Study of aggregation in therapeutic monoclonal antibodies subjected to stress and long-term stability tests by analyzing size exclusion liquid chromatographic profiles

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Abstract

Research into stress and stability is essential during the development of therapeutic proteins to ensure quality and safety of the final medicine. Greater knowledge of the effects of stress on aggregation can help avoid undesirable conformational and colloidal instabilities. With this in mind we investigated five marketed therapeutic monoclonal antibodies (mAbs) namely bebacizumab (BVZ), cetuximab (CTX), infliximab (IFX), rituximab (RTX) and tratuzumab (TTZ) in their innovative medicines. These were submitted to different controlled stresses, to freeze/thaw cycles and used for long-term stability studies once the vials were opened. Aggregate formation was tracked by analyzing the mAbs chromatographic profiles by size-exclusion liquid chromatography coupled with diode array detection. Results indicated that the tendency to aggregate depends on the particular stress conditions and on the concentration and nature of the mAb, even though all share similar IgG1-structure. Fragmentation of the mAb produced by the stress was probably due to the rupture of cystines between the two heavy chains. Regarding stability study, BVZ, RTX and TTZ proved to be the most stable when stored at 4 °C and in freeze/thaw cycles with no tendency to form aggregates. INF tends to form aggregates at 0.5 mg/mL, while in CTX, the most unstable, degradation was detected.

Introduction

Protein aggregation is one of the most challenging problems in pharmaceutical monoclonal antibody (mAb) drug formulations [1]. The self-association of protein entities to form aggregates is directly related with final product quality in terms of its efficacy and safety, in that large aggregates can induce immunogenicity [2, 3]. The dynamic nature of the structure of the mAbs, typical of proteins, leads to a natural tendency to form aggregates, the disruption of the fragile balance of interactions that maintains the three dimensional structure (which is held together by a combination of Van der Waals forces, hydrogen bonds, disulfide linkages, and hydrophobic interactions). This promotes the exposition of the internal hydrophobic amino acid residues to the aqueous medium, which interacts with other protein entities forming higher order aggregation [4]. Nevertheless, aggregation can arise from both covalent and non-covalent interactions of protein molecules [5]. In solution, the native structure of mAbs is easily affected by a wider group of physical and chemical stresses such as heat, pH, pressure, organic solvents, oxidative conditions, light etc., that modify the protein's three-dimensional structure [6]. In addition to promoting the loss of biological functions, these modifications could lead directly to the formation of undesirable irreversible aggregates (not natural aggregates). As aggregation can affect a therapeutic mAb at any stage of its life, from its development and production to its final intravenous administration to the patient in solution, it is important to control this process throughout this period.

Among the most widely used therapeutic mAbs in the world, are bevacizumab (BVZ), cetuximab (CTX), infliximab (IFX), rituximab (RTX) and trastuzumab (TTZ). Each of these five mAbs is the active principle ingredient of medicines used in hospitals. This means that they are handled and/or prepared in solutions in the hospital pharmacy unit before delivery to patients by infusion. In this process, they can be submitted (often by accident) to stress factors, such as those cited above, in which there is a risk of aggregate formation. BVZ (Avastin®) is indicated for the treatment of several kinds of cancer (metastatic carcinoma of the colon or rectum, breast cancer, lung cancer, etc.) [7] and also frequently used off-label in intravitreal injections for treating age-related macular degeneration (AMD) [8, 9], choroidal neovascularisation [10], diabeticretinopathies [11] and neovascular glaucoma [12]. CTX (Erbitux®) is a potent chimeric mouse and/or human mAb, approved worldwide for the treatment of colon, head and neck cancers [13]. IFX (Remicade®) is mainly used for the treatment of psoriasis, Crohn's disease, ankylosing spondylitis, psoriatic arthritis, rheumatoid arthritis, and ulcerative colitis [14]. RTX (Mabthera®) is indicated for use in the treatment of non-Hodgkin's lymphoma [15, 16], rheumatoid polyarthritis [15, 17] and chronic lymphoid leukemia [18]. TTZ (Herceptin ®) is indicated for the treatment of patients with metastatic breast cancer whose tumors overexpress HER2 (25% of the patients) [19]. These mAbs belong to the subclass IgG1, with a shared general structure (Fig. 1) differing mainly in the variable region, where the complementary determining region is located. They also have different glycation patterns, leading to specific characteristics that could be related with their tendency to form aggregates [20], either natural or unnatural.

