Structural modifications of muAb after light exposure

The exposure of the murine antibody formulated in 20 mM histidine to light entailed an increasing yellow staining of the samples. The staining derived from the placebo formulation, since the control samples of placebo solution exposed to light were equally colored. With prolonged incubation time the intensity of the staining increased. Since this phenomenon was not observed in the human antibody samples that were formulated in a citrate-phosphate buffer with mannitol, it can be assumed that the coloring is related to oxidation of the histidine in the placebo formulation. This side effect of the irradiation interfered with several analytical methods, like fluorescence and UV absorbance spectroscopy measurements. Fluorescence measurements could not be conducted at all, whereas 2^nd derivative UV absorbance spectroscopy was possible for the samples after 24 h and 48 h of light exposure. Already within this incubation time a tremendous alteration in the a/b ratio was detected suggesting a distinct modification in tertiary structure of light exposed muAb. Furthermore, 2^nd derivative FTIR spectroscopy revealed significant changes in the secondary structure such as the formation of α-helices in the antibody.

82 4.4 DISCUSSION

Four different stress methods aiming to generate aggregates of a murine IgG2c antibody were investigated. All methods elicited aggregation of the antibody and thus are evaluated to be suitable for preparative generation of larger amounts of aggregates.

Table 4-5 provides an overview of the classification of physico-chemical changes on the muAb molecule caused by the stress conditions.

Table 4-5 – Overview of resulting physico-chemical alterations in muAb after stress studies.

Stress method

Enhanced number of particles

Formation of soluble aggregates

Formation of fragments

Altered secondary

structure

Altered tertiary structure /

unfolding

Stirring ++ - - + +

Shaking + - - + +

Heat + + + ++ ++

Light + ++ + ++ ++

- No substantial alterations detected + Moderate alterations detected ++ Strong alterations detected

Agitation of muAb by stirring and shaking particularly induces the generation of particulate protein aggregates. At a similar agitation speed stirring with PTFE – coated stir bars was found to be more efficient in aggregation than shaking, resulting in faster formation of higher particle numbers, accompanied by a loss of soluble protein content. Furthermore, no increased particle loading was detected in the buffer formulation (either stirred or shaken).

Therefore, it can be concluded that the majority of particles is composed of protein. Though no soluble aggregates were detected by SEC in either sample after agitation modifications in the protein`s secondary and tertiary structure were indicated. Again the stirred samples revealed stronger changes after 24 hours than the shaken samples after 48 hours of incubation. After stirring the most severe disparities in the parameters for protein conformation were discovered in the ANS fluorescence spectra. Already after 4 h of stirring a considerable increase in ANS fluorescence was detectable, suggesting the exposure of hydrophobic moieties and thus unfolding of the antibody. At the same time it was found from the 2^nd derivative FTIR spectra that the unfolding is accompanied by alterations in secondary structure. Interestingly, the shaken muAb samples show similar changes in 2^nd derivative FTIR spectra, but no differences in ANS fluorescence and 2^nd derivative UV absorbance spectra were detected at all. Therefore, it can be assumed, that shaking the protein for up to 48 h does not imply substantial unfolding expressed by the enhanced exposure of hydrophobic patches. However, a minor quenching of the tryptophane fluorescence was found which might be considered as precursor for unfolding when shaking is prolonged. Regarding the five aggregation mechanisms widely accepted in biopharmaceutical research [Philo et al., 2009], both stress methods, shaking and stirring, are

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assumed to be at least partly based on the interaction between the protein and surfaces. The air-water interface enables an enrichment of protein molecules that potentially can involve unfolding [Chi et al., 2003a; Manning et al., 1995]. During stirring the PTFE surface of the stir bars might additionally serve as areas of protein enrichment. Such resulting assemblings of mostly native antibody molecules in literature are assumed to be weak and might be resolvable.

Kiese et al. reported that insoluble aggregates of a monoclonal antibody vanished during storage, indicating the reversibility of the assembling [Kiese et al., 2010].

