Novel Bioconjugation Strategy Using Elevated Hydrostatic Pressure: A Case Study for the Site-Specific Attachment of Polyethylene Glycol (PEGylation) of Recombinant Human Ciliary Neurotrophic Factor

Abstract

In this paper, we reported a novel strategy for the site-specific attachment of polyethylene glycol (PEGylation) of proteins using elevated hydrostatic pressure. The process was similar to the conventional one except the reactor was under elevated hydrostatic pressure. The model protein was recombinant human ciliary neurotrophic factor (rhCNTF), and the reagent was monomethoxy-polyethylene glycol-maleimide (mPEG-MAL). PEGylation with mPEG (40 kDa)-MAL at pH 7.0 under normal pressure for 5 h achieved a less than 5% yield. In comparison, when the pressure was elevated, the PEGylation yield was increased dramatically, reaching nearly 90% at 250 MPa. Furthermore, the following phenomena were observed: (1) high-hydrostatic-pressure PEGylation (HHPP) could operate at a low reactant ratio of 1:1.2 (rhCNTF to mPEG-MAL), while the conventional process needs a much-higher ratio. (2) Short and long chains of PEG gave a similar yield of 90% in HHPP, while the conventional yield for the short chain of the PEG was higher than that of the long chain. (3) The reaction pH in the range of 7.0 to 8.0 had almost no influence upon the yield of HHPP, while the PEGylation yield was significantly increased by a factor of three from pH 7.0 to 8.0 at normal pressure. Surface accessibility analysis was performed using GRASP2 software, and we found that Cys17 of rhCNTF was located at the concave patches, which may have steric hindrance for the PEG to approach. The speculated benefit of HHPP was the facilitation of target-site exposure, reducing the steric hindrance and making the reaction much easier. Structure and activity analysis demonstrated that the HHPP product was comparable to the PEGylated rhCNTF prepared through a conventional method. Overall, this work demonstrated that HHPP, as we proposed, may have application potentials in various conjugations of biomacromolecules.

Microwave-Induced Transient Heating Accelerates Protein PEGylation

“…Curiously, PEGylation at 60 °C (conventional heating) yielded a value of ∼0.6, only slightly closer to this practical maximum of 0.7. This could suggest that heat may contribute to increasing the accessibility of the hindered Cys-34 by modifying conformation, in line with the arguments of others . Overall, transient heating with microwaves appears to be most amenable to enhancing the rate of reactions that are intrinsically slow and for which irreversible deactivation processes do not exist.…”

“…Intriguingly, the high degree of PEGylation suggests an influence of transient heating on the conformation or dynamics of the protein or mPEG-Ald, or both. Regarding conformation, Wang et al 24 demonstrated that partially and reversibly unfolding a protein using hydrostatic pressure improved the surface accessibility of a reduced cysteine residue buried in a hydrophobic patch, which improved its PEGylation. Thus, the result obtained herein could indeed reflect reversible structural deformations caused by transient heating.…”

Amphiphilic Nanoaggregates with Bimodal MRI and Optical Properties Exhibiting Magnetic Field Dependent Switching from Positive to Negative Contrast Enhancement

“…In optical imaging, research is mainly focusing on the near-infrared (NIR) window (650 to 1350 nm) for emissive probes, as maximum depth penetration is observed in this region . Nanoparticles (NPs) and molecular complexes have been reported as bimodal MRI/optical probes. ,,− Conjugation of iron oxide NPs to fluorescent probes has been reported as a strategy toward creating negative MRI CAs with additional optical properties. − Further development has included upconverting/paramagnetic lanthanide NPs which can be excited with laser wavelengths in the NIR window and exhibit negative MRI contrast enhancement. , Various organic probes have been conjugated to paramagnetic materials to investigate their usefulness in bimodal applications. , Similarly, luminescent transition metals and lanthanides have been linked to DTPA or DOTA or have been aggregated into magnetofluorescent liposomes and nanoparticles. − Organic fluorescent dyes are limited by very short fluorescence lifetimes (several ns at most), small Stokes’ shifts, and poor resistance to photobleaching. On the other hand, lanthanide-based systems combining magnetic and optical properties do not suffer from these drawbacks and represent an attractive alternative.…”

Continuous flow microreactor for protein PEGylation

“…There are two main challenges in protein PEGylation, getting high conversion yield (i.e., high productivity) and increasing the selectivity of mono-PEGylation. Conversion and selectivity depend on a variety of factors such as the properties of the protein molecule, the PEGylation reagent, 12,13 as well as the reaction conditions such as protein:PEG molar ratio, 14 pH, 15 temperature, 16 pressure, 17 and salt concentration. 18 Consequently, there is a wide range of a) Author to whom correspondence should be addressed: rghosh@mcmaster.ca 1932-1058/2018/12(4)/044114/10/$30.00…”