An Automated Two-Column Antibody Purification Method with Modifications for Inline Neutralization

Antibody-based therapeutics, such as monoclonal antibodies (mAbs), have transformed the outcomes for many patients by offering new and effective treatments for numerous diseases, from cancer to infectious diseases and a broad range of inflammatory and autoimmune diseases. The benefits of these therapies include high specificity, affinity, potency, and long in vivo half-life. Many therapeutic antibodies are biologic drugs; drugs made in living cells in a controlled way. However, their complex structures and production processes make them expensive to produce, and they can cost over 22 times that of alternative small molecule drugs, increasing the pressure on healthcare systems and limiting patient access.

Biosimilars are chemically synthesized, structurally similar alternatives to biologics that produce the same therapeutic function. Designed to shift the balance within drug discovery and development from effective yet expensive biologics towards making more accessible therapies, biosimilars offer an innovative and more affordable approach to treatment.

Despite being a comparatively low-cost alternative to traditional biologics, the development and manufacture of biosimilars can still be complex and costly. Recent innovations in biosimilars research and a rise in their clinical application have resulted in a growing demand for the development of more efficient, affordable, and flexible solutions for biosimilar production, as well as for more traditional therapeutic antibodies during discovery research. A large portion of these production costs are taken up in purification processing.

Purification plays a critical role during the production of biosimilars and biologics to achieve product purity and often involves multiple steps, including capture, intermediate, and polishing column chromatography, ultrafiltration, and diafiltration. Purification also contributes to ensuring the safety of therapeutic antibodies by avoiding any adverse effects from impurities. Purification plays a similarly important step in research laboratories looking to produce pure antibodies at the tens-of-milligrams scale for further study and development. Consequently, there is considerable interest in improving the efficiency of protein purification processes both at research- and production-scale.  

Protein purification and the challenges to overcome

At production scale, the purification process for biologics and biosimilars commonly begins with a protein capture step, which typically involves a Protein A or G column, followed by a series of polishing steps to remove any remaining product and process-related impurities. For mAb purification in research and discovery, however, the standard method only uses one polishing step; size exclusion chromatography (SEC). In this approach, the captured antibody is washed and eluted by a low pH buffer and fractionated. The fractions with the monodispersed target biologic or biosimilar then require neutralization to a physiologically appropriate pH for stability and to maintain their native properties. The sample may then require concentration prior to loading onto an SEC column. The SEC column separates the monomeric fraction into the final buffer of choice.

Although robust and highly effective, this approach still faces critical pain points. For example, while Protein A resins allow for high target specificity and affinity, resulting in high-purity end products, they are associated with high production costs. Consequently, there is also a demand for low-cost alternatives to Protein A for more cost-effective biosimilar production. In addition, limitations and inefficiencies in the antibody purification workflow are introduced by steps that require an acidic elution step, such as recovery of the bound antibody from the affinity matrix. This sudden drop in pH is associated with aggregate formation due to changes in the physiochemical properties of the product, which results in product loss and has implications for the safety and purity of the final product.

In manufacturing, the affinity elution pool is often held at an acidic pH for viral inactivation. However, this can result in an increase of aggregates requiring removal before the biosimilar is generated in its final therapeutic format. While advantageous at the industry level, where production scales are large, research laboratories producing small quantities of biosimilars have a greater need for the development of rapid purification techniques that can minimize aggregation and isolate the prominent monomer fraction from aggregates.

Another critical pain point in current purification processes is that cumulative hands-on processing and manual intervention increase the potential for inconsistencies in antibody purity and yield. These issues become particularly prevalent when researchers are processing multiple antibodies. Consequently, this necessitates the need for a flexible and automated protocol that integrates pH neutralization and downstream aggregate removal into the purification process to minimize the effect of the acidic elution step, improving antibody yield and consistency while freeing up researchers’ time.

Modifying two-column tandem purification to streamline biosimilar purification process development

Modification of traditional two-column tandem purification offers researchers a solution to the formation of aggregates during the acidic elution step. This can be achieved through the integration of an inline neutralization step by adding additional valves and accessories to a researcher’s base chromatography system. The ability to automate two-column tandem purification workflows supports a hands-off approach for researchers and permits the reproducible production of high-quality antibodies at the tens-of-milligrams scale. This helps overcome inconsistencies in antibody yield and purity between day-to-day runs and among operators. Streamlining the entire process into a single automated workflow also has important implications for overall costs, which are considerably reduced, while also reducing the labor intensity of the process.

     

One such modification of the traditional two-column purification setup combines affinity purification, pH neutralization, and SEC in a single automated chromatographic run, minimizing the exposure of antibodies to acidic pH. This approach was adopted to achieve optimal purification efficiency for a biosimilar of the biologic drug trastuzumab.

The workflow begins with an antibody affinity capture and elution purification step and is followed by rapid inline neutralization of the eluate before finishing with a polishing step to isolate the monomeric fraction from antibody aggregates using SEC (Figure 1). As demonstrated in this workflow, the configuration of the tubing on the base chromatography system allows simultaneous elution from the column used for affinity purification and subsequent inline pH neutralization prior to loading on the SEC column (Figure 2). The system used was modified using a low mixing volume Y-mixer to deliver the neutralization buffer (1 M Tris, pH 8.0), allowing the eluate to be neutralized and filtered immediately, prior to loading onto the SEC column to separate antibody monomers from fragments and oligomeric aggregates. The final amount of purified biosimilar recovered was 30 mg from a sample loading volume of 500 ml of trastuzumab-containing supernatant. This workflow allowed the entire two-column tandem purification process to be completed within five hours and yielded pure monomeric antibodies suitable for use at the research level.

To enable automation of multiple antibody purification runs, for example, an additional inlet valve can be placed before the sample pump, enabling the sequential processing of multiple samples while permitting automated washing of the sample pump after each sample is introduced. Incorporating multi-sample automation increases throughput to increase the amount of final product generated for further study and development.

A streamlined, automated solution for research-scale antibody purification

In line with the rise of antibody-based therapeutics, there is a growing demand for the development of more efficient and affordable solutions for the purification of therapeutic antibodies used in drug discovery research. Chromatography systems that are easily adaptable and can be configured to enable automated and high-throughput processing allow for the production of consistent and reproducible pure monomeric antibodies, improving the efficiency of the process while driving down the cost of purification. Similarly, modifications enable inline neutralizations to overcome common inefficiencies associated with traditional purification methods to streamline biologic and biosimilar process development workflows.