Bioconjugation, i.e., coupling biomolecules with (usually) small molecules, is of increasing interest for the pharmaceutical industry, since it enables designing molecules with special, highly interesting properties. A well-known example of bioconjugates are antibody-drug conjugates (ADCs), which are used, e.g., for the treatment of cancer (Chau et al., 2019, Khongorzul et al., 2019, Perez et al., 2014, Sievers and Senter, 2013). Binding anticancer drugs to a specific antibody thereby allows a targeted release of the drug into the tumor cells only, while healthy cells remain unharmed (Flygare et al., 2012), which strongly reduces side effects. This is achieved by the specific affinity of the antibody, which binds to tumor cells only. Drugs may also be coupled with other biomolecules than antibodies. For instance, low molecular weight proteins, like lysozyme, are used as drug carriers in the treatment of renal-specific diseases (Haas et al., 1997, Haselberg et al., 2011, Wang et al., 2013), since lysozyme selectively accumulates in proximal tubular cells (Zhou et al., 2014), which allows a reduction in the required drug dose and extrarenal toxicity (Haverdings et al., 2001).

A further application of bioconjugation is the monitoring of the behavior of proteins by fluorescent labeling, which is an established technique in different areas of life sciences. It is, e.g., used for detecting bacteria in medical applications (Arabski et al., 2015, Zheng et al., 2016). A common fluorescing molecule used for such applications is fluorescein isothiocyanate (FITC). The lysozyme-FITC conjugates that are studied in the present work are not only directly relevant in life science (Sugahara et al., 2000, Takano et al., 2004, Zheng et al., 2016) but they can also be considered as a convenient, non-toxic proxy for lysozyme-drug conjugates or even ADCs, as the size, structure, and hydrophobicity of FITC are similar to the respective properties of many common drugs, which are usually highly toxic and therefore challenging to handle (Arakawa et al., 2016, Müller et al., 2020).

While downstream processing of biomolecules is basically always demanding, special challenges arise when bioconjugates have to be separated, as usually multiple different bioconjugates are obtained during the synthesis, which subsequently have to be separated. The synthesized bioconjugates may differ in the number of (small) molecules that are bound to the biomolecule, and in the positions where this binding takes place (Sadiki et al., 2020). This can have a significant impact on the physiological properties of the bioconjugates (Strop et al., 2013). For instance, only some of the conjugates obtained in ADC synthesis meet the drug efficacy and safety requirements and are, hence, of interest for pharmaceutical applications (Matsuda, 2021), so that they have to be separated from the other ADC conjugates. The separation of bioconjugates is, however, hampered by the fact that the molecular size and physico-chemical properties of the synthesized bioconjugates are often very similar. Hence, purifying bioconjugates is an important, but challenging task.

An established technique for the purification of biomolecules is hydrophobic interaction chromatography (HIC). It has, e.g., been proposed for the purification of ADCs (Matsuda et al., 2020, Rodriguez-Aller et al., 2016) and lysozyme-drug conjugates (Goszczyński et al., 2017). HIC can be carried out at mild, and thus non-denaturing, conditions (Fekete et al., 2016), but its performance depends on many influencing parameters, such as salt type, salt concentration, and pH value, as well as on the temperature. While there are many studies in the literature on the influence of salts in HIC of biomolecules (for an overview see, e.g., (Lienqueo et al., 2007)), the adsorption behavior of biomolecules can significantly change if small molecules are bound to them (Bingaman et al., 2003, Romanowska et al., 2015). Romanowska et al. (2015) have studied the adsorption of lysozyme-FITC conjugates on charged surfaces with docking simulations and Teske et al. (2005) have shown that the conjugation of lysozyme can have an influence on the adsorption in HIC. However, systematic studies of adsorption isotherms of lysozyme-FITC conjugates are still missing and the same holds for models describing the adsorption of lysozyme-FITC conjugates.

Therefore, in this work, the adsorption of lysozyme-FITC conjugates on the hydrophobic resin Toyopearl PPG-600 M was studied in batch adsorption experiments, which were carried out at pH 7.0 and 25 °C. Equilibrium adsorption isotherms were investigated for solutions containing either sodium chloride or ammonium sulfate with ionic strengths up to 2000 mM and compared to the corresponding results of lysozyme. Lysozyme-FITC conjugates cannot be purchased in pure form. Therefore, the studied lysozyme-FITC conjugates were synthesized and separated in the present work. The separation was achieved by HIC, i.e., by the process on which we want to learn more with our study. Furthermore, a mathematical model was developed in order to describe the equilibrium adsorption isotherms of lysozyme-FITC conjugates on the studied HIC material. In the following, for brevity, the term ‘protein’ is used both for lysozyme and the lysozyme-FITC conjugates, when a distinction is not needed.