Solving Key Challenges in (Bio)pharmaceutical Analyses

Common challenges in (bio)pharmaceutical analyses include ionic interactions, metal contamination, and analyte loss often observed with conventional stainless-steel high performance liquid chromatography (HPLC) columns. These can be overcome by the use of bioinert column hardware, which can be utilized across a wide range of applications, from oligonucleotide and lipidomic analyses to antibody and small-molecule studies. By reducing analyte interaction with the column hardware, recovery can be improved, peak tailing reduced, and compatibility with native mass spectrometry (MS) conditions ensured. This article will discuss several practical examples using bioinert columns, demonstrating their ability to deliver reliable, high-resolution results with precision and reproducibility.

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The number of bioanalytical applications continue to grow and along with them interest in (bio)inert high performance liquid chromatography (HPLC) columns. Conventional ultrahigh-pressure liquid chromatography (UHPLC) columns are made of stainless steel, providing mechanical resilience and compatibility with most solvents. However, eluents such as methanol and acetonitrile can cause corrosion and metal erosion. The resulting positively charged surface can lead to undesired ionic interactions with electron-rich analytes (1). These interactions compromise analytical results by causing non-specific adsorption, resulting in low recovery, peak broadening, and carryover, particularly under low to neutral pH conditions, as metals are more electropositive under these conditions (2).

To reduce adsorption on metal surfaces, several workflows can be applied as alternatives. A common method is to passivate the system and column with an overnight flush using 0.5% phosphoric acid in 90:10 acetonitrile—water (3). Another approach is to passivate the system with a treatment using an ethylenediaminetetraacetic acid (EDTA) solution (4). However, the durability of these procedures is limited and requires regular repetition. Passivation can last anywhere from a few months to only a few hours.

If the column is not stable under passivation processes, selected additives in the mobile phase or sample diluent can mask the active sites. Commonly used additives include phosphoric acid, citric acid, or EDTA (5). Nevertheless, these additives can adversely affect detection with mass spectrometry (MS) because they are not volatile or can lead to ion suppression.

Another effective treatment to prevent ionic interactions with metal surfaces is to condition the system, including the column, with the target substance or a similar compound. In this case, the metal surface is saturated with the substance, preventing further adsorption. While effective for some analytes, this method is not a permanent solution. The effect can diminish as a result of sample changes; for example, longer oligonucleotides interact more strongly with the metal surface (6). In addition, direct comparisons of pre-conditioned stainless‑steel columns with bioinert coated columns often still show a reduced peak area and therefore reduced recovery.

Using a bioinert (U)HPLC system and column is therefore advantageous. Since the column body and frits represent more than 70% of the surface in contact with the analytes (5), significant improvements can be achieved by using bioinert column hardware.

Alongside PEEK column hardware, several more pressure-stable concepts are available: a bioinert coating of the stainless-steel column body and frits, PEEK-lined stainless-steel columns combined with PEEK frits, and columns applying titanium.

PEEK and PEEK-lined hardware are widely used in bioanalytical applications but have limitations. They are non-compatible with certain organic solvents and lack the mechanical stability of stainless steel. Further, special connectors are required when working with PEEK or PEEK-lined columns. PEEK is also quite hydrophobic, which can lead to further interactions.

Titanium-lined column hardware is known for its biocompatibility; however, it is not bioinert because metal erosion can still occur, resulting in contamination of the silica bed. The eluted titanium is capable of causing ionic bonding with free, remaining silanol groups, leading to the adsorption of analytes onto the stationary phase. This effect is particularly pronounced for stationary phases without or with insufficient endcapping (7).

A bioinert coated stainless-steel column is resistant to commonly used solvents while maintaining the mechanical resilience of conventional columns. However, differences exist between available bioinert coated stainless-steel columns. Variations in coating composition and thickness,
often not fully disclosed by manufacturers, can significantly affect the quality of analytical results.

Bioinert columns can be used for a wide range of applications, such as oligonucleotides, different lipid classes, antibodies, and small coordinating compounds, utilizing HPLC modes like reversed-phase (RP), ion exchange (IEX), hydrophilic interaction (HILIC), or size‑exclusion chromatography (SEC).

These analytes have electron-rich or coordinating moieties that can interact with the metal surface or contain metal-induced oxidizable parts. Specific examples of these substances and their applications will be detailed in the following sections.

Oligonucleotide Analysis

One of the best known applications of bioinert columns lies in the analysis of oligonucleotides. Their electron‑rich backbone is prone to irreversible adsorption on metal surfaces, leading to low recovery and significant peak tailing.

