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Pharmaceutical Manufacturing and Packing Sourcer

Easy Protein Processing

Protein-based drugs are notoriously difficult to formulate, but if their properties are assessed correctly, an effective formulation screening can be performed to render a successful drug substance and final product

Recombinant biologics represent a large and important class of modern therapeutics.The development of a successful biologic medicinal product is the result of optimised process and formulation development activities.The goal is to obtain a stable product during manufacture, storage and use. As biologics have a high molecular weight with almost no permeation through biological membranes, combined with their susceptibility to digestive fluids, the parenteral route is still the preferred route of application. Due to their size and specific structures, physical stability is just as important as chemical stability, and content does not equate to potency as with small molecules.

Taking all this into consideration, biologics are complex molecules and successful formulation requires special strategies.These strategies have to address both, limited availability in the early phases of drug development as well as less time for carrying out experiments due to short development timelines. The goal of early stage drug development is to develop a physically and chemically stable product, addressing not only shelf life, but also manufacturability and compatibility with the processes later in the project. All efforts at this stage result in the first application form (liquid or a lyophilisate) and allow moving an investigational new drug into clinics quicker to test its therapeutic value.

Since ‘the process makes the product’, the impact of process parameters in the quality of the final product has to be assessed early in the development process. Although it is possible to use a service formulation up to proof-ofconcept, early identification of the final robust formulation has the advantage of avoiding changes in the formulation, or requiring repeated studies to prove comparability, safety and efficacy. The typical timepoint for the initiation of a formulation screening is when the project enters the preclinical development phase (see Figure 1).

In around 12 to 14 months, the drug substance manufacturing process has to be developed and a formulation identified. Apart from this, a manufacturing process for the drug product also has to be developed and GLP material for toxicity and stability studies has to be manufactured.Taking this into consideration, the time for a formulation development is limited to six to nine months. Subtracting the time required for performing stability studies, the time left to decide on a formulation for clinical studies is limited to three to six months (see Figure 2).

In developing the same, or at least very similar, protein drugs, a formulation platform might be useful in reducing development time and the risk of failure. Typically, development portfolios show some heterogeneity either in the substructure of protein drugs or more significant differences due to different protein formats; for example with antibodies, where IgGs are accompanied by fragments and engineered with more artificial species like BITEs. A few examples of different antibody formats currently in clinics can be seen in Table 1.

Performing the Formulation

Taking all the factors described into consideration (complexity, different formats, short development timelines and limitations with the availability of the API), an effective and reliable screening procedure for the identification of an optimal formulation is desirable. One approach to master this challenge is to separate physical and chemical stability optimisation, as they are both prerequisite for a successful product. Following this approach, the protein will be screened in a first step for formulation conditions (pH, ionic strength, surfactants, carbohydrates) leading to a physically stable product.This first screening can be done using Thermofluor technology, where the impact of external factors on the stability of a protein in solution can be determined by measuring its unfolding temperature (1).This technology, based on the interaction of a hydrophobic fluorescent dye with hydrophobic domains upon heating, requires only very small amounts of protein and can be done in a short time and using microtitreplates, allowing highthroughput screening experiments. Figure 3 (page 38) shows how this approach allows the screening of several formulation options with small amounts of API at the start of the development, when often research process are available only having non-optimised protein titres.

The Thermofluor technology allows the selection of external factors for the proteins for optimised protein stability in terms of a physical constant. Consequently, when promising formulation candidates have been preselected, they can be tested for physical stability in typical temperature/shear stress experiments. Since continous improvements are made on the API side in parallel, sufficient amounts of API are available to make these experiments in original container closure materials; either at the original scale or downsized. These experiments add real physical stability information, since the protein is stressed under worst-case conditions and stability can be assessed, for example in terms of aggregation (SEC or larger aggregates). Even at this stage, subvisible particles (1-10μm) can be assessed and taken into consideration for further narrowing the list of formulation candidates (3).These experiments allow the identification of ideal formulation parameters, as well as setting the base for a selection of candidate formulations for an orienting stability study to assess chemical stability as well. Figure 4 illustrates the process of the formulation selection process.

Following this approach will require specific amounts of API (as illustrated in Table 2) and can be used for compatibility experiments as well.Adding NaCl to the test solutions provides early information about the stability after a saline dilution or adding solvents; the compatibility in manufacturing processes requiring these solvents (drug delivery systems) may also become predictable.

Besides the low amounts of API to start with, another advantage of this procedure is the high-throughput testing capability, since the Thermofluor test can be carried out in 96 microtitre plates. Throughput can be increased by using liquid handling equipment or a fraction sampler for the preparative SEC for the preparation of the protein in different buffer solutions.


The approach described in this article allows us to address the main challenges in the formulation of protein drugs. Both physical and chemical stability optimisations of formulations are dealt with, and each allows the effective use of resources and time to obtain a stable protein formulation. In several development projects, this approach has been shown to be very effective and efficient, leading to reliable selection decisions in the development process.The concept can be followed with limited resources, and allows scientific support of formulation decisions,backing early proofof- concept. Another advantage of this concept is that the selected formulation will have the potential to be the final formulation, and no formulation related comparability exercises will be required.

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Carsten Olbrich studied Pharmacy at the University of Heidelberg and continued his qualification at the Free University Berlin, completing a PhD in Pharmaceutical Science. Carsten joined Schering AG in 2000 as a Laboratory Head in diagnostic research working on antibody attachment to surfaces of intravenous injectable polymeric particles, and quality methods for targeting specific imaging agents. In 2004, he joined the Pharmaceutical Development Department, where he acted as Associated Director in the CMC Technology Office and Drug Delivery Systems department as a scout for new technologies for CMC. In 2006, he joined the Parenteral Development Group of Bayer Schering Pharma AG as a director and is now a Senior Scientist in the Liquid Dosage Form development department, where he is responsible for drug product development of parenterals, especially biologicals.
Carsten Olbrich
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