Effect of Degraded Polysorbate 20 on the Thermal Stability of Chymotrypsinogen

Introduction

My research topic will focus on the effect of degraded polysorbate 20 on the thermal stability of chymotrypsinogen, there have been no detailed studies regarding this topic. I will review the literature of the wider subject area regarding protein stability and forced degradation studies. Information from a board range sources including but not limited to peer-reviewed journal articles, books, and published dissertations will be used, the credibility of authors will be assessed via citation databases. The areas to be discussed are relevant to my research topic and include surfactants specifically polysorbates, looking at their structure, relevance in the pharmaceutical industry, their degradation mechanisms and how they stabilise proteins in general. Also looking at a basic overview of the structure and function of proteins specifically chymotrypsinogen, proteins used in medical treatment and the destabilisation of proteins. The reason for conducting the literature review is to get a better understanding of previous work regarding my chosen topic and to then allow to build on that knowledge.

Surfactants

The basic structure of surfactants is of a hydrophilic polar head and hydrophobic hydrocarbon tail; this amphiphilic nature allows surfactants to adsorb at interfaces or to form structures like micelles and bilayers. Surfactants accumulate at the interfaces as a result of the hydrophobic effect explained by Gibbs free energy equation. This equation represents the thermodynamics of transfer for a hydrocarbon from an organic solvent into water:

$∆\mathit{Gt}=∆\mathit{Ht}–T∆\mathit{St}$

$∆S$

t is negative and therefore unfavourable, the greater the relative size of the hydrocarbon the more negative $∆S$

t becomes. Near room temperature $∆H$

is zero, as the temperature increases when the solution is in the waterbath, $∆H$

will becomes more positive and $∆S$

becomes less negative (Kerwin, 2008). It requires less energy for the hydrocarbon chains to bond with themselves than the water.

Depending on the type of polar group, surfactants can be classified as either anionic, cationic, non-ionic, amphoteric or zwitterionic (Pletnev, 2001). Surfactants are used extensively in the pharmaceutical industry, they can act as detergents, emulsifiers and wetting agents in formulations (Lee et al., 2011). The most common type of surfactant used for internal formulations is non-ionic surfactants, due to a favorable toxicity profile and a resistance to ionic damage. 

Polysorbates

Polysorbates are the most common non-ionic surfactant used in the industry currently, therefore due to the prevalence and gaps in credible research regarding the effect of different stresses on the efficacy of polysorbate. Polysorbates are cheap and easy to produce.  

Polysorbate molecules work well at low concentrations because of their high hydrophile‐lipophile balance and low critical micelle concentration (Šilha et al., 1989). They are made up of esterified fatty acid derivates of polyoxyethylene (POE) sorbitan (Kishore et al., 2011).  There are many commercially available polysorbate molecules all with varying units of poly (ethylene glycol), referring to figure 1 polysorbate 20 would have a total unit (w+x+y+z) of 20 units and would be the subject of the study (Kishore et al., 2011). The hydrophobic character is a result of the hydrocarbon chain and the hydrophilic character is a result of the ethylene oxide units.

Micelle structures form above the critical micelle concentration (CMC), the ethylene oxide units’ position to be in contact with the water and the tails position away, the micelles are spherical. The CMC is not fixed and is affected by the solution environment. Increasing the temperature when in the water bath would lower the CMC this can consequently cause phase-separation when the cloud point (cp) is reached. (Kerwin, 2008)

Polysorbate solutions sold by companies have a percentage of different fatty acid esters, the European pharmacopeia defines these amounts, but the united states pharmacopeia doesn’t (Kerwin, 2008). It states in the EU pharmacopeia that for polysorbate 20 the lauric acid content should be around 40-60%, the myristic acid content of 14-25%, caproic acid of less than 1% and linoleic acid of less than 3%. The percentages vary with different polysorbate mixtures (COUNCIL OF EUROPE 2001).

Figure 1 structure of polysorbate 20 (Hoffmann et al., 2009).

How polysorbates work

Polysorbates act as detergents, stabilising protein solutions mainly through inhibiting protein aggregation, a common cause of protein instability.

