MilliporeSigma’s SAFC^® Raw Material Experts Nelli Erwin and Balu Guduri met with Pharma’s Almanac Editor in Chief David Alvaro, Ph.D., to discuss protein instability and aggregation in biologic drug formulation and the range of stabilizer solutions to overcome these challenges.

David Alvaro (DA): What are the main formulation issues with respect to protein instability?

Nelli Erwin (NE): During the development of a biopharmaceutical product, a number of challenges exist. This includes the inherently limited stability of proteins during manufacturing processes and long-term storage. The protein structure is held together by relatively weak bonds and can be easily destabilized by storage conditions and the stresses that occur during manufacturing. This can lead to the denaturation of proteins, where hydrophobic patches of the protein are exposed to its surface and interact with one another, leading to the formation of larger aggregates. The conformational stability of the protein is mainly affected by extreme pH and/or elevated temperatures.

Similarly, protein aggregation can be induced by the exposure to interfaces, such as water–air or water–container interfaces, in the primary packaging or air bubbles introduced by agitation. Proteins tend to adsorb at these interfaces, which can also result in unfolding and aggregation.

However, proteins can also self-associate in their native or folded states. In general, this can occur at high ionic strength or at pH that is close to the isoelectric point (pI) of the protein. Here, attractive protein–protein interactions are favored due to the lack of electrostatic repulsion forces, and solubility of the protein is reduced. Oxidative stresses caused by exposure to light or the presence of metals and/or oxygen can also lead to protein degradation and aggregation in some cases.

Most proteins are prone to being affected by these types of stresses. In general, though, the stability depends on the nature of the protein itself. The main objective of formulation development is to improve protein stability and prevent aggregation against these stresses. 

DA: What are the ultimate consequences of aggregation and these other stability issues, both from a manufacturing perspective and with respect to efficacy and patient safety?

NE: Regarding efficacy, aggregation may have a negative impact, since protein aggregates generally have reduced or even no biological activity. Patient safety can also be negatively impacted by aggregates, as they are often recognized as foreign entities by the immune system and can thus induce adverse immune responses in patients.

Manufacturability is also an issue. Aggregates can clog membranes during filtration steps and columns during chromatographic purification. They can also block pumps and filling machines.

Balu Guduri (BG): It is worth adding that these manufacturing issues ultimately lead to higher manufacturing costs.

DA: While the goal is obviously to minimize and ideally eliminate aggregates from biologic drug products, is there a defined acceptable level of aggregation or denaturation?

NE: According to regulatory agencies, including the U.S. FDA and the EMEA, injectable solutions need to be practically free of visible particles, and a maximum allowable level of sub-visible particles is described. This is also applicable for protein-based pharmaceutical products and includes not only foreign particles but also protein aggregates. If manufacturers demonstrate that it is not possible to remove all protein aggregates, acceptable levels may be authorized, given that suitable safety measures have been implemented. 

DA: What aspects of the manufacturing process should be targeted to minimize protein aggregation?

NE: As already mentioned, proteins experience different conditions during manufacturing. That includes a wide range of pH, ionic strength, and contact materials during multiple chromatography steps and increased protein concentrations during ultrafiltration/diafiltration, when the final buffer exchange takes place to generate the final product formulation. These steps can critically affect the level of aggregation, but of course other factors, such as mechanical stresses and air–liquid or solid–liquid interfaces, can impact aggregate formation throughout the manufacturing process. Beyond optimization of the pH and the ionic strength of solutions, the use of appropriate excipients significantly increases protein stability. 

DA: Can you explain the different mechanisms by which excipients can help stabilize proteins?

NE:  There are different mechanisms available for different types of excipients. Surfactants are surface-modifying agents that help minimize the adsorption of proteins at interfaces. Sugars, polyols, and amino acids are also commonly used stabilizers; they operate by preferential-exclusion and preferential-binding mechanisms.

In the former, sugars are preferentially excluded from the surface of proteins. Interactions between excipient and protein are not favored. This means that any protein state that has a larger surface area is thermodynamically less favorable, and equilibrium is shifted toward states with smaller surface area, which is typically the folded protein state. In this way, the thermodynamic stability of the protein is increased with respect to unfolding. 

Other excipients, such as some amino acids, interact favorably with proteins via hydrogen bonds or by forming electrostatic and/or hydrophobic interactions with protein side chains. Such interactions can improve protein solubility. Many amino acids can have both stabilizing and destabilizing effects on proteins, depending on solution conditions, amino acid concentration, and the protein itself. Although proteins can (partially) unfold in the presence of these excipients, aggregation can be prevented due to minimized protein–protein interactions. Since this is generally a concentration-dependent effect, the protein refolds to its native state once the excipient is removed. 

DA: With so many types of excipients available for protein stabilization, how does one choose which to use for a given protein? Is there a strategy for determining the optimum formulation?

BG: Most of the time, drug developers have certain categories of excipients that they use for protein stabilization, mostly based on their experience. They often start with sugars or polyols. Depending on the extent of the stability required, they determine the concentrations of these ingredients and choose any additional excipient categories — amino acids, surfactants, salts — to use in conjunction with them and maintain the balance of the formulation. Excipient combinations may also be chosen, considering the route of administration.

NE: Overall, it is quite an empirical approach that is used. Selecting an appropriate formulation composition to improve protein stability can be done step-by-step or more systematically via Design of Experiment (DoE). Assessing stability under stress conditions by using various analytical techniques can help predict long-term stability of proteins. However, there is always a risk that these data sets don’t correlate well.  Due to the complex protein aggregation behavior and its various influencing factors, formulation development remains a significant challenge in the pharmaceutical industry.

