On the Aggregation and Nucleation Mechanism of the Monoclonal Antibody Anti-CD20 Near Liquid-Liquid Phase Separation (LLPS)

Abstract

The crystallization of Anti-CD20, a full-length monoclonal antibody, has been studied in the PEG400/ na 2 So 4 /Water system near Liquid-Liquid Phase Separation (LLPS) conditions by both sitting-drop vapour diffusion and batch methods. In order to understand the Anti-CD20 crystallization propensity in the solvent system of different compositions, we investigated some measurable parameters, normally used to assess protein conformational and colloidal stability in solution, with the aim to understand the aggregation mechanism of this complex biomacromolecule. We propose that under crystallization conditions a minor population of specifically aggregated protein molecules are present. While this minor species hardly contributes to the measured average solution behaviour, it induces and promotes crystal formation. The existence of this minor species is the result of the LLPS occurring concomitantly under crystallization conditions. A thorough understanding of protein aggregation behaviour in solution is crucial for the controlled manufacture and formulation of therapeutics and for the development of crystallization-based purification strategies for biological drugs 1-4. Unfortunately, while the state diagram of protein-solvent systems can provide indications about the phase behaviour of biomacromolecules and their stability in solution, the intimate mechanism underlying protein nucleation and crystal growth have not been fully understood yet and, in many cases, it remains elusive. Protein aggregation is directly related to protein-protein interactions and may be strongly related to protein crystallization behaviour 5. Since different types of intermolecular forces are involved in protein interactions, such as electrostatic, van der Waals and hydrophobic 6 , environmental factors like pH, ionic strength and the presence of additives, can drastically alter the intermolecular interaction profile of proteins in solution 7,8. Colloidal and conformational stabilities are the two main factors that govern the existence of a protein aggregate in a solution 9,10. Colloidal stability is a delicate balance between repulsive and attractive forces between proteins, while conformational stability is related to the free energy difference between two molecular states, folded and partially or totally unfolded. Several investigations have found that the melting temperature (T m) and the osmotic second virial coefficient (B 22) can respectively account for the protein conformational 11 and colloidal 12 stability. Both T m and B 22 can be experimentally determined by using correspondingly Dynamic Light Scattering (DLS) and Static Light Scattering (SLS) methods 13. Aggregation induced by thermal unfolding can be monitored by measuring the average hydrodynamic diameter value as a function of temperature, while B 22 describes the first deviation from ideal behaviour of dilute colloidal solutions 14. A positive or negative value of B 22 accounts for a net repulsive or attractive interaction balance, respectively, and could therefore be used t...

Review of Liquid–Liquid Phase Separation in Crystallization: From Fundamentals to Application

“…B 22 and k d measured under dilution are generally considered as predictors of high concentration behavior. Nevertheless, the high concentration behavior may differ from the behavior observed under dilution conditions because the “crowding” effect, i.e., increased opportunities for molecular interactions, deviations from thermodynamic ideals, and higher order interactions (e.g., B 23 , B 222 ), can significantly alter the net protein–protein interaction. , On the basis of the experimental observation of LLPS in the nucleation region, the authors suggest that it is likely that only a small number of protein molecules distributed at the droplet interface can experience protein–protein short-range attraction interactions induced by local environmental factors, without affecting the average B 22 value …”

“…With the passage of time, the anisotropic interaction may be the reason for the evolution of the system from a phase separation state to a reversible aggregation state. In this case, LLPS is presumed to be metastable during nucleation. ,, However, when Mastropietro et al studied the crystallization of anti-CD20 (full-length monoclonal antibody) in a PEG400/Na 2 SO 4 /H 2 O system near LLPS, they found that second virial coefficient B 22 (positive value means repulsive interaction, negative value means attractive interaction) was not associated with protein interaction. The authors explain that in a complex mixture containing several components, the B 22 value cannot predict the LLPS since it may be associated with the protein-free polymer–salt phase separation.…”

Template-Assisted Crystallization Behavior in Stirred Solutions of the Monoclonal Antibody Anti-CD20: Probability Distributions of Induction Times

“…Moreover, previous studies on Anti-CD20 crystallization showed that when an LLPS occurs in the crystallization solution, the nucleation of mAbs crystals occurs at the same moment. 32 , 45 These two phenomena being closely correlated, we assume the occurrence of one triggers the occurrence of the other, making it hard to discriminate which phenomenon is actually detected when the light transmission through the sample reduces.…”

Alginate Bioconjugate and Graphene Oxide in Multifunctional Hydrogels for Versatile Biomedical Applications

“…Static light scattering measurements (Zetasizer Nano ZS instrument, Malvern Panalytical, Malvern, UK) were performed on Alg and AlgCF water solutions with a concentration between 0.1 and 5.0 mg mL −1 in order to determine the average molecular weight [ 69 ]. All the solutions were filtered using a 0.22 μm filter (Millex ® -GV syringe-driven filter unit, Merck/Sigma Aldrich, Darmstadt, Germany), and then placed in quartz cuvettes.…”