Polysorbates ability to stabilise proteins is not predictable, for example, Hoffmann et al., 2009 using DSC concluded that PS 20 has no effect on the thermal stability of lysozyme, the results showed that PS20 didn’t change the Tm, heat capacity or denaturation enthalpy of lysozyme. However, the author’s results regarding the effect of PS 20 on thermal stability of bovine serum albumin (BSA) showed increases in heat capacity and denaturation enthalpy. Stabilisation ability also depends on the stress applied, Bam et al., 1998 found using DSC PS 20 had an insignificant on the thermal stability, but inhibiting aggregation via agitation. The reason for the unpredictability is unclear but potential differences in protein-polysorbate interactions and protective mechanisms have been hypothesised. One of the more understood mechanism is PS ability to adsorb onto interfaces, the air/liquid and the solid/liquid interface. Increasing the concentration PS 20 would decrease the surface tension of the protein and thus increase stability (chymotrypsinogen) aqueous system till the critical micelle concentration is reached, after this point increasing the concentration has no effect on stability as the interfaces become saturated and micelles form, which is undesirable.Second potential protective mechanism involves PS 20 acting like a chaperone, which assists protein refolding. The equilibrium of the protein shifts towards the denatured state to the native state (Bam et al., 1998). Furthermore, the hydrophobic regions on the protein interact with hydrophobic regions of the polysorbate stopping the protein denature due to its own hydrophobic interactions. The concentration of detergent required to achieve stabilisation is different for different proteins, there is an optimum concentration, below this amount protein aggregation is not sufficiently reduced and above this point, proteins start to aggregate (Hoffmann et al., 2009).

Degradation of polysorbates

Polysorbates undergo thermal autoxidation resulting in breakage of the ethylene bonds represented by w,x, and y on figure 1. The end products of radical reaction result in the formation of acids like formic and acetic acid, this will increase the acidity of the polysorbate-chymotrypsinogen solution, but the solution will the buffered with potassium phosphate to limit this. The peroxide concentration is low at first but increases along with the duration of the autoxidation reaction, the rate of peroxide formation is increased with temperature and therefore the rate of degradation  (Kishore et al., 2011). During the initial stage of propagation, the rate of peroxide formation is greater than termination, reaching a short-lived steady state where the rate of peroxide formation and decomposition are equal then finally decomposition dominates. TGA-DSC studies carried out by Kishore et al., 2011 found that during initiation of radical the reaction the rate of autoxidation is first order with respect to radical formation, where the radical formation is the rate determining step. Previous studies have shown peroxide causing oxidative damage to subject proteins, there will no antioxidants present in the polysorbate-chymotrypsinogen solution and therefore oxidative damage of chymotrypsinogen will occur. The reaction is initiated with light and therefore polysorbate 20 mixtures will be stored in a dark place.  Donbrow et al 1978 analysis the chain reactions occurring:

$\mathit{RH }\to R.+H.\mathit{ }$

Initiation

$R. +O2\to \mathit{ROO}.$

$\mathit{ROO}. +\mathit{RH}\to \mathit{ROOH}+R.$

Propagation

$–\left(\mathit{OCH}2\mathit{CH}2\right)O– +O2– \to –\left[\mathit{OCH}2C\left(\mathit{OOH}\right)O\right]–$

 oxyethylene oxidises to form peroxides

$2\mathit{RO}2.\to \mathit{inactive\; products}$

$\mathit{RO}2.+R. \to \mathit{inactive\; products}$

$2R.\to \mathit{inactive\; products}$

Termination

Polysorbates also undergo hydrolysis at the ester bond forming a carboxylic acid and alcohol. The kinetics of the hydrolytic degradation calculated by Bates, Nightingale and Dixon, 1973 suggest it follows a pseudo-first order. The study looked at the hydrolysis of polysorbate 80 (PS80), but because the structures are so similar the pathway still applies. The study found PS80 was stable between pH 3 to 7.6, also that hydrolytic degradation, the pH of my test solution would be buffered to 7.4. Furthermore, increasing concentration of the PS 80 decreases the rate of hydrolysis, due to shielding of the ester bonds by micellar PS80 (Bates, Nightingale and Dixon, 1973). Type of degraded products

Protein Therapeutics 

Protein therapeutics make up an ever-increasing share of pharmaceutical products this is due to advances in biotechnology, proteins have never been cheaper to produce. Current medical treatments include, but not limited to fusion proteins, replacement enzymes and interferons. Protein therapeutics are increasingly used as second- or first-line treatments in the NHS due to their reduced cost (Lee et al., 2011). Proteins are made up of single units called amino acids, there 20 different amino acids, each amino acid has a different side chain. Based on the interactions of the amino acids the polypeptides fold into various three-dimensional structures. it’s the 3d structure of proteins dictate that function of the protein. If the 3d structure of any protein therapeutics is altered through denaturation, aggregation or adsorption the internal interactions between amino acids will change and therefore its 3d structure, this would result in loss of biological activity (Lee et al., 2011).

