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Three case studies illustrate some analytical methods important for stability testing.
Stability testing of biopharmaceuticals must be performed according to regulatory requirements for drug development programs to establish the re-test/expiry date for drugs. During such studies, however, it is not uncommon that atypical or unexpected data may arise, and given the crucial need for these studies, effective and rapid response is necessary to prevent impact on drug-development timelines. The cause of any issue must be identified, and any newly observed impurities/degradants must be characterized and assessed for any risk to safety, quality, and efficacy.
This article presents three common scenarios observed during stability testing of biologics and the use of orthogonal analysis, stability experience, and troubleshooting measures to identify causes, assess risk, and define subsequent steps in the development of a biopharmaceutical.
Protein product and acidic species
In the first study, Molecule X was a lyophilized formulation of a protein, a sterile drug product contained in a vial with an intended long-term storage condition of +5 °C ± 3 °C. The material had been tested by gel isoelectric focusing in the early phase of drug development. The method that was used was standard and had not been further reviewed or optimized during the program. The isoelectric focusing (IEF) gel profiles showed unclear banding with poor resolution.
To provide greater understanding of charged-based variants of the molecule, a method with greater sensitivity was developed using imaged capillary isoelectric focusing (cIEF). As expected, cIEF indicated that the poorly resolved bands on the IEF gel actually represented four charged-based variants. These consisted of the main product isoform that focused at the expected isoelectric point of the product (i.e., at pI [isoelectric point] 6.8), two acidic isoforms, and one basic isoform (Figure 1). This test was validated and incorporated into the release and stability programs for the product. During routine testing at the three-month time point, however, an atypical isoform was observed that had not been detected during previous testing procedures or during method development/validation. The data indicated that the change in cIEF profile was accompanied by a change in the ELISA results for the molecule, thus showing that the atypical peak impacted product activity.
To further characterize this atypical peak, the sample was tested against a frozen retained sample from T=0, by electrospray mass spectrometry quadrupole-time-of-flight (ESI MS Q-ToF) analysis. The data confirmed that the T=3 sample had an increased level of deamidation of approximately 15% (Figure 2). In addition, MS/MS sequencing confirmed that the site of deamidation was located at an asparagine residue that was located in the complementarity-determining region (CDR) of the molecule.
These data correlated with tertiary structural changes identified by near-ultraviolet circular dichroism (near-UV CD), where changes in the spectral profile were observed. Supporting data from Fourier transform infrared (FTIR) analysis showed that secondary structural changes in the reduction of β-sheets had occurred.
The investigation therefore showed that the molecule had an amino acid residue that had an increased propensity to deamidate in the active domain. Unfortunately, this was not identified in early development, as the original method had insufficient resolution to distinguish between charged variants.
Based on the results, the product sequence was reviewed and modified to remove the susceptible residue. The development program was significantly delayed as various additional supporting studies were required. This delay could have been prevented if an optimized method had been developed upfront in the early development phase.
A forced degradation study during early development would confirm key degradation pathways, and determine whether such a method is stability-indicating for the drug molecule or not. These degradation data should also be used in the validation of the chosen method for charge-based variant analysis of the product.
Monoclonal IgG drug substance and particulates
In a second study, Molecule Y was a recombinant protein drug substance in a liquid formulation. The drug product itself was produced following the vialing and lyophilization of the drug substance. The drug substance had an intended long-term storage condition of +5 °C ± 3 °C, and data had been used to establish a two-year shelf life.
During ongoing and established stability studies, activity testing of the drug substance unexpectedly failed at T=6 months. This result did not correlate with historical data or any of the other data available. During the investigation, it was noted that all testing for the sample was performed at site A with the exception of activity testing, which was performed on a small aliquot of the sample at site B. In this case, the shipping procedure for the sample used in activity testing differed from the one normally used. The courier had intended to ship the sample to the testing lab directly, as had been done previously; however, this time the courier had made an error and shipped the sample to an incorrect location. The sample then had to be transported to the correct location. The temperature monitoring data indicated that the cold chain had been adequately maintained throughout the shipment. The only item of note was that because of the need to correct the delivery of the sample, the product had been subjected to additional eight hours of transport by truck, which had not occurred in previous shipments.
Based on this, a small-scale agitation study was performed in which the product was subjected to agitation at 30 rpm over 0, 4, 8, 16 and 24 hours. Samples were tested using low-volume methods to assess levels of visible particles by visual appearance, sub-visible particles by light obscuration and smaller size particles by size exclusion chromatography (SEC) and dynamic light scattering (DLS).
DLS was used to assess the levels of higher-molecular-weight (HMW) species at particle sizes from 10nm to 1μm. The results showed significant levels of higher MW species in the later time points compared with the control sample at T=0 hours, which was demonstrated in a series of correlograms (Figure 3). The control sample showed a steep sigmoidal correlogram as a result of the correlogram function, indicating small monomeric-type species, whereas the degraded sample showed a broad, slow decay, indicating the formation of multiple, large, slowly diffusing, high-MW species. It was also observed in the sample wells that no particles were evident in the T=0 hours well, but it was obvious that there were particles in the T=16 hours sample well. These particles were identified as consisting of dimers, trimers, and multimers of the drug molecule, and it was concluded that the material was sensitive to agitation. This meant that shipment as a liquid was not a suitable procedure to use for this drug product.
Following confirmation that the product was stable after a freeze/thaw test procedure, the shipment conditions for the material were selected as frozen. This issue could have been identified during early development had an agitation assessment been performed. It is recommended that an agitation assessment be performed as part of an early-phase forced-degradation study or that it is incorporated into the first stability study.
Hormone drug product temperature excursion
In a third study, Molecule Z, a hormone drug product in a liquid formulation, had an intended long-term storage condition of +5 °C ± 3 °C. The product was in ongoing Phase II clinical trials, and during shipment of a batch for use in the next stage of clinical trials, the temperature that the sample was being held at dropped to 1 °C, outside of the required range.
During shipment, customs had retained the product at the airