AKTA pure micro chromatography system - Cytiva (2024)

Introduction

Monoclonal antibodies (mAbs) have transformed modern medicine over the past 25 years, revolutionizing treatments and potential cures of diseases including cancer and autoimmune disorders. However, ensuring safety, efficacy, and consistency of these complex molecules is a significant challenge. Tools to assist in identifying critical quality attributes (CQA) are crucial.

The journey towards obtaining an approved biotherapeutic, like a mAb, is lengthy and complex. These large proteins undergo extensive engineering to achieve desired characteristics such as high specificity for their targets, low toxicity, and favorable pharmacokinetics. They also need to be manufacturable, meaning they must be purified to homogeneity and remain soluble and stable throughout purification, manufacturing, storage, and transport until they reach the patient.

Unfortunately, during the manufacturing process, mAbs often undergo chemical and enzymatic post-translational modifications (PTMs), resulting in a population of variants that differ in size, charge, and hydrophobicity. These modifications can impact stability, immunogenicity, solubility, binding affinity, and aggregate formation of your biotherapeutic.

The role of analytical chromatography

Researchers use analytical chromatography to obtain valuable insights into the characteristics of mAbs, including purity, stability, and structural integrity. This information is crucial for optimizing production, purification, and storage processes.

One significant concern in development and manufacturing of therapeutic proteins, such as mAbs, is aggregation. Aggregates can negatively impact stability, shelf life, and efficacy of the product. They can also potentially create immunogenicity and safety risks for patients. By monitoring aggregation levels throughout the manufacturing process, scientists can take corrective steps to optimize production and minimize aggregate formation. Size exclusion chromatography (SEC) is a technique that is used to quantify and characterize mAb aggregates. It allows for the separation and measurement of aggregates within a sample based on their size.

Another important aspect to consider is charge variants, also known as charge heterogeneity. This refers to the presence of different charge variants within a mAb sample. Charge variants may result from post-translational modifications like deamidation, oxidation, glycation, or C-terminal lysine clipping. These charged variants can significantly influence mAb stability, pharmacokinetics, and immunogenicity—underscoring the need for their thorough characterization. Ion exchange chromatography (IEX) exploits the difference in net charge among mAb variants, allowing for separation and quantification. This information is critical to ensure consistency across batches and optimizing formulation strategies.

In this study, we established a laboratory-scale process to assess the critical quality attributes (CQAs)of a mAb expressed in CHO cells. The mAb was purified in two steps, affinity chromatography using MabSelect PrismA™ resin and IEX using Capto™ S ImpAct resin. Analytical chromatography was performed after each purification step, to guide fraction pooling (Fig 1).

Fig 1. Flow scheme of the purification process of a mAb and the analysis with analytical chromatography. Aggregation was assessed after affinity purification using analytical SEC. The affinity purified mAb was further purified using IEX and fractions were run on both analytical SEC and IEX to estimate aggregate content and charged variants. Fractions corresponding to lowest content of aggregation and main mAb variants were pooled. Final pool was analyzed with both analytical SEC and IEX.

Analytical SEC

Fractions from the affinity purification were neutralized and run on analytical SEC using the ÄKTA pure micro system (Fig 2). ÄKTA pure™ micro system is developed to have low hold up volume flow-paths and is well suited for both preparatory and analytical chromatography. Samples were injected with the ALIAS™ bio autosampler intended for microscale and analytical applications.

Fig 2. Analytical SEC for aggregate analysis on Superdex™ 200 Increase 3.2/300 column using ÄKTA pure™ micro system and the ALIAS™ autosampler. *The column was pre-equilibrated with 1.5 CV before the first run.

The aggregate content was determined by integrating the peak area using UNICORN™ software. Analyzed fractions after affinity purification were determined to contain 1-2% aggregates (Fig 3).

Fig 3. a) Chromatogram showing the affinity purification of the mAb. b) Four fractions from the affinity chromatography, representing the start, mid and end of the peak were run on analytical SEC using the ÄKTA pure™ micro system. Peak area integration suggested an aggregate content between 1-2% for each fraction in the main peak. c) Enlarged image of analytical SEC chromatogram. The minor peak preceding the main peak in each chromatogram corresponds to mAb aggregates. d) Aggregate content (%) shown for each fraction in the peak of the affinity chromatogram.

Analytical IEX chromatography

Affinity chromatography fractions collected between 85-93 mL were pooled and purified on a Tricorn™ 10/100 column packed with 8 ml Capto™ S ImpAct resin. The shape of the main IEX peak indicated the presence of charged variants and aggregates (Fig 4).

To evaluate aggregate content, samples from every other fraction covering this peak were analyzed with analytical SEC (Fig 4). Peaks were integrated and aggregate content was estimated to < 2% for fractions collected between 110 to 125 mL.

Fig 4. a) The shape of the chromatogram showing the IEX purification indicated the presence of charged variants and fragments. b) Selected fractions from the IEX purification were analyzed with analytical SEC. c) Aggregate (gray) and fragment (yellow) content (%) across the peak.

Prior to analytical IEX, 50 µl of each fraction was desalted using Amersham™ MicroSpin™ G-25 columns and diluted 1:1 in buffer A. 50 µl desalted and diluted sample were analyzed with high-resolution IEX using Capto™ HiRes S 5/50 column with a purification protocol using both step and linear gradient (Fig 5).

Fig 5. Protocol for analytical IEX for analysis of charged variants on Capto™ HiRes S 5/50 column using the ÄKTA pure™ micro system and ALIAS™ autosampler.

Peaks, corresponding to fragmented, acidic, main, and basic antibody variants (Fig 6), were integrated manually and peak areas were calculated.

Fig 6. a) Chromatogram showing the IEX purification. b) Three fractions from the main peak after IEX were analyzed. Based on previous data (not shown) this peak usually consists of fragments, acidic, main peak, and basic variants. c) The distribution and amount (%) of these variants (aggregates in blue, acidic in green and basic in yellow) is shown in the bottom chromatogram.

Combining the information on aggregate content, charge variants and main peak concentration, resulted in a more focused and narrower pooling strategy than relying solely on aggregate content analysis. The fractions collected between 115-125 ml were pooled and stored for further use. This final pool was analyzed with analytical SEC and IEX as previously described (Fig 7).

Fig 7. a) Chromatogram showing the IEX purification. b) Three fractions from the main peak after IEX were analyzed. Based on previous data (not shown) this peak usually consists of fragments, acidic, main peak, and basic variants. c) The distribution and amount (%) of these variants (aggregates in blue, acidic in green and basic in yellow) is shown in the bottom chromatogram.

Summary and Conclusion

• A highly expressed monoclonal antibody (mAb) was purified from CHO supernatant using affinity chromatography and ion exchange chromatography on an ÄKTA pure™ 25 M system.
• After each purification step, 10 µl of neutralized fractions were run on analytical SEC on a Superdex 200 Increase 3.2/300 column using the ÄKTA pure™ micro system to determine aggregate content.
• After preparative ion exchange chromatography, 50 µl of desalted and diluted fractions were subjected to analytical IEX on a Capto™ HiRes S 5/50 column using the ÄKTA pure™ micro system to assess the mixed population of charged variants.
• By combining the information obtained from analytical SEC and IEX, a purified sample with minimal levels of aggregates, fragments, and charged variants was obtained.
• This study demonstrates the use of the ÄKTA pure micro™ system combined with the ALIAS™ bio autosampler to evaluate CQA for a mAb.

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ÄKTA pure, ALIAS, Amersham, Capto, MabSelect PrismA, MicroSpin, Superdex, Tricorn, and UNICORN are trademarks of Global Life Sciences Solutions USA LLC or an affiliate doing business as Cytiva.

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