Enzymes

Enzymes are biological catalysts increasing the rate of chemical reactions without being altered structurally themselves. Enzymes are vital to allow reactions to take place at sufficient rates in the cell, important for life. Most, but not all enzymes are proteins, enzymes work by lowering the activation energy needed to start a reaction. A substrate binds the enzymes active site forming an enzyme-substrate complex, the substrate undergoes catalytic change and the product dissociates off. The shape of the substrate is complementary to the active site, enzymes are specific, and the shape of the active site depends on the structure one the enzyme. Enzymes are classified based on their function. There are six different classes of enzyme: Oxidoreductase, transferases, Hydrolases, Lyases, Isomerases, and ligases.

Chymotrypsinogen

Chymotrypsinogen is made up of 245 amino acids, it has a molecular mass of 25.7 kDa (Romero et al., 2015). There are five disulfide bonds between the amino acids and the secondary structure mainly consists of beta-sheets (Romero et al., 2015). Previous studies have shown Chymotrypsinogen is thermally-denatured at acidic pH (Romero et al., 2015). Current research indicates the denaturation process is between two states native and denatured. (Romero et al., 2015). There are 3 different types of chymotrypsinogen A, B, and C. Type A has a similar primary structure to type B, but type C differs from both as it has a higher molecular weight (Freer et al., 1970).

Chymotrypsinogen Is created and secreted in the pancreas, it is a precursor enzyme to chymotrypsin. Referred to as a zymogen it has no enzymatic activity. Chymotrypsinogen is activated by trypsin another proteolytic digestive enzyme, trypsin does this by cleavage of the covalent bond between arginine 15 and isoleucine 16.  Cleavage forms an unstable indeterminate π-chymotrypsin which dimerizes to form the stable form α-chymotrypsin.   Chymotrypsin is a protease, a type of hydrolases enzyme that breaks down large protein molecules into smaller amino acids in the intestine allowing for easier absorption, it works by hydrolysis (Berg, Tymoczko and Stryer, 2002). Based on a previous study “chymotrypsinogen forms soluble, non-covalent aggregates at elevated temperatures” (Andrews, Weiss and Roberts, 2008).

Denaturation of proteins

Denaturation of proteins is defined as an alteration of the native protein resulting in changes in the properties of the protein, ultimately denaturation occurs due to intermolecular disruptions (Neurath et al., 1944). There are many mechanisms governing this process, understanding of such mechanisms is important in industry as preventative measures can be used during development to inhibit such events and prolong the shelf life.  One such mechanism can be concentration governing, proteins form aggregates in higher concentrations. Initially, the protein oligomerises which is reversible, but at higher concentrations, the complex has the possibility to irreversibly associate (Lee et al., 2011). A study by Ismail, Mantsch and Wong, 1992 which studied the structural changes of chymotrypsinogen when the temperature is decreased using infrared spectroscopy concluded the two bands at 1627cm-1 and 1564cm-1 respectfully weren’t dependant on the concentration of chymotrypsinogen. Another such mechanism is through a conformational change, this occurs due to the denature sate of a protein having a greater affinity with each other. Conformational change can be encouraged by stresses like heat, chemically or by degradations like oxidative damage (Lee et al., 2011). Potentially as the peroxide number of the polysorbate-chymotrypsinogen solution increases this can result in chymotrypsinogen undergoing oxidative damage and aggregating via this mechanism. Lastly, through interface-induced mechanisms where proteins adsorb to an interface, polysorbates are effective at inhibiting this mechanism (Kishore et al., 2011).

Aggregation can either be physical, the linking of the polymer chain without alternating the sequence or chemical, where new covalent bonds form between residues.

These mechanisms result in changes that may a decrease in solubility of the protein, decreased biological activity this is important in protein therapeutics or and changes in molecular size and shape it’s this property that would be analysed via DLS.

Proteins in an aqueous environment are in equilibrium between a native active state (N) and the inactive denatured state(D).  Increase in temperature would shift the equilibrium towards the inactive state. The position of equilibrium at a specific temperature can be expressed as keq , the concentration of the native over the denatured. Tm is the temperature where the native and denatured are equal in concentration and Keq equals one. 

