Interfacial Adsorption of Monoclonal Antibody: a Combined Study of Spectroscopic Ellipsometry and Neutron Reflection

  • Zongyi Li

Student thesis: Phd


Therapeutic monoclonal antibodies (mAbs) are a rapidly growing class of biopharmaceutical products that are used in treating serious diseases such as cancers, autoimmune diseases and Alzheimer's disease. The interfacial adsorption of mAb is a crucial issue in product manufacturing, shipping and storage, as the process can trigger antibody aggregation and loss of bioactivity. A non-ionic surfactant, Tween 80 (Polysorbate 80), is commonly used to prevent interfacial adsorption and stabilise products. In work presented in this thesis, the interfacial adsorption behaviour of a mAb, COE-3, was studied at air/water interface and SiO2/water interface, in the presence and absence of Tween 80. The influence of fragment crystallisation (Fc) and antigen-binding fragment (Fab) on the intact mAb adsorption at the air/water interface were investigated by Neutron Reflection (NR). By taking advantage of isotopic contrast variation, the thickness, adsorbed amount, molecular orientation and immersion of the adsorbed layers could be determined. The results indicated that Fab adsorption was slower and had lower adsorbed amount than Fc, but could dominate the time and concentration dependency of adsorption from the intact mAb. Moreover, change of buffer pH has little effect on its adsorption at the air/water interface. Tween 80 with 20 ethoxylates (Tween 80 20EO) can start to remove antibody from the water surface at the surfactant concentration of 1/100 CMC. The complete replacement of COE-3 was achieved when the surfactant concentration reached 1/10 CMC. The adsorbed Fc, Fab and the intact COE-3 all retained their globular structures on the water surface, in the presence and absence of Tween 80 20EO. The adsorption dynamics of COE-3 at SiO2/water interface was studied using Spectroscopic Ellipsometry (SE) under various buffer conditions (pH and ionic strength). A simulation based on the DLVO theory helped interpret the data, indicating that electrostatic interaction played an essential role in mAb adsorption on the SiO2 surface. The equilibrated adsorption layer was characterised by NR, implying the adsorbed COE-3 molecules adopted a ‘flat-on’ position with the short axes of their segments perpendicular to the interface. Tween 80 20EO and Tween 80 7EO could not prevent COE-3 adsorption under the conditions studied. However, Tween 80 7EO can form a well-defined surfactant bilayer on the adsorbed antibody layer via self-assembly. The surfactant bilayer formed regardless of the mode of mixing, i.e., from a mixed mAb-surfactant solution or from the binding of surfactant to pre-adsorbed mAb layer. This work has demonstrated the power of the combined approach to unravel the detailed interfacial molecular processes and will benefit the future search for more effective surfactants to alleviate mAb adsorption.
Date of Award1 Aug 2018
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorJian Lu (Supervisor) & Thomas Waigh (Supervisor)

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