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Achieving high concentration protein/antibody formulations

1. Introduction

As a multitude of protein pharmaceuticals are introduced to the market, investigations are focusing on various opportunities to improve the competitive aspects of the products. As research regarding other routes of delivery generally conclude that protein therapeutics require parenteral delivery until further innovations become available, the fields for advancement are limited to delivery devices and formulations. Competitive formulation improvement can be effective in earning market preference by offering convenient storage, user-friendly preparation, patient self-administration, availability to attractive injection devices, etc. This article introduces a unique approach to achieving more stable formulations with lower viscosity by using combinations of amino acids. The benefits of this approach are demonstrated in lyophilized and liquid formulations for intravenous and subcutaneous delivery.

While proteins typically demonstrate good solubility in an aqueous environment, issues such as irreversible aggregation, irreversible precipitation, and/or high viscosity emerge at elevated concentrations. The practical feasibility of whether proteins can be concentrated beyond protein and water weight ratios of 1:3 (about 350 mg/mL), due to volumetric contributions of the protein, is unclear. Even if stability could be managed at such high concentration, bulk viscosity would exceed the capabilities of current manufacturing practices or available parenteral delivery methods. Therefore, this article will focus on practical solutions for achieving commercially viable protein formulations with concentrations up to 350 mg/mL.

1.1 General observations at higher protein concentrations

Consider that a typical aqueous protein solution has an approximate density of 1.0 mg/mL. In a 200 mg/mL formulation, 0.2 mL of solution volume is occupied by protein molecules. In other words, 200 mg of protein is dissolved not in 1 mL of water but in 0.8 mL of water to make the 200 mg/mL solution (water to protein ratio of 4:1). As protein concentrations increase, i.e., the relative volume occupied by protein molecules increases, the amount of water required to dissolve the protein effectively decreases (Figure 1).

Figure 1. Volumetric contribution of water at various protein concentrations 

In such a congested environment, the kinetics of various intermolecular interactions among protein molecules would shift, resulting in changes in large scale physical properties. Multiple weak interactions can collectively contribute to molecule-dependent intermolecular associations (Connolly et al, 2012). Such interactions may promote undesirable degradations, e.g., aggregation and precipitation during manufacturing and storage, and/or undesirable bulk physical properties such as increased viscosity, which can be prohibitive in terms of manufacturing and delivery (Shire et al, 2004).

Examples of issues experienced from protein formulations at high concentration are shown in Figure 2.

Figure 2. Examples of high concentration protein formulations (from left: turbid, precipitated, gelled with high viscosity)

1.2 Screening formulations at higher concentrations

The leading practical challenges encountered in developing high concentration formulations arise from limitations in available experimental and analytical tools for identifying leading formulations. Another practical challenge involves the large drug substance consumption required during formulation development.

Often, for formulations with little prior development, higher concentrations may not initially appear attainable. Performing basic formulation screening at lower concentration identifies optimal protein-specific formulation conditions that may sustain higher concentrations. In addition to basic formulation screening, it is essential to address relevant issues that arise at higher concentration. Major degradations that dominate at lower protein concentrations may not necessarily represent the major degradations that occur at higher concentrations. In other words, the leading formulations or stabilizers identified at lower concentration may differ from the leading formulations or stabilizers appropriate at higher concentrations.

A commonly used experimental tool for high throughput formulation screening is the use of elevated temperature to stress the protein. Theoretically, accelerated stability studies are designed to expedite the major degradation pathways to identify optimal formulation candidates in a short period of time. However, the elevated temperatures used in these studies require careful testing to target only relevant degradants and degradation pathways. At high temperatures, similar degradation products, e.g., aggregation or precipitation can be produced by vastly different underlying mechanisms that may not be relevant to the real-time stability of the protein. Despite the efficiency and convenience of high temperature high throughput screening platforms, it is important to note that if appropriate stress conditions are not implemented, the leading formulations may be obscured by formulations, which stabilize irrelevant degradation pathways.

Ideally, analytical methods for characterizing high concentration samples should maximize the efficiency of screening and also minimize the total amount of sample required for analysis. For example, a method which can support the simultaneous processing and measurement of 50 to 100 samples, e.g., 96 plate format, with minimal sample volume, e.g., less than 100 μL, would be ideal. However, many of the degradants and formulation properties discussed above require product-specific methods developed to detect specific stability issues. Lengthy analytical times, e.g., HPLC, and or larger volume sample requirement, e.g., subvisible particle counting or viscosity measurements, may be unavoidable for some proteins.

2. Current approaches for developing high concentration formulations

Since the increase of demands for high concentration formulations starting in the early 2000’s, numerous high concentration formulations have successfully been achieved through various techniques. These include optimization of pH, use of salts, or addition of individual amino acids befitting the protein of interest. However, these techniques have proven somewhat limited in effectiveness, achieving moderate concentration ranges within a small range of proteins. The highest concentration protein products introduced to market have been limited to albumin (250 mg/mL), immunoglobulin (200 mg/mL), and certolizumab pegol (200 mg/mL). Novel technologies or screening processes that can achieve higher concentration and be applied to a large variety of proteins are desperately needed at the time. 