$N\leftrightarrow D$

$\mathit{Keq}=\left[U\right]/\left[D\right]$

Differential scanning calorimetry

Differential scanning calorimetry (DSC) is an analytical technique used to study the thermal stability of biological molecules including proteins. It is commonly used in the pharmaceutical industry in research and development, quality assurance and quality control. The melting point (Tm) is measured, an indicator of thermal stability, the greater the melting, the more stable the protein. It does this by investigating how a change in temperature effects a samples heat capacity relative to a reference. The sample is heated along with the reference at a constant rate, the reference cell in the study would consist of the potassium phosphate buffer and polysorbate 20 in solution, excluding the test protein chymotrypsinogen. The sample and reference are both heated when a protein denatures heat is absorbed resulting in a difference in temperature between the sample and reference cell. (GROENEWOUD, 2001).

Dynamic light scattering

Dynamic light scattering (DLS) is a technique used in physics and biological labs, a monochromatic laser is shone at particles such as polypeptides in solution. Particles in solution exhibit random movement as a result of colliding with molecules in the medium this is called Brownian motion. When the beam of light hits the particles exhibiting Brownian motion this causes the wavelength of the light to change. The greater the size of the particle, the greater the change in wavelength, using this information, the shape and size about the particle can be calculated along with its diffusion coefficient. This technique is useful in the pharmaceutical industry as these properties are useful in drug design and development processes. One useful application of DLS is that it can be used to analyse thermal stability of the protein.  As a protein undergoes thermal denaturation the size and thus scatting intensity increases. (Arzenšek, Podgornik and Kuzman, 2010).

Conclusion

In conclusion surfactants or surface-active molecules are widely used in the pharmaceutical industry in several applications, there are different types of surfactants, but one commonly used non-ionic surfactant is polysorbate. There are different types of polysorbate all with varying ethylene oxide subunits, polysorbate 20 would be the focus of the study. There are a lack of studies looking at polysorbate 20 degradation on the effect of protein stability, furthermore current knowledge on how polysorbates stabilise protein is incomplete, there are only a few hypothesised mechanisms. Also based on current knowledge whether polysorbates stabilise proteins are unpredictable, stabilising some proteins but not others, this is an area for future study. Polysorbates degrade via autoxidation and hydrolysis, autoxidation being the major pathway. As a result of the degraded by-products of polysorbate, chymotrypsinogen will undergo oxidative damage and consequently denaturation. Denaturation can be observed via DLS.

