminary Screening: Select 3-4 affinity resins of this type for screening, and use platform chromatography methods. For example, antibody affinity chromatography usually uses 100 mM sodium acetate-acetic acid/100 mM glycine buffer to elute the target product, and detect the yield and purity of the target product.

• Process Optimization: Further optimize resin based on the preliminary screening results, and the process parameters such as loading, leaching, and elution in combination with the corresponding DOE experimental design.

Ion Exchange Chromatography Screening Strategy

• Determine the Chromatography Purpose and the Isoelectric Point of the Target Product: Define the chromatography purpose and the isoelectric point of the target product. The isoelectric point of the target product can be obtained by calculating the target product sequence or by capillary isoelectric focusing electrophoresis.

• Resin and Mode Selection: For impurities whose isoelectric point is lower than that of the target product, anion exchange chromatography in flow-through mode can be used for purification; For impurities whose isoelectric point is higher than that of the target product, the cation exchange chromatography in binding/elution mode can usually be used for purification.

• Screening Method: Resin screening in flow-through mode can usually use DOE combined with 96-well plate for high-throughput screening; Column chromatography screening is the preferred method for resin screening in binding/elution mode.

Hydrophobic Chromatography Screening Strategy

• Hydrophobicity Pre-experiment: The hydrophobicity of the target molecule is determined by high salt gradient pre-experiment, and the hydrophobic ligand density is optimized by gradient elution, which can predict the hydrophobicity of the target product.

• Optimization of Loading Salt Concentration: The target product will be combined with hydrophobic resins in a high-salt system and eluted under low-salt conditions. Therefore, when the optimization of loading salt concentration in the chromatography process, ammonium sulfate is usually used as the loading pretreated salt.

• Resin Screening: After preliminarily determining the type of ligand and the salt concentration required for loading, the gradient elution method from high salt to low salt can be used for resin screening and process optimization, and the subsequent elution can be further optimized to step elution through experimental design.

Gel Filtration Chromatography Screening Strategy

• Molecular Weight Evaluation: Generally understand of the molecular weight of the target product and impurities, which can be obtained by gel electrophoresis, capillary electrophoresis and other methods.

• Resin Selection: Mainly focus on the pore range of the resin. Generally speaking, the larger the pore diameter, the larger the separation range, such as Geldex® 75pg has smaller pores and resin separation ranges from 3-70 kda, while Geldex® pores of 200pg are large, and the resin separation range is 10-600 kda, so it is necessary to select the appropriate resin according to the purification needs first.

• Specific Adsorption: Pay attention to the specific adsorption effect of the resin on the target product, and reduce the specific adsorption between the target product and the resin as much as possible.

Buffer Screening

Buffer Type Selection

Select the appropriate buffer type such as phosphate buffer, Tris-HCl buffer, acetate buffer, etc., according to the nature and purification requirements of the target protein.

Parametric Gradient Design

Set the pH value, ionic strength, salt concentration and other parameters of the buffer solution in a gradient. For example, the pH value can be set to 5.5, 6.0, 6.5, 7.0, 7.5, etc., and the ionic strength can be set to 0.1 M, 0.2 M, 0.3 M, etc.

High-Throughput Screening Experiments

Use buffers with different parameters for the steps of balancing, loading, washing and elution of resins, and screen the optimal buffer conditions by detecting the purity, recovery and impurity removal rate of protein.

Screening of Process Parameters

Identify Critical Parameters

Such as loading, flow rate, elution gradient, etc. These parameters have an important influence on the purification effect and process stability.

Experimental Protocol Design

Use DOE methods such as orthogonal experiment and single factor experiment, combine and design the key process parameters to determine the experimental conditions.

Data-Driven Optimization

Carry out the experiment on a high-throughput platform, collect the purification results under various experimental conditions, analyze the data, and screen the best combination of process parameters.

Case Application: Development of Purification Process for a Yeast Secreted Protein

Secretion expression supernatant of a certain yeast:

• Molecular weight: 27 kda,

• Isoelectric point: 9.90,

• Moderate hydrophobicity,

• Screen appropriate experimental conditions through high-throughput,

• Screen pre-experimental conditions through 96-well plates.

Resin Strategy

The resins of BioLink are MaXtar® S, SP Chromstar® FF, MaXtar® MMC, MaXtar® MMC HR, MaXtar® Phenyl HR, MaXtar® butyl HR.

Chromatography Condition Strategy

Ion Chromatography Buffer

A：PB+NaCl, pH7.0, 5 mS/cm；

B：PB+NaCl, pH7.5, 5 mS/cm；

C：PB+NaCl, pH7.0, 7 mS/cm；

D：PB+NaCl, pH7.5, 7 mS/cm；

E：PB+NaCl, pH7.0, 9 mS/cm；

F：PB+NaCl, pH7.5, 9 mS/cm.

Hydrophobic Chromatography Buffer

G：PB, pH7.0；

H：PB, pH7.5；

I：PB, 0.5 M (NH4)2SO4, pH7.0；

J：PB, 0.5 M (NH4)2SO4, pH7.5；

K：PB, 1 M (NH4)2SO4, pH7.0；

L：PB, 1 M (NH4)2SO4, pH7.5；

M：PB, 1.5 M (NH4)2SO4, pH7.0；

N：PB, 1.5 M (NH4)2SO4, pH7.5.

Through the separation effect and process control parameters of cation chromatography, composite chromatography and hydrophobic chromatography, the appropriate chromatography combination strategy was selected, and finally the buffer solution and binding/elution mode were optimized to determine the best purification process of protein purification.

Conclusion

Process development for high-throughput platform significantly improves the efficiency and reliability of biological purification processes through systematic screening strategies and data analysis. The strategies and cases in this article can provide reference for the process development of similar projects and help achieve efficient and low-cost industrial production.