Modeling the competition between aggregation and self‐assembly during virus‐like particle processing

Abstract

Understanding and controlling aggregation is an essential aspect in the development of pharmaceutical proteins to improve product yield, potency and quality consistency. Even a minute quantity of aggregates may be reactogenic and can render the final product unusable. Self-assembly processing of virus-like particles (VLPs) is an efficient method to quicken the delivery of safe and efficacious vaccines to the market at low cost. VLP production, as with the manufacture of many biotherapeutics, is susceptible to aggregation, which may be minimized through the use of accurate and practical mathematical models. However, existing models for virus assembly are idealized, and do not predict the non-native aggregation behavior of self-assembling viral subunits in a tractable nor useful way. Here we present a mechanistic mathematical model describing VLP self-assembly that accounts for partitioning of reactive subunits between the correct and aggregation pathways. Our results show that unproductive aggregation causes up to 38% product loss by competing favorably with the productive nucleation of self-assembling subunits, therefore limiting the availability of nuclei for subsequent capsid growth. The protein subunit aggregation reaction exhibits an apparent second-order concentration dependence, suggesting a dimerization-controlled agglomeration pathway. Despite the plethora of possible assembly intermediates and aggregation pathways, protein aggregation behavior may be predicted by a relatively simple yet realistic model. More importantly, we have shown that our bioengineering model is amenable to different reactor formats, thus opening the way to rational scale-up strategies for products that comprise biomolecular assemblies.

Engineering batch and pulse refolding with transition of aggregation kinetics: An investigation using green fluorescent protein (GFP)

“…9A), the pure 2nd order model gave a better prediction than transition model. However this prediction was reactor specific (Ding et al, 2010) because using alternative refolding strategies like pulse refolding for Fig. 9B-D showed weaker prediction for the pure 2nd order model.…”

“…To ensure that transition model can be applied to other reactor formats (Buswell and Middelberg, 2003;Ding et al, 2010), pulse refolding was performed experimentally from 1 pulse (batch) to 4 pulses, reaching a final protein and residual urea concentration of 200 mg/ml and 0.9 M respectively. Consistent with pulse refolding on human proinsulin (Winter et al, 2002) and the autoprotease 6His-EDDIE-sGFPmut3.1 (Pan et al, 2014), experimental results showed yield improvements from batch to pulse refolding (Fig.…”

“…For example, a kinetic scheme for lysozyme refolding and aggregation that involved a sequential polymerization with the folding intermediates and the native protein (Buswell and Middelberg, 2003) as well as the competition between aggregation and self-assembly during viruslike particle processing (Ding et al, 2010).…”

Expression of unique chimeric human papilloma virus type 16 (HPV‐16) L1‐L2 proteins in Pichia pastoris and Hansenula polymorpha

“…They are not infectious as they do not contain any genetic material and therefore serve as excellent vaccine candidates (Chackerian, ). Recombinant expression of HPV type 16 major capsid protein, L1, results in the spontaneous self‐assembly of dimers to which free L1 monomers subsequently associate until a closed icosahedral VLP is formed that resembles native virions (Ding, Chuan, He, & Middelberg, ; Kirnbauer, Booy, Cheng, Lowy, & Schiller, ). Although the minor capsid protein, L2, is a prerequisite for infectious virions, it is not required for VLP formation (Fahey, Raff, Da Silva, & Kast, ).…”

Virus-like particle bioprocessing: challenges and opportunities

“…The in vitro assembly of other VLPs including for rotavirus [9], cowpea chlorotic mottle virus [10] and bacteriophages MS2 [2] and Qβ [3] has also been examined. Cell-free VLP assembly approaches are expected to mature as understanding of VLP assembly and its control improves, and as bioprocess-relevant mathematical models that capture not only the ideal assembly reaction, but also off-pathway aggregation, are developed [11].…”