3. Using of amino acid combinations for developing high concentration formulations

To date, overall results from studies suggest that most issues observed for protein formulations at high concentration are related to increases in intermolecular interactions. This is not unexpected, as the purpose of many proteins is to functionally interact with other biological materials, including other proteins. The primary, secondary, tertiary, and quaternary structures all chemically and physically contribute to form unique surface structure(s) to facilitate or disrupt these interactions. When many proteins are forced to occupy a small space, weak intermolecular interactions can form and cumulatively introduce the various problems discussed previously. These interactions form at protein surfaces, which have sites of varying charge, structure, and hydrophobicity. By adding a free amino acid to the formulation, the amino acid would interact with protein surface sites with complimentary characteristics and mask the site, obstructing the formation of intermolecular interactions. Combinations of amino acids would be even more effective, as they could cover a variety of protein surface sites with different characteristics, further inhibiting the formation of intermolecular interactions.

The combinatorial effects of amino acids in stabilizing high concentration protein formulations can be observed for the protein infliximab. Infliximab forms insoluble aggregates at high concentration (Figure 3). The rate of precipitation is substantially reduced when single stabilizing amino acids are added. When the stabilizing amino acids are added in combination, at the same collective amino acid concentration (w/v), significantly improved stability was observed (Figure 3).

Figure 3. Stabilization of infliximab by multiple amino acids


The benefit of using amino acid combinations to address separate stability and viscosity issues can be observed for the protein trastuzumab (Figure 4). Under stress, trastuzumab at 100 mg/mL forms insoluble aggregates. The addition of a stabilizing amino acid effectively reduces the rate of precipitation while another amino acid, L-proline, is not as effective (Figure 4-a). However, L-proline is effective in reducing the viscosity of trastuzumab at high concentration, while the stabilizing amino acid is not effective (Figure 4-b). By combining the stabilizing amino acid and L-proline, the stability of trastuzumab was improved while simultaneously reducing formulation viscosity (Figure 4).

Figure 4. Use of amino acids for high concentration trastuzumab formulation

(a) Stabilization by amino acids

(b) Reduction of viscosity by amino acids

Similar experiments were conducted using various commercially available protein pharmaceuticals, including cytokines, hormones, enzymes, fusion proteins, plasma derived proteins, antibodies, etc. Various combinations of amino acids successfully addressed most issues experienced at higher protein concentrations (data available under CDA).

3. Protein-Specific High Throughput Screening

Due to the limited availability of resources, time, and material, developing a high concentration formulation can be very expensive, if not impossible, unless a very efficient screening process is available. As the use of amino acid combinations has been effective with a variety of proteins, the screening process can be focused on amino acids. Even so, the matrix size can become rather large considering the many possible concentrations and combinations of amino acids. Therefore, developing efficient screening protocols, i.e., rapid preparation, treatment, analyses of samples, that minimize the amount of drug substance, needs to be developed. It is crucial to design the screening conditions to reflect relevant stability issues, as rate-limiting steps may change when formulations are subjected to high temperature accelerated stability conditions. Likewise, it is preferred that the screening studies are carried out at the highest feasible concentrations to elicit degradation pathways and issues, which occur at higher concentrations.

The accurate and reproducible quantification of bulk viscosity, as the name suggests, typically requires significant sample volumes when compared to other analytical methods. This equates to large amounts of drug substance required for high concentration sample preparation. Numerous experimental techniques and instrumentations have been developed to require less than 100 μL of sample volume. These include small volume viscometers like RheoSense’s m-VROC™ viscometer (www.rheosense.com), custom-designed injectability techniques using electromechanical testers, use of dynamic scattering (Smidt and Crommelin, 1991; Neergaard et al, 2013), and molecular rotor dyes (Haidekker et al, 2010).

The potential benefits of high concentration formulations, both for biopharmaceutical companies and patients, have made it an alluring goal for many protein products. With the continued development of high concentration formulation technologies, high throughput formulation screening techniques, and innovative analytical methods, more biopharmaceutical products can be introduced to the market at high concentrations.

REFERENCES

Brian D. Connolly, Chris Petry, Sandeep Yadav, Barthe´lemy Demeule, Natalie Ciaccio, Jamie M. R. Moore, Steven J. Shire, and Yatin R. Gokarn (2012) Weak Interactions Govern the Viscosity of Concentrated Antibody Solutions: High-Throughput Analysis Using the Diffusion Interaction Parameter Biophys. J. 103: 69–78

Mark A. Haidekker, Matthew Nipper, Adnan Mustafic, Darcy Lichlyter, Marianna Dakanali, and Emmanuel A. Theodorakis. (2010) Dyes with Segmental Mobility: Molecular Rotors. Advanced Fluorescence Reporters in Chemistry and Biology I: Fundamentals and Molecular Design, A.P. Demchenko (ed.), Springer Ser Fluoresc 8: 267–308

Martin S. Neergaard, Devendra S. Kalonia, Henrik Parshad, Anders D. Nielsen, Eva H. Møller, Marco van de Weert (2013) Viscosity of high concentration protein formulations of monoclonal antibodies of the IgG1 and IgG4 subclass – Prediction of viscosity through protein–protein interaction measurements. Eur. J. Pharm Sci. 49: 400-410

Steven J. Shire, Zahra Shahrokh, Jun Liu (2004) Challenges in the Development of High Protein Concentration Formulations. J. Pharm Sci. 93: 1390-1402

Jan H. De Smidt and Daan J.A. Crommelin (1991) Viscosity measurement in aqueous polymer solutions by dynamic light scattering. Int. J. Pharm. 77: 261-264

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