The storage of the murine IgG2c antibody at an elevated temperature of 50°C entails the formation of soluble as well as insoluble aggregates. The distribution of protein species detected by SEC indicates the growth of aggregates gaining an intermediate size of soluble oligomers before exceeding the benchmark and forming insoluble oligomers. Since the monomer content is only slightly reduced during 27 d of incubation, it is suggested that the formation of conformationally altered structures leads to aggregation and later on small oligomers tend assemble to large aggregates. The protein`s structure was found to be considerably altered to an increasing extent during storage at 50°C. The characteristic secondary structure of the antibody changed and substantial unfolding including the exposure of hydrophobic moieties to the surface was proven. Hence, the aggregation mechanism based on conformationally altered structures is definitely involved in the formation of aggregates during storage at 50°C [Kendrick et al., 1998; Krishnamurthy et al., 2002; Krishnan et al., 2002;

Wang, 2005]. However, the generation of remarkable amounts of aggregates took several weeks which is disadvantageous for the aimed repeated preparation of aggregate samples for in vivo studies.

Similar to the human model antibody (see previous Chapter 3) light exposure induces vast structural modifications and strong aggregation in the murine antibody as well. Huge percentages of the total amount of protein were incorporated in aggregates during light exposure. Size exclusion chromatograms showed a strong reduction of monomeric species coincidental with a steady elevation of the amount of soluble oligomers. At the same time the total recovery of soluble protein species drastically decreased. Besides, the exposure to light results in alterations of the histidine containing formulation buffer as well. Presumably the histidine is oxidized by light. The distribution of protein species after light exposure is different to the muAb samples stored at 50°C, where the oligomer content in the soluble fraction is reduced with increasing formation of insoluble species and the monomer content remains rather stable.

Therefore, the mechanisms behind differ as well. The nucleation theory reported in literature [Chi et al., 2003b; Philo et al., 2009] is assumed to be part of the underlying aggregation mechanism during light exposure. The soluble oligomers are assumed to serve as nuclei interacting with monomeric antibody. This interaction leads to growth and subsequently the formation of particulate aggregates. At the same time, the amount of oligomers in the remaining soluble fraction does not drop but slightly increase, indicating that no association between oligomers occurred. However, the immense structural modifications proven by spectroscopic

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methods indicate the involvement of altered conformations in aggregation too. A combination of several aggregation mechanisms has to be assumed.

Regarding the previous study performed with the model antibody (see Chapter 3), the behavior of both immunoglobulines under similar stress conditions is concluded to be very much comparable. The human IgG1 as well as the murine IgG2c are prone to the formation of subvisible particles by stirring. Both agitation procedures performed during these studies, stirring and shaking, had only minor impact on the conformation of the IgG`s, whereas heating and light exposure resulted in significant alterations of their secondary and tertiary structures and unfolding. The most impressive formation of soluble protein aggregates was triggered by light exposure in both cases. However, some substantial differences between both antibodies have to be mentioned as well. Storage at 50°C for several weeks resulted in strong fragmentation of the human antibody but not in the murine antibody. This finding can be explained by the higher susceptibility of the IgG1 isotype to fragmentation as described by Ishikawa et al. [Ishikawa et al., 2010]. Instead, in the murine antibody higher amounts of soluble aggregates were detected after storage at 50°C. These discrepancies can be assumed to depend on the different Tm and thus the unfolding of the proteins. The different formulation buffers also have to be taken into consideration when comparing the aggregation behaviors of both IgG`s. It can be concluded that the histidine in muAb formulation is oxidized during light exposure [Huvaere et al., 2009]. The degradation products of histidine have a major impact on the analytical methods to evaluate the modifications appearing in muAb samples during light exposure.

4.5 CONCLUSION

A variety of methods to reproducibly generate aggregates in a human model IgG1 antibody was successfully transferred to a murine IgG2c antibody. Furthermore, the analytical tools to detect changes in protein structure were transferred as well.

Comparing the results of the murine IgG2c antibody to the aggregation studies on the model human IgG1 antibody in chapter 3, it can be concluded that the characteristics of the formulations after stressing are very similar. Both molecules form more or less only insoluble aggregates during agitation stress, with stirring being much more efficient than shaking. Both molecules are prone to conformational alterations by light exposure, forming huge amounts of soluble oligomers amongst others. After storage at 50°C some differences can be discriminated between huAb and muAb. The murine antibody seems to be more susceptible for the elevated temperature and revealed more distinct changes. This can be explained by the lower Tm of the murine molecule.

In the murine antibody formulation, which is aimed to be used in subsequent in vivo studies, all investigated stress procedures induced the formation of aggregates. After certain

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incubation time all four methods led to the formation of insoluble aggregates, namely subvisible particles. Besides, heavy modifications of protein structure were detected after storage at 50°C and after light exposure. The latter was also found to be the only procedure leading to high amounts of soluble aggregates. The next chapters will deal with the preparative separation of these species from monomer and fragments.