Previous ion-pair reversed-phase liquid chromatography (IP-RPLC) studies with phosphorothioated RNA have shown that peak area and height can be up to twice as high when using a bioinert column compared to a regular stainless-steel column (complete results can be found in reference 8). Alongside selecting the appropriate ion-pairing agent, using bioinert column hardware is a key factor for achieving sharp peaks.

If the use of ion-pairing agents must be avoided, Hayashi and Sun have described a RPLC–MS method without the use of ion pairing agents. In this method, ammonium bicarbonate was used as an additive in the mobile phase, and a bioinert coated C18 column was employed to analyze several commercially available oligonucleotides with high resolution and sensitivity (9).

Alternatively, HILIC can be used to avoid ion-pairing agents. Similar results regarding column hardware can be observed using this technique. Figure 1 shows a comparison of a stainless‑steel column, a PEEK-lined column, and
a bioinert coated column, all packed with the same stationary phase and in the same dimensions. In addition to the short DNA oligonucleotides shown (dT15–dT35), longer DNA and short RNA oligonucleotides (dT40–100, rU15–30) were analyzed with similar results (data not shown). The stainless-steel column exhibited significant tailing and reduced peak height and area. Using
the PEEK‑lined column improved the results, but the highest recovery and sharpest peaks were achieved with the bioinert coated column, even after conditioning.

Unlike IP-RP, bioinert columns require some conditioning in HILIC mode, though the cause is not yet known (6). While 20 injections were necessary to condition the stainless-steel column and 14 for the PEEK‑lined column, the bioinert coated column was pre-conditioned after only eight injections, with minimal differences (less than 10%) between the initial and final peak areas (10).

Bioinert coated column hardware is also applied in ion exchange chromatography, specifically anion exchange chromatography (AEX), for oligonucleotide analysis. In AEX methods, PEEK column hardware is often used due to its bioinert properties. However, using less hydrophobic bioinert coated column hardware can positively affect recovery.

A comparison study has shown that a bioinert coated column could be used immediately with the first injection, whereas a PEEK column required some conditioning, likely because of hydrophobic interactions with the PEEK hardware (data not shown, 11).

Lipidomic Applications

Various lipid markers contain phosphate groups, which can cause significant tailing and low recovery when in contact with metal surfaces. Using a bioinert column can notably improve these results. Rubenzucker et al. (12) demonstrated this phenomenon by comparing conventional stainless-steel, PEEK-lined, and bioinert coated columns for the comprehensive analysis of signaling lipids. By using the bioinert-coated column, long-term reproducible results across different matrices were achieved.

A fast lipidomic analysis in under 10 min is possible using a bioinert coated C8 column (Figure 2). A well-defined separation between classes of lysolipids, phospholipids, and triglycerides—typical for reversed-phase lipidomic analysis—can be achieved.

Even with the less hydrophobic C8 modification, molecular species of complex lipids can be effectively separated in a fast run, as demonstrated by a total ion chromatogram (TIC) (Figure 2). The bioinert-coated hardware ensures stable recovery for routine analyses. During this fast analysis, approximately 700 distinct molecules can be reliably identified, depending on the MS/MS performance of the spectrometer.

Bioinert Column Hardware in Antibody Analyses

Although antibodies are not typically the focus of bioinert column hardware, there are applications where its use is beneficial. For example, Roche developed a native IEX-MS method for the analysis of intact antibodies using a bioinert coated cation exchange column and ammonium acetate solutions containing 2% acetonitrile for gradient elution. The starting gradient was 0–100%B over 55 min but was adjusted for specific monoclonal antibodies (mAbs). For instance, a gradient of 47–58% B performed best for trastuzumab (Figure 3). Using a smaller internal diameter of 2.1 mm, which is typically not available for PEEK columns, helped to reduce the flow required for detection with nano‑electrospray ionization MS.

When using SEC, especially in combination with MS detection, bioinert column hardware is required to mitigate interactions between the column hardware and mAbs, antibody–drug conjugates (ADCs), and their fragments
or nanobodies. Figure 4 demonstrates the excellent separation of a single‑chain variable fragment (scFv) and its conjugated species (FDC) under MS conditions, using a PEEK-lined column with diol modification and a 12 nm pore size.

