It’s a craft many scientists learn early on in their careers: chromatography. Taken from two Greek words, chroma (color) and graphein (to write), the term initially described separation of pigments from a complex mixture. Contemporary chromatography, however, has progressed from crude science to a near art form. Yet as new drug modalities and delivery systems continue to evolve, fresh challenges arise, especially for bioprocessing.

Here at GEN, we’ve spoken with several companies that are pioneering next-generation approaches, and they’ve given us an update on the new variations and refinements they’ve been bringing to chromatography, a workhorse of the laboratory that is starting to look more like a thoroughbred.

For example, new multimodal chromatography methodologies for biomolecules combine various media into a single step to enhance efficiency of separation. Software is also evolving. More sophisticated scouting software can more rapidly and robustly optimize chromatographic conditions.

According to the experts that we consulted, the separation of complex biomolecules—always a tricky business—is being improved because instrumentation is starting to incorporate elements that remain bioinert over a wide range of pH and salt levels. However, the experts also sound a cautionary note: new drug modalities will require even more sophisticated solutions and closer cooperation between clients and providers.

salt concentration levels
At Rice University, the laboratory led by Christy Landes, PhD, studies how proteins behave on surfaces such as permeable membranes in chromatographic columns. For example, her team has discovered how salt concentrations can tune two distinct interactions between a nylon stationary phase support and a model protein, transferrin. This work suggests that a combination of experiment and simulation can be used to optimize separation conditions.

Solving complexity issues

One of the many challenges facing contemporary chromatography is “complexity,” states Diab Elmashni, global director, protein purification, Bio-Rad Laboratories. “Complexity is introduced from many sources,” he continues, “such as new and challenging molecules, new and ever-changing viruses and diseases, manufacturing scalability, and procurement of raw materials.”

To meet this challenge Bio-Rad introduced mixed-mode chromatography resins. According to Elmashni, these resins “offer unique separation properties and unparalleled selectivity and resolution for a variety of molecules.”

How does it work? Elmashni explains, “Multimodal chromatography combines the properties of anion or cation exchange media and metal affinity media, often enabling the separation of biomolecules that appear homogeneous using other chromatographic methods. By combining multiple modes of separation into a single step, the user gains efficiency in their processes such as reduced complexity and greater return on investment.”

Applications for the new technology are numerous. “Mixed-mode resins can be used in applications such as purification of enzymes, monoclonal and polyclonal antibodies of various classes, antibody fragments, and bispecific antibodies,” Elmashni points out. “They can also be utilized for efficient isolation and purification of viruses and virus-like particles (VLPs) and separation of supercoiled DNA from linear duplexes as well as single-stranded DNA from double-stranded DNA.”

Another advantage and use is for both up- and downstream biomanufacturing. “The mixed-mode resins are ideal for large-scale bioprocessing applications,” Elmashni asserts. “These media can be used at any stage in a purification process from initial capture to final polishing.”

The company continues to eye even more possible innovations for this approach. Elmashni summarizes, “The new resins recently launched and those in development focus on overcoming the complexity in new and challenging molecules. Our resins have risen to the challenges of the COVID-19 virus in that they are employed in the purification of proteins used in the manufacturing of COVID-19 vaccines and treatments.”

Jump-starting method development

One of the major challenges facing any chromatography analysis is accurately determining which conditions would provide the best results. Thus, the use of the method development solution can help to more quickly and robustly fine-tune the optimal conditions.

“The use of statistical software to arrive at the best solution for an experiment is not a new approach,” notes Nivesh K. Mittal, PhD, HPLC product manager, marketing, Shimadzu Scientific Instruments. “However, the data from chromatographic software had to be ported into statistical software, and the software had to be trained on identifying the best chromatographic parameters. This was time consuming and needed a high level of expertise from both a chromatographic as well as a statistical standpoint.”

To help solve that problem, the company developed LabSolutions MD (Method Development) software. It can seamlessly capture chromatographic data and design analysis methods, giving users the ability to identify the best chromatographic separation.

Shimadzu’s Method Development Solutions are comprised not only of LabSolutions MD software, but also of the Nexera Method Scouting System (MSS). “The MSS allows a user to scout up to 6 columns and 16 mobile phases, for a total of 96 methods tested in a short time automatically,” Mittal details. “But the question remains: How can a user automate the selection of the best method?”