DA: What expertise does MilliporeSigma have in this space under the SAFC^® brand? And what is the scope and vision for the Emprove^® portfolio?

BG: Under its SAFC^® portfolio, MilliporeSigma aims to provide pharmaceutical manufacturers with GMP-grade excipients for the formulation of all types of biopharmaceutical products in conjunction with comprehensive regulatory support. All products under the Emprove^® Essential & Expert portfolio are manufactured under IPEC-PQG GMP conditions and are multi-compendial and meet the highest purity standards.

Emprove^® products also come with extensive documentation, including material qualification data, quality management dossiers, and operational excellence dossiers. The latter two facilitate risk assessment and process optimization, enabling our customers to assess the quality with exact values. Overall, we help our pharma customers mitigate any supply risks with respect to their excipients.

The Emprove^® Program includes over 400 pharmaceutical raw and starting materials and a selection of filtration and single-use products. There are three Emprove^® product groups, including Emprove^® API for drug substances, Emprove^® Essential Products, which include excipients intended for use in low-risk (orally delivered) formulations, and Emprove^® Expert, which includes excipients intended for use in high-risk (injected delivery) applications.

Emprove^® Expert grade excipients are suitable for use in parenteral formulations and meet requirements for endotoxins, microbial contamination, and other criteria. They also come with elemental impurity information as determined according to ICH Q3D. As a result, drug manufacturers know exactly what is in their formulation with respect to the excipients they are using.

Our SAFC^® protein stabilizer portfolio under the Emprove^® Expert umbrella includes products like sucrose and trehalose (sugars); mannitol and sorbitol (polyols); glycine, histidine, and arginine (amino acids), EDTA (Triplex^® chelating reagent); and polaxamer 188 (surfactant). With  a comprehensive line of products supported by comprehensive regulatory assistance, we are very much like a one-stop-shop for this aspect of biologics formulation.

DA: Can you take me through the various Emprove^® Expert grade excipients and discuss the advantages of each?

NE: For our Emprove^® Expert grade sucrose, we have developed a purification method that removes nanoparticulate impurities (NPIs) because it has been reported in the literature that NPIs can destabilize proteins and induce protein aggregation and fragmentation. Of course, the purification process also reduces endotoxins (< 0.3 I.U. per gram) and bioburden, so there are many benefits to the Emprove^® Expert sucrose product.

BG: We use the same manufacturing practice as sucrose for our Emprove^® Expert trehalose product, so it is also of the highest purity.

Crystalline glycine often shows a tendency toward caking, clumping, and dust formation, which can reduce process efficiency and safety. In contrast, our granulated glycine is free-flowing and easy to handle. Reduced caking and clumping result in enhanced processability, improving both manufacturing speed and operator safety.

Poloxamer 188 Emprove^® Expert is an attractive alternative to polysorbate, which is known to be susceptible to hydrolysis and oxidation, the degradation products of which are impurities that negatively affect the stability of proteins.

All of the other excipients in the Emprove^® Expert line also have very low endotoxin and microbial specifications compared with other excipients on the market. Apart from the extensive regulatory support we provide, that is one of the primary differentiating factors from other suppliers. 

DA: Is MilliporeSigma working on any new SAFC^® protein stabilizing excipients that you can talk about?

NE: We are continuously working on new technologies that we can provide to our customers to overcome their challenges with protein aggregation — this is one of our primary R&D missions. I can’t provide any details yet, but, on the one hand, we are continuously working on improving the quality of our excipients that are already on the market, and on other hand we are developing new technologies for our customers.

Some of this work is driven by the novel modalities being developed by biopharma manufacturers, such as antibody–drug conjugates, which have very different requirements with respect to protein stability and the prevention of aggregation. Different modalities require different excipients.

DA: Where do you see Emprove^® excipients positioned within the broader excipient market?

BG: The excipient market is highly fragmented. Nevertheless, our SAFC^® portfolio and Emprove^® Program has been on the market a long time and has become quite synonymous with IPEC-PQG GMP grade.

I would also like to note that there are additional differentiators beyond the high quality and regulatory support of our Emprove^® raw and starting materials, such as packaging flexibility. Whatever the customer requires in terms of packaging sizes and packaging material, we can generally tailor a solution to meet their needs.

In addition, based on the requirements of the different pharmacopoeia, we have internally developed single, cross-validated analytical methods for each specification on the certificates of analysis (CoAs) for Emprove^® grade excipients. As a result, rather than having unnecessary long CoAs with multiple tests for each specification value, CoAs are simplified, and our customers have much less QC work to do.

Similarly, in our Emprove^® Operational Excellence Dossiers, we provide trend data based on actual batch results, which can help customers reduce the level of testing required for Emprove^® raw and starting materials. Reduced testing equates to reduced time and cost, which are increasingly important in the biopharma industry, making these aspects of our Emprove^® Program even more differentiating. 

DA: Are there any advantages for customers that purchase additional SAFC^® products from MilliporeSigma along with Emprove^® grade excipients?

BG: Many pharmaceutical manufacturers are looking to simplify their supply chains and purchase the majority of products from one source where possible. MilliporeSigma has the entire range of products, from single-use bioreactors to cell culture media to all of the downstream processing equipment, whether single-use or multiple-use. We also supply all of the chemicals from upstream to final formulation, and we also sell laboratory equipment. Layered on top are all of the services we offer to support each and every unit operation involved in drug production.

For raw material consumables used in GMP drug manufacturing, the majority within our SAFC^® portfolio have been qualified for the Emprove^® Program, and those that haven’t been are undergoing qualification and the construction of their regulatory dossiers.