The Role of Bioinert Hardware for Small Molecules

Various small molecules with coordinating characteristics require metal-free analysis hardware. Examples include mycotoxins such as fumonisins, chelating compounds like hinokitiol, and several metabolites involved in tryptophan metabolism (13). Consequently, using bioinert hardware is advisable for applications such as non-targeted metabolite screening. Figure 5 illustrates the screening of polar metabolites in human plasma under HILIC conditions. The method covered a wide range of polar compounds with excellent peak capacity (average baseline peak width of 0.25 min). Additionally, it resolved important critical pairs such as leucine and isoleucine, as well as asymmetric and symmetric dimethylarginine (ADMA and SDMA, respectively). Combined with high sensitivity, this method ensured the reliable generation of biological hypotheses.

Summary

Stainless-steel columns can cause non‑specific adsorption of various substance classes, resulting in low recovery, peak tailing, and carryover. Passivating or pre-conditioning the system is only temporarily effective, making the use of bioinert columns and systems the best solution. Substances such as oligonucleotides, lipids, proteins, peptides, mAbs, and various small molecules can be affected by metal surfaces.

This article presents several application examples where bioinert columns are used to provide sharper peaks and higher recoveries. Furthermore, bioinert hardware is valuable for different HPLC modes, particularly when native MS conditions are required (for example, SEC).

Acknowledgment

Data courtesy of Institute of Pharmaceutical Sciences of Western Switzerland (University of Geneva), Switzerland (Figure 1, 2, 5), Roche Diagnostics, Germany (Figure 3), and Antikor, UK (Figure 4).

References

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(2) Guimaraes, G. J.; Bartlett, M. G. The Critical Role of Mobile Phase pH in the Performance of Oligonucleotide Ion-Pair Liquid Chromatography–Mass Spectrometry Methods. Futur. Sci. OA. 2021, 7 (10).
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(7) De Pra, M.; Greco, G.; Krajewski, M. P.; et al. Effects of Titanium Contamination Caused by Iron-Free High Performance Liquid Chromatography Systems on Peak Shape and Retention of Drugs with Chelating Properties. J. Chromatogr. A 2020, 1611, 460619.
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(8) Eßer, D.; Rojahn, A. M.Improving Chromatographic Results for Oligonucleotides with Column Hardware. The Column 2023, 19 (2), 2–8.

(9) Hayashi, Y; Sun, Y. Overcoming Challenges in Oligonucleotide Therapeutics Analysis: A Novel Nonion Pair Approach. J. Am. Soc. Mass Spectrom. 2024, 35 (9), 2034–2037. DOI: 10.1021/jasms.4c00270

(10) YMC, HILIC Analysis of Oligonucleotides Using Different Bioinert
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(11) YMC, The Supremacy of Bioinert Coated Hardware in Oligonucleotide Analysis Using YMC Accura BioPro IEX QF, YMC Technical Note, 2024.

(12) Rubenzucker, S.; Manke, M.-C.; Lehmann, R.; et al. A Targeted, Bioinert LC−MS/MS Method for Sensitive, Comprehensive Analysis of Signaling Lipids. Anal. Chem. 2024, 96 (23), 9643–9652. DOI: 10.1021/acs.analchem.4c01388

(13) Eßer, D.; Rakotondravao, M.; Higuchi, Y.; et al. Enhanced Analysis of Coordinating Compounds in Tryptophan Metabolism Using Bioinert Coated Hardware in HPLC. Poster presented at the 34th International Symposium on Chromatography Conference, Liverpool, United Kingdom, 2024.

Ann Marie Rojahn studied chemistry and biotechnology in combination with an apprenticeship as a chemical laboratory assistant at the University of Applied Sciences in Krefeld, Germany. After receiving a bachelor’s degree, she pursued a master’s programme in chemistry at the University of Düsseldorf, Germany, with a focus on organic chemistry. Since 2019, she has worked for YMC Europe in Dinslaken as a product specialist in the analytical chromatography team.

Daniel Eßer studied chemistry at the University of Applied Sciences Bonn Rhein-Sieg, in Rheinbach, Germany, with a focus on pharmaceutical and analytical chemistry. He received his PhD in pharmaceutical and medicinal chemistry at the University of Düsseldorf, Germany. During his postdoc at the Institute of Pharmaceutical and Medicinal Chemistry of the University of Düsseldorf, he established a nanoLC–MS system. In 2013 he joined YMC Europe in Dinslaken, Germany, as a product specialist for analytical chromatography. Since 2017, he has been responsible for YMC’s analytical (U)HPLC column portfolio as the product manager for analytical chromatography.

Direct correspondence to: a.rojahn@ymc.eu