Several analytical techniques can be used to track aggregation in therapeutic mAbs [1]. An interesting review of the techniques currently in use in the biopharmaceutical field for aggregation quantification and sizing and for structural analysis of aggregates is provided by S. Oshinbolu et al. [21]. Dynamic light scattering (DLS) is commonly used to estimate the size and relative percentage of aggregates and their hydrodynamic radio [22] between 1 nm and 5 μm [23]. Nevertheless, size exclusion chromatography (SEC) is the most commonly used analytical technique for quantifying and sizing soluble aggregates (<50 nm) in the biopharmaceutical field. This is due to its high precision, robustness and simplicity. This well-known, well-established technique bases its analysis on the column separations of mAb monomers and aggregates by size. SEC coupled with diode array detection (DAD) can also provide information about modifications in the structure by tracking changes in the UV mAb spectra [24].

In view of all the above, the goal of this research is to find out more about the aggregation of the five most widely used therapeutic mAbs, i.e. BVZ, CTX, INF, RTZ and TTZ (all marketed worldwide), in order to provide important, valuable information about their behavior when used and handled in hospitals. Stability studies were performed over a period of 15 days on open vials of these medicines. The medicines were stored refrigerated (at 4 °C) and frozen (at −20 °C). Several dilutions of INF and RTX within the concentration ranges for clinical use were also included in the study. In this way, we obtained data to help us evaluate the possibility of reusing the daily surplus of these very expensive medicines in hospitals, so contributing to the sustainability of health care systems. SEC High Performance Liquid Chromatography (HPLC) coupled with DAD was used to track aggregation throughout the study. In previous research this analytical technique was used for tracking light-induced aggregation [25]. A SEC-HPLC/DAD profile was characterized for each fresh mAb and later compared with those obtained in the stress study to detect aggregation and/or disruption of the protein chain into smaller pieces. Once we had corroborated the ability of this method to track aggregation by analyzing stressed samples, we began conducting the stability studies.

Section snippets

Materials

Five marketed therapeutic mAbs -all of them IgG1- were used in our study, in their commercially available medicine format. The daily surplus was kindly supplied by the Pharmacy Unit of the University Hospital “San Cecilio” (Granada, Spain). The mAbs and their corresponding medicines are as follows:

BVZ - Avastin® (Roche Pharma AG, Grenzach-Wyhlen, Germany) is a recombinant humanized mAb with a molecular weight of 149 kDa. The medicine, Avastin©, is a solution for perfusion that indicates a

Aggregation profile by SEC of non-degraded therapeutic mAbs

The five therapeutic mAbs were analyzed by SEC-HPLC/DAD in order to obtain their aggregate profile in the conditions in which they are normally used in clinical practice. This profile therefore corresponds to the full therapeutic activity of the mAbs and can be considered an effective quality control. To this end, the medicines were analyzed immediately after opening or preparation as in the case of Remicade® (lyophilized powder for dissolution in water), and always within the expiry date. This

Conclusion

This paper offers an in-depth study of the aggregation process in the five most commonly used therapeutic mAbs worldwide. The mAbs were tested in their innovative medicine formulations, i.e. BBZ (Avastin®), CTX (Erbitux®), INF (Remicade®), RTX (Mabthera®) and TTZ (Herceptin®.) For this purpose, we used chromatographic aggregation profiles by SEC-HPLC/DAD, the most frequently used analytical technique for quality control in biopharmaceutical production. Firstly, control SEC aggregation profiles

Conflict of interest

The authors confirm that there is no conflict of interest in the content of this article.

Funding

This work was supported by the Spanish National Institute of Health (Instituto Carlos II), and was therefore also partially supported by European Regional Development Funds (ERDF) [FIS PI10-00201].

Acknowledgments

This project was funded by Project FIS: PI10/00201 from the Instituto Carlos III, Ministerio de Economía y Competitividad, Spain. It was also partially supported by European Regional Development Funds (ERDF). The authors would like to thank the Hospital Pharmacy Unit at the University Hospital of “San Cecilio” for kindly supplying all the medicine samples and the Biomedical Research Foundation “Alejandro Otero” (FIBAO) for the support given during the course of this research.

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