Proteome wide evaluation of the separation ability of hydrophobic interaction chromatography by fluorescent dye binding analysis

Abstract

Hydrophobic interaction chromatography (HIC) is an important tool in the industrial purification of proteins from various sources. The HIC separation behavior of individual (or model) proteins has been widely researched by others. On the contrary, this study focused on the fractionation ability of HIC when it is challenged with whole proteomes. The impact of the nature of three different proteomes, that is, yeast, soybean, and Chinese hamster ovary cells, on HIC separation was investigated. In doing so, chromatography fractions obtained under standardized conditions were evaluated in terms of their overall hydrophobicity--as measured by fluorescence dye binding. This technique allowed for the calculation of an average protein surface hydrophobicity (S(0)) for each fraction; a unique correlation between S(0) and the observed chromatographic behavior was established in each case. Following a similar strategy, the effect of three different ligands (polypropylene glycol, phenyl, and butyl) and two adsorbent particle sizes (65 and 100 µm) on the chromatographic behavior of the yeast proteome was evaluated. As expected, the superficial hydrophobicity of the proteins eluted is correlated with the salt concentration of its corresponding elution step. The findings reveled how--and in which extent--the type of ligand and the size of the beads actually influenced the fractionation of the complex biological mixture. Summarizing, the approach presented here can be instrumental to the study of the performance of chromatography adsorbents under conditions close to industrial practice and to the development of downstream processing strategies.

Synthesis of adsorbents with dendronic structures for protein hydrophobic interaction chromatography

“…As expected, the profile of proteins in each fraction is different suggesting that the proteins from the proteome from S. cerevisiae present different hydrophobicity and therefore can be separated according to this property. It should be noted that HIC fractionation of a yeast proteome using various commercial adsorbents with ligands with different hydrophobicity strength exhibited similar results as compared to RG3 [3]. Even more, with these results the practical capability of these novel dendron-based adsorbents is shown giving step to their incorporation in different processes once optimization of both synthesis and operation is achieved.…”

“…HIC is a powerful and widely used technique for purification of proteins [3,4]. It is a key methodology when purification of monoclonal antibodies [5][6][7] is required.…”

“…HIC relies on the interaction between the hydrophobic regions on the surface of the biomolecules and the ligands on the support under high concentration of salts i.e. ammonium sulfate or sodium chloride [3]. There are numerous commercially available adsorbents with different types of hydrophobic ligands i.e.…”

Biological nanoparticles: Relevance as novel target drug delivery systems and leading chromatographic isolation approaches

“…Protocols using poly(ethylene terephthalate) (PET) capillary‐channeled polymer (C‐CP) fibers in HIC have been developed with results comparable to the ones obtained with traditional methods like UC and commercial available kits [57]. Exosomes were successfully purified from culture milieu and human urine [57,79]. These fibers are melt‐extruded from commodity polymers (e.g., nylon 6, polypropylene, and poly(ethylene terephthalate) (polyester, PET)), having a unique cross‐sectional profile consisting of eight legs on the periphery.…”

“…Considering this, chromatographic techniques have emerged as a selective and highly accepted method for the purification of these biological nanoparticles and other naturally occurring nano-vesicles due to the multiple chemistries and geometries involved, besides the high purity and yields that can be obtained. In the last few years, other methods besides size-exclusion such as ionic-exchange, hydrophobicinteraction, and AC have been used for the isolation and purification of these types of nanoparticles all with their respective advantages and disadvantages (Table 1 [14,15,[56][57][58][59]). More recently, the use of monolithic chromatography has enhanced the efficiency of the protocols with remarkable results and additionally, it has even reduced the number of separation operations used in their purification (Table 2 [54,[60][61][62][63][64][65][66][67]).…”