When it comes to complex downstream purification challenges, time spent optimizing mixed-mode chromatography (MMC) results in a cost-effective and efficient solution that scales with your bioprocess.

By Chris Sadler

The 1950s and 1960s were arguably great times to be a bioseparation nerd. Pioneers like Peterson, Sobers, Porath, Flodin, Hjertén, Axén, Cuatrecasas, and Sjöqvist were all busy changing the game for those of us who followed–not least with the development and optimization of Protein A-based affinity chromatography. But that’s not to say bioseparation science has been sitting on its hands ever since. 

Next-generation Protein A products brought higher caustic stability, higher binding capacities, and improved base matrix characteristics. But the constant balancing act of cost, efficiency, and selectivity has played out in myriad ways. 

In more recent years, we’ve seen the emergence of newer technologies like mixed-mode chromatography products (see Footnote: Mixed mode 101), which sit between traditional ion exchange or hydrophobic interaction chromatography media and affinity   absorbents. In short, mixed-mode chromatography offers either cost-effective target selectivity or, in the case of flow-through operation, more efficient removal of complex impurities.

One person’s affinity chromatography is another person’s mixed-mode treasure 

Given Astrea Bioseparations’ successful history of using its proprietary triazine scaffold technology to develop custom affinity adsorbents for specific targets within defined bioseparation steps, we found ourselves with an increasing number of interesting but highly targeted resins. Could these products, similar to drug repurposing, have multiple applications hidden within their small-molecule chemistry?

The answer to that question lay in a science “sandpit.” And we’ve very much enjoyed digging around to discover that these high-performing custom adsorbents do indeed have utility in often unexpected places, for completely different targets and in other positions within purification workflows.

The MiMode™ product family was born.

MiMode™: Your advantage

I’d argue that the defining advantage of the MiMode™ product family is the flexibility or “tunability” it affords when tackling complex purification challenges, from stubborn host cell protein distributions to complex aggregates. The ability to screen against an array of mixed-mode products,  each of which occupies a different physico-chemical space,  under a range of chromatographic conditions, makes purification puzzles a lot less daunting.

For example, bispecific antibodies represent a significant purification challenge. Unlike “vanilla” monoclonal antibodies, which benefit from a standard platform approach to purification, bispecifics often result in awkward impurities (mispaired species, diverse aggregates) and demand bespoke solutions. Notably, Protein A captures all species with Fc regions and not just the target molecule.

With bispecifics, and with other complex mixtures, the tunability of mixed-mode chromatography,  and, specifically, MiMode™ products,  really comes into its own. By screening for the best MiMode™ product and manipulating parameters like pH, conductivity, lyotropic character, buffer ionic strength, you can selectively target complex impurities in a way that is simply not possible with a standard affinity interaction or traditional ion exchange or hydrophobic interaction chromatography.

To make screening for MiMode™ products and parameters more accessible, we’ve developed the MiMode™ PuraBead® 96-well plates in two configurations: i) multiple MiMode™ products on a single plate to interrogate the entire design space, ii) single product plates to further fine-tune process conditions or investigate different product formats.

In the background, we’re trying to generate as much data as we can to give developers a head start on product and parameter choice when it comes to challenges that present themselves more frequently. For example, we’ve recently been working on exploring the best product and parameters for host cell DNA removal from lentivirus vectors for the gene therapy developers. More broadly, we continue with more fun in the mixed-mode sandpit, uncovering new applications of the technology within different bioprocesses and expression systems. 

Ultimately, the exciting and fun thing about mixed-mode chromatography is that it’s extremely hard to predict what will work surprisingly well for a given target and process. We invite you to jump into the sandpit to find out for yourself! Just know that Astrea Bioseparations’ R&D and the Field Application Scientist (FAS) teams are both more than happy to help solve your particular purification puzzle.

Footnote: Mixed mode 101

When thinking about bioseparations in bioprocessing, the predominant modes of chromatography media can be grouped into two camps: i) the more “generic”: ion exchange (net surface charge-based separation), hydrophobic interaction (hydrophobicity-based separation), and size-exclusion chromatography (size- and shape-based separation), and ii) the highly specific: affinity chromatography.

Unsurprisingly, combining (mixing!) different generic modes–for example, cation exchange and hydrophobic interaction–within the same resin facilitates multiple interactions with target molecules or impurities (or a distribution of impurities). The result: increased selectivity and high caustic stability without the expense and headache of working with protein-based affinity products.

Welcome to mixed-mode (sometimes called multimodal) chromatography (MMC). 


Meet the author: Chris Sadler

I studied chemistry at university (hello, UEA alumni!), and my first science job after leaving university in 2005 was as a research assistant in the chemistry team at Prometic Bioseparations; 15 years later, the company rebranded to Astrea Bioseparations (referencing the Greek goddess of purity). 

I was initially drawn to Prometic because it combined synthetic chemistry with biomolecule purification. Though out my academic career, biochemistry including protein structure and engineering were topics I was particularly interested in. The position at Prometic was therefore a perfect fit for my interests then and now. My first “proper boss” was an experienced chemist called Stuart Jordan, who sadly passed away earlier this year (2025). I learned a great deal from him in the field of organic synthesis, small molecule purification and the art of brewing!

Over the years, I’ve grown and evolved with the company, working my way through various roles within the R&D team. But I’ve also gained a great understanding of the overall bioprocessing landscape thanks to colleagues in our DSP team and working closely with end users of our products. Today, I find myself in a broader role, overseeing R&D, new product development, and applications projects for Astrea Bioseparations’ entire resin portfolio.