References

• Andrews, J., Weiss and Roberts, C. (2008). Nucleation, Growth, and Activation Energies for Seeded and Unseeded Aggregation of α-Chymotrypsinogen A†. Biochemistry, [online] 47(8), pp.2397-2403. Available at: https://pubs.acs.org/doi/abs/10.1021/bi7019244 [Accessed 22 Nov. 2018].
• Arzenšek, D., Podgornik, R. and Kuzman, D. (2010). Dynamic light scattering and application to proteins in solutions. Ljubljana: University of Ljubljana, pp.1-19.
• Bam, N., Cleland, J., Yang, J., Manning, M., Carpenter, J., Kelley, R. and Randolph║, T. (1998). Tween protects recombinant human growth hormone against agitation-induced damage via hydrophobic interactions. Journal of Pharmaceutical Sciences, [online] 87(12), pp.1554-1559. Available at: https://ac.els-cdn.com/S0022354915507211/1-s2.0-S0022354915507211-main.pdf?_tid=41396958-9f2b-4a0d-8c54-0bb9fea1dbe1&acdnat=1543442710_0b4abd36540c871b8c93623febde16b6 [Accessed 29 Nov. 2018].
• BATES, T., NIGHTINGALE, C. and DIXON, E. (1973). Kinetics of hydrolysis of polyoxyethylene (20) sorbitan fatty acid ester surfactants. Journal of Pharmacy and Pharmacology, [online] 25(6), pp.470-477. Available at: https://onlinelibrary.wiley.com/doi/epdf/10.1111/j.2042-7158.1973.tb09135.x [Accessed 25 Nov. 2018].
• Berg, J., Tymoczko, J. and Stryer, L. (2002). Biochemistry. 5th ed. New York: W.H. Freeman and Co., pp.200-215.
• COUNCIL OF EUROPE. (2001). European pharmacopoeia. Strasbourg, Council of Europe
• Donbrow, M., Hamburger, R., Azaz, E. and Pillersdorf, A. (1978). Development of acidity in non-ionic surfactants: formic and acetic acid. The Analyst, 103(1225), p.400.
• Freer, S., Kraut, J., Robertus, J., Wright, H. and Nguyen-Huu-Xuong (1970). Chymotrypsinogen: 2,5-Å crystal structure, comparison with α-chymotrypsin, and implications for zymogen activation. Biochemistry, [online] 9(9), pp.1997-2009. Available at: https://pubs.acs.org/doi/pdf/10.1021/bi00811a022 [Accessed 3 Dec. 2018].
• GROENEWOUD, W. (2001). DIFFERENTIAL SCANNING CALORIMETRY. Characterisation of Polymers by Thermal Analysis, pp.10-60.
• Hoffmann, C., Blume, A., Miller, I. and Garidel, P. (2009). Insights into protein–polysorbate interactions analysed by means of isothermal titration and differential scanning calorimetry. European Biophysics Journal, [online] 38(5), pp.557-568. Available at: https://link.springer.com/article/10.1007%2Fs00249-009-0404-6#CR69 [Accessed 22 Nov. 2018].
• Kerwin, B. (2008). Polysorbates 20 and 80 Used in the Formulation of Protein Biotherapeutics: Structure and Degradation Pathways. Journal of Pharmaceutical Sciences, 97(8), pp.2924-2935.
• Kishore, R., Pappenberger, A., Dauphin, I., Ross, A., Buergi, B., Staempfli, A. and Mahler, H. (2011). Degradation of Polysorbates 20 and 80: Studies on Thermal Autoxidation and Hydrolysis. Journal of Pharmaceutical Sciences, [online] 100(2), pp.721-731. Available at: https://onlinelibrary.wiley.com/doi/full/10.1002/jps.22290 [Accessed 21 Nov. 2018].
• Ismail, A., Mantsch, H. and Wong, P. (1992). Aggregation of chymotrypsinogen: portrait by infrared spectroscopy. Biochimica et Biophysica Acta (BBA) – Protein Structure and Molecular Enzymology, [online] 1121(1-2), pp.183-188. Available at: https://www.sciencedirect.com/science/article/pii/016748389290353F?via%3Dihub [Accessed 4 Dec. 2018].
• Lee, H., McAuley, A., Schilke, K. and McGuire, J. (2011). Molecular origins of surfactant-mediated stabilization of protein drugs. Advanced Drug Delivery Reviews, [online] 63(13), pp.1160-1171. Available at: http://wolfson.huji.ac.il/purification/Course92632_2014/Aggregation/Lee2011.pdf [Accessed 2 Dec. 2018].
• Neurath, H., Greenstein, J., Putnam, F. and Erickson, J. (1944). The Chemistry of Protein Denaturation. Chemical Reviews, [online] 34(2), pp.157-265. Available at: https://pubs.acs.org/doi/pdf/10.1021/cr60108a003 [Accessed 3 Nov. 2018].
• Pletnev, M. (2001). 1. Chemistry of surfactants. Studies in Interface Science, [online] 13(1), pp.1-97. Available at: https://www.sciencedirect.com/science/article/pii/S1383730301800624 [Accessed 3 Dec. 2018].
• Romero, C., Abella, J., Velázquez, A. and Sancho, J. (2015). Thermal denaturation of α-chymotrypsinogen A in presence of polyols at pH 2.0 and pH 3.0. Journal of Thermal Analysis and Calorimetry, [online] 120(1), pp.489-499. Available at: https://link.springer.com/article/10.1007/s10973-014-4374-2 [Accessed 3 Dec. 2018].
• Wang, W., Wang, Y. and Wang, D. (2008). Dual effects of Tween 80 on protein stability. International Journal of Pharmaceutics, [online] 347(1-2), pp.31-38. Available at: https://www.sciencedirect.com/science/article/pii/S0378517307005212 [Accessed 21 Nov. 2018].
• Šilha, J., Bareš, M., Zeman, I. and Šmidrkal, J. (1989). The HLB number determination of polyoxyethylene surfactants. Collection of Czechoslovak Chemical Communications, [online] 54(4), pp.945-952. Available at: http://cccc.uochb.cas.cz/54/4/0945/ [Accessed 21 Nov. 2018].