Regarding all analytical results characterizing the muAb samples, tremendous discrepancies between the stress methods were discovered, which was strongly desired concerning the planned in vivo studies. However, separation of the aggregated species and an affiliated characterization of these aggregate need to be done prior to animal studies.

4.6 REFERENCES

[Chi et al., 2003a], Roles of conformational stability and colloidal stability in the aggregation of recombinant human granulocyte colony-stimulating factor, Protein Science, 12, 903-913 [Chi et al., 2003b], Physical Stability of Proteins in Aqueous Solution: Mechanism and Driving Forces in Nonnative Protein Aggregation, Pharmaceutical Research, 20, 1325-1336

[Datamonitor, 2010] Monoclonal Antibodies: 2010,

http://www.datamonitor.com/store/Product/monoclonal_antibodies_2010?productid=HC00029-00002

[Huvaere et al., 2009], Light-Induced Oxidation of Tryptophan and Histidine. Reactivity of Aromatic N-Heterocycles toward Triplet-Excited Flavins, Journal of the American Chemical Society, 131, 8049–8060

[Ishikawa et al., 2010], Influence of pH on heat-induced aggregation and degradation of therapeutic monoclonal antibodies., Biological and Pharmaceutical Bulletin, 33, 1413-1417 [Janeway et al., 2004], Immunobiology: The Immune System in Health and Disease, 6th Edition,

[Kendrick et al., 1998], Aggregation of Recombinant Human Interferon Gamma: Kinetics and Structural Transitions, J. Pharm. Sci., 87, 1069-1076

[Kiese et al., 2010], Equilibrium studies of protein aggregates and homogeneous nucleation in protein formulation, J. Pharm. Sci., 99, 632-644

[Krishnamurthy et al., 2002], The stability factor: importance in formulation development, Current Pharmaceutical Biotechnology, 3, 361-371

[Krishnan et al., 2002], Aggregation of Granulocyte Colony Stimulating Factor under Physiological Conditions: Characterization and Thermodynamic Inhibition, Biochemistry, 41, 6422-6431

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[Manning et al., 1995], Approaches for increasing the solution stability or proteins, Biotechnology and Bioengineering, 48, 506-512

[PhEur 2.2.1., 2011], Clarity and degree of opalescence of liquids, European Directorate for the Quality of Medicine (EDQM), 7th edition,

[Philo et al., 2009], Mechanisms of protein aggregation, Current Pharmaceutical Biotechnology, 10, 348-351

[Roskos et al., 2004], The clinical pharmacology of therapeutic monoclonal antibodies, Drug Development Research, 61, 108-120

[Wang, 2005], Protein aggregation and its inhibition in biopharmaceutics, International Journal of Pharmaceutics, 289, 1-30

[Wang et al., 2006], Antibody structure, instability, and formulation, J. Pharm. Sci., 96, 1-26

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5 F RACTIONATION OF P ^ROTEIN A GGREGATES OF A H ^UMAN M ^ONOCLONAL A NTIBODY BY AF4

5.1 INTRODUCTION

The aim of this study was to develop a preparative AF4 method to separate soluble aggregates of a monoclonal antibody from the concurrently existing monomeric species and fragments in preparation of an in vivo study. A human monoclonal antibody of IgG1 isotype (huAb) should first be utilized for method development and optimization. This step aims to achieve a suitable concentration of a certain protein aggregate that is stable during several days of storage. The final concentration of the protein aggregates after fractionation should at least exceed 250 µg/mL, due to limited volumes that can be administered subcutaneously. The separated fractions were characterized as far as possible. Furthermore, it was checked whether those aggregates separated from concomitant protein species show a tendency to dissociate during storage. Later on, the entire procedure was transferred and reassigned to a mouse monoclonal antibody (muAb) and the immunogenicity of these aggregates was evaluated in an animal study. -80°C and 2-8°C storage were chosen as appropriate conditions for the preparation of in vivo studies.

Due to the investigations on various methods capable of inducing aggregation in the model huAb (see chapter 3 of this thesis), light exposure was utilized to provide a suitable distribution of soluble aggregates for this study. It was shown that the conditions of irradiation resulted in abundant amounts of huAb-oligomers that were differentiable by SEC. Therefore, a separation via asymmetrical flow field-flow fractionation seemed to be feasible as well.

5.1.1 The use of asymmetrical flow field-flow fractionation (AF4) to separate