Stress and Protein Instability During Formulation and Fill/Finish Processes

Biopharmaceutical product manufacturing is a complex process that involves many steps, including drug substance and bulk formulation, filtration, shipping, pooling, filling, lyophilization, inspections, packaging, and storage. During these processes, proteins are subjected to many different forms of stresses, such as agitation, temperature, light exposure, and oxidation. These stresses can lead to protein denaturation and aggregation, which compromises the product quality and can even lead to loss of a production batch in some cases. Testing the impact of these stresses on the drug products and developing the corresponding mitigation strategies are essential to maintaining protein stability during the formulation and fill/finish processes.

Agitation
Agitation is one of the common physical stresses that protein therapeutics are subjected to during the routine manufacturing process. Agitation occurs during mixing, ultrafiltration/diafiltration, pumping, shipping, and filling. The exact mechanism for agitation-induced protein denaturation remains to be established. It has been postulated that agitation can cause protein unfolding at the air-liquid interface to irreversibly expose the interior hydrophobic core of a protein and lead to intermolecular association of nonpolar residues. Polysorbates, despite their inconsistent protective effects against other stresses, are usually effective against agitation-induced protein aggregation. Increase in protein concentration also is reported to decrease aggregation with respect to agitation.

The protein susceptibility to agitation stress can be assessed by numerous methods. Besides vortexing and stirring, the shaking study, which is easy to set up and requires a small volume of sample, is commonly used. The shaking study is initiated by placing the protein solution in a container, such as bag, tube, or vessel, and shaking it at an appropriate speed for up to a few days. Samples are taken at different time points to monitor the key critical quality attributes (CQAs) of the protein formulation.

Some process development teams prefer use of a pumping study, which resembles the actual agitation stress in the pharmaceutical manufacturing process. In this type of study, the protein formulation is placed in a vessel and circulated in a closed loop by a peristaltic pump with samples taken at different time points. Lastly, a mock shipping study, which simulates the agitation and other stresses during transportation, may also be used. The results from this mock study are also useful to define the shipping logistics and to support future shipping validation studies.

Freeze/thaw and time-out-of-refrigeration
Freeze/thaw is another common stress encountered during protein therapeutic manufacturing. Despite all the precautions, accidental freezing during bulk storage or shipment does happen. The storage of a frozen process intermediate, drug substance, or formulated bulk substance demands demonstration of the protein freezing stability. For freeze-dried drug products, freezing stability is a prerequisite for a successful lyophilization process. Also, for a lot of multinational pharmaceutical companies, the drug substance manufacturing, drug product fill/finish, and product release are often located at different sites across the globe. The samples taken during the formulation and fill/finish process need to be shipped from one place to another for characterization or release. Once the samples are received, it may take days for some complex assays, such as peptide mapping by liquid chromatography–mass spectrometry (LC–MS), to be completed. For labile products, these characterization or release samples have to be frozen to maintain their integrity before analyses.

Some proteins are prone to freeze/thaw stress, and the process results in protein aggregation, while others are much more resistant. Studies have shown that freeze/thaw-induced protein aggregation is mainly attributed to interfacial adsorption of protein at ice-liquid interfaces. Other factors, such as freeze-induced buffer pH shift and localized cryoconcentration of excipients and protein, may also contribute to the protein denaturation process. Because most of these protein formulations contain amorphous excipients and their glass-transition temperatures (Tg’) are usually -20 ºC or lower, the -20 ºC freezers, even though they are convenient and widely available, should be avoided for protein freezing and storage. Cryoprotectants and surfactants, such as sucrose and Polysorbate 80, are commonly used to stabilize proteins against freeze/thaw stresses.

A freeze/thaw study, as the name implies, can be used to assess the susceptibility of protein formulation to the freeze/thaw stress. It is commonly done by filling a sterile plastic tube, freezing to -80 ºC then thawing at 2–8 ºC or room temperature for up to 10 cycles. Samples are taken after each cycle and analyzed for protein CQAs.

Like freeze/thaw, thermal stress can also impact protein stability, which happens when protein formulations are removed from the 2–8 ºC storage temperature and are placed at room temperature for formulation or fill/finish processing. At the elevated temperature, proteins may partially unfold, which often leads to protein degradation and aggregation. For a temperature-sensitive formulation, the time-rtout-of-refrigeration should be recorded and tabulated to limit the combined room temperature exposure during manufacturing processes. An accelerated stability study and a temperature cycling study between 2–8 ºC and room temperature can be used to set such a limit.

Oxidation
Protein oxidation is the covalent modification of a protein either by reactive oxygen species directly or by oxidative stress indirectly. Several amino acids, such as methionine, cysteine, histidine, and tryptophan are more sensitive to oxidation than other amino acid residues. Protein oxidation often leads to structural change, loss of activity, and a possible increase in immunogenicity. It is important to monitor the degree of oxidative modification of therapeutic proteins during the formulation process.

It is known that protein oxidations can be induced by the trace metal ions leached from production equipment, as well as oxygen, which is either from the air or dissolved within the buffer. The most common protein oxidation in biotherapeutic manufacturing processes is caused by peroxides, such as hydrogen peroxide (HP) and organic peroxide. Peroxides can be generated after the formulation is exposed to light. Peroxides can also be introduced into protein formulations by water for injection (WFI), which contains up to ~50 ppb HP based on the author’s testing, and by impurities from some of the commonly used excipients, including polysorbate, sucrose, and sorbitol.

In addition, HP can be introduced into protein formulations during the filling process in isolators, which are now widely used due to their increased environmental quality and lower operating cost. The isolator decontamination is normally accomplished by using vaporized hydrogen peroxide (VPHP) at ~700 ppm before being purged extensively with the sterile clean air. However, a trace amount of HP, usually at sub-ppm concentrations, remains in the isolators after the purge and can be absorbed into protein formulations. It is therefore necessary to establish the VPHP exposure limits for protein formulations prior to the filling process.

The VPHP exposure limits can be established empirically by exposing protein samples to the theoretical residual VPHP levels in the isolators and monitoring the real-time and accelerated protein stability. Alternatively, a HP-spiking study, where different concentrations of HP (up to a few ppm) are spiked into the protein formulations to evaluate their impacts on protein stabilities, can be performed. If the protein therapeutic is sensitive to oxidation, it may be necessary to lower the residual VPHP level in the isolators by extending the purge cycle or to limit the total residence time of the filled drug vials in the isolators.

Light exposure
Light exposure is inevitable for protein therapeutics throughout the manufacturing processes, including formulation, filling, inspection, and packaging. Exposure to light can directly cause oxidation of specific amino acid residues in the protein, such as tryptophan, tyrosine, phenylalanine, and serine, and can lead to protein aggregation and inactivation. Indirectly, light exposure can cause protein oxidation by generating reactive oxygen species after first reacted with the buffer or excipients.

It is important to assess the photostability of protein drug substance and drug product to ensure that they can endure the normal manufacturing lighting conditions. Photostability is evaluated by exposing a drug product to both visible and UV lights following International Conference on Harmonization (ICH) guidelines, which recommend an exposure dosage of equal or greater than 1.2 million Lux hr and 200 Watt hr/m² for visible and UV lights, respectively. A systematic photostability testing should consist of both forced degradation and confirmatory tests. Drug product can be presented in the final container/closure system, with or without marketing package, during the exposure. For liquid drug substances or formulated bulks, they should be exposed in chemically inert and transparent containers.

About the Author
Mark Yang is director, late stage process development at Genzyme.

Article DetailsBioPharm International
Vol. 28, No. 6
Pages: 46–47, 49
Citation: When referring to this article, please cite it as M. Yang, "Stress and Protein Instability During Formulation and Fill/Finish Processes," BioPharm International28 (6) 2015.