The answer, Mittal says, is to use LabSolutions MD: “It automatically selects the best method by assessing something called the ‘Evaluation Value.’ The Evaluation Value is based on automated tracking of chromatographic parameters along with statistical calculations to provide the best results.”

Mittal stresses that the major advantage of this solution is its hybrid approach. “This reduces the expertise required on the part of the user and reduces the time required to transfer data from the chromatography software to the statistical evaluation software,” he relates. “Additionally, the time saved from not needing to teach the disjointed statistical software as to what is important is another major benefit.”

Biological molecule challenges

Compared with small molecules purified for pharmaceutical applications, biological molecules often require more harsh and complex conditions for chromatographic analyses. Biomolecules include proteins, carbohydrates, lipids, and nucleic acids. In the case of biomolecular drugs such as monoclonal antibodies (mAbs), antibody-drug conjugates, and mAb subunits, there are many levels of characterization required depending on their makeup, as well as on variables such as post-translational modifications and aggregation states.

More detailed comments on these challenges are offered by Ulrich Eberhardinger, PhD, product manager, strategic marketing, Agilent Technologies. “Solvents and reagents of these applications are often demanding and require inertness of instrumentation over a wide pH range and a tolerance against high salt concentrations,” he says. “Further, biomolecules such as nucleotides can stick to stainless-steel surfaces, which is the standard material in the liquid chromatography (LC) flow path.”

Agilent recently announced that it has launched new systems and upgraded existing systems to expand and enhance its next-generation chromatography portfolio. For example, Agilent has introduced the 1290 Infinity II Bio 2D-LC, and the company has upgraded the OpenLab CDS data system.

Eberhardinger asserts that Agilent’s two-dimensional liquid chromatography (2D-LC) technology represents an improvement over traditional approaches: “The 2D-LC technology offers superior resolution for co-eluting compounds like isomers and higher peak capacity for complex samples. While 2D-LC has been applied successfully to such measurements for a while, its complexity often limited the use to academic researchers. This new 1290 Infinity II system combines two ideally orthogonal measurements and can therefore increase separation efficiency, as well as simplify and automate multiple steps of sample preparation and analysis.”

Eberhardinger notes that the “Bio” in the name of the instrument stands for a biocompatible solution that uses MP35N as a noble material in the flow path. With this biocompatible nickel-cobalt alloy, potential corrosion from high-salt-containing buffers as well as protein modifications can be reduced to a minimum. He reiterates, “This is robust under demanding conditions and strongly reduces binding of precious samples to surfaces.”

Cornelia Vad, senior product manager, strategic marketing, notes that the overall system can incorporate the company’s InfinityLab 2D-LC Protein A Size Exclusion (ProtA-SEC) Kit to provide a complete workflow solution for mAbs. She explains, “Titer and aggregation as two critical quality attributes are determined in one run, which saves more than 80% of time, gives more reliable results, and minimizes the risk of mAb modification during Protein A purification.”

Eyeing the future

As therapeutic markets continue to shift toward biomolecule solutions, the challenges facing chromatographers shift as well. Brian Domanski, strategic collaborations scientist, pharmaceuticals, Shimadzu Scientific Instruments, provides an example: “Analytical chromatography of biomolecules highlights potential deficits to the traditional stainless-steel construction of HPLC and UHPLC systems, which can be prone to peak shape distortion or adsorption of target analytes, as well as corrosion from a high-salt mobile phase.”

Domanski says the company’s Nexera Bioinert XS system has helped solve that challenge by providing bioinert UHPLC performance, and with the Prominence Bioinert HPLC system, “offers a range of options for analyses such as mAb aggregation, adeno-associated virus titer, and oligonucleotide assay.”

Another issue confronting companies is the on-column resolution of biomolecules. “This is often difficult to obtain, and integration or purification of unresolved peaks is more typical,” Domanski elaborates. “To overcome this, a feature was developed to automatically divide designated peaks into multiple fractions, allowing the user to decide between maximizing purity or yield.

“When combined with unique software like our PDA Peak Deconvolution algorithm, co-eluting impurities can be spotted and excluded from recovery. The same software can be used in R&D to reveal unknown impurities or in quality control to enhance the accuracy of integration for peaks.”

Domanski also reflects on the future of the field: “With each new drug modality and delivery system, the needs of chromatography evolve, but the principal functions remain the same: analysis and purification.” Thus, he advises companies to continuously work with their customers and opinion leaders to provide solutions to current and emerging chromatography challenges.