This document discusses protein aggregation, which is a major challenge in bioprocessing. Protein aggregation can occur through different pathways and is influenced by environmental conditions like pH, temperature and salt concentration. The document presents an study that aimed to understand how adding polyelectrolytes or changing pH impacts protein aggregation. Turbidity experiments were used to analyze aggregation of myoglobin formulations with salts like NaCl and MgCl2, with and without the polyelectrolyte PSS. The results showed that PSS reduced aggregation in myoglobin-MgCl2 solutions. Lowering pH also decreased aggregation for myoglobin-MgCl2, but increased it for myoglobin-NaCl, due to changes in intermolecular forces. The

1. Introduction and
Background
• Aggregation is arguably the most common hurdle
encountered during bio processing and is one of the main
challenges still faced behind widespread
commercialization of bio-therapeutics. The reason why
protein aggregation is so problematic is because it can
occur throughout the process in bio processing and is very
often occurring irreversibly [1].
• Experimental and theoretical studies suggest that
protein aggregation usually forms from intermolecular
interaction between destabilized or unstable proteins.
Protein aggregation occurs when physiological conditions
such as pH, temperature, addition of denaturants etc, are
changed, which in turn destabilize the native proteins.
Addition to these protein-protein interactions, proteins and
polyelectrolytes can interact to form complexes under
chosen solvent conditions [2],[3].
• There are several pathways for protein aggregation to
occur. One pathway of aggregation is when native protein
goes directly to aggregated form. However, since the
interactions in the aggregated state are weak, the process
is highly reversible and does not usually causes the
reduced yields. The irreversible process of aggregation
occurs when native protein goes to an intermediate state
(reversible) but then further grows to become an aggregate
(irreversible) from that intermediate state, I, shown in
Figure 1. The rate limiting step is the formation of
intermediate partially folded state from native protein and
therefore most study methods of protein aggregation are
centred on determining that rate limiting step and how that
can influence the formation of partially folded state [2].
• A widely studied method used to prevent aggregation
is by addition of small molecules additives to formulation,
commonly referred to as co-solvents (such as salts, sugars,
amino acids, alcohols etc) to prevent aggregation.
• Protein aggregation can also be prevented by changing
physiological conditions such as pH, temperature, addition
of denaturants etc.
• Therefore, main objective is to study effects of
aggregation upon addition of additives to protein
formulations and furthermore, how changing the
environmental conditions change aggregation. If there is
repulsion between partially folded proteins, it can prevent
aggregation.
Materials and Methods
• There are several experimental methods that can be
deployed to detect protein aggregation, both in terms of
quality and quantity. Among the most popular methods
are by using static light scattering and turbidity titrations.
However, only turbidity titrations were used in the current
study.
• In a turbidity experiment, the decrease in the intensity
of a light beam incident on the sample of fluid is
measured. The decrease in intensity of incident light is
due to light scattered by aggregates in solution, the
amount of which is proportional to the product of
aggregate concentration and aggregate molecular weight
(Figure 2). Turbidimetric titrations were carried out by
inserting a turbidity probe coupled with a pH probe into
the sample.
• Firstly, effect of polyelectrolyte (PSS) addition into
protein (myoglobin) formulation was explored at different
ionic strengths. NaCl and MgCl2 were used at 4M and 2M
respectively. For both salt cases, aggregation was
observed via turbidity, with and without PSS addition.
• Secondly, effect of pH change at different ionic
strengths of NaCl and MgCl2 were explored around pI of
myoglobin.
• It can be seen that addition of salts at higher
concentrations lead to aggregation (Figure 3 & Figure 4),
however this can be suppressed with addition of
polyelectrolyte, such as PSS in some cases (Figure 4).
• It can be seen in Figure 5, that as we decreased pH
below pI in case of magnesium chloride at various ionic
strengths, turbidity decreases ,meaning it stabilizes the
system against aggregation as intermolecular forces are
repulsive. However, in case of sodium chloride with the
same type of experiment, Figure 6, this effect is not
observed and turbidity generally increases with lowering
the pH below pI, meaning that electrostatic repulsions are
attractive.
Conclusions
It can be concluded that addition of polyelectrolyte (PSS)
in protein formulation can in some cases lead to reduced
electrostatic forces within formulation; thus decreasing
aggregation. This effect was found for myoglobin solution
containing PSS and magnesium chloride. Aggregation can
also be successfully controlled by changing other factors
in formulations, such as salt concentration, pH etc. In case
of magnesium chloride in myoglobin formulation, at
various ionic strengths, turbidity decreased ,meaning it
stabilized the system against aggregation as
intermolecular forces were repulsive.
Abdullah Ahmad
Supervisor: Dr Robin Curtis
School of Chemical Engineering and Analytical Science, The University of Manchester
Figure 2 Visiual change observed in turbidity due to
aggregation of the solution.
Acknowledgment
I would like to take this opportunity to thank Dr. Robin
Curtis for his immense support and assistance throughout
the project.
References
[1] WANG, W. 2005. "Protein aggregation and its inhibition in
biopharmaceutics", International Journal of Pharmaceutics v289: 1
[2] WANG ET AL., 2010. “Protein aggregation-Pathways and influencing
factors”, Journal of Pharmaceutical Sciences v390: 1
[3] WANG, W. & ROBERTS C.J. 2010. Aggregation of therapeutic proteins.,
New Jersey: Willey Press, 2010
Results
Effect of PSS addition into myoglobin formulation
Effect of pH change at different ionic strength of salts
Figure 3 Showing turbidity titration of Myoglobin-NaCl
system, with and without PSS.
Understanding Protein-Protein & Protein-Polyelectrolyte
Interactions in Protein Formulations
Figure 1 Showing different protein aggregation
pathways in typical processing conditions, where N
refers to the native protein, A* is the aggregate of the
native protein, I is the partially folded protein and U is
the totally unfolded protein. Ac is the critically sized
aggregate which can spontaneously grow on to the larger
aggregate denoted by An [2].
Figure 4 Showing turbidity titration of Myoglobin-MgCl2
system, with and without PSS.
Figure 5 Showing turbidity vs pH of MgCl2 system at
different ionic strengths
Figure 6 Showing turbidity vs pH of NaCl system at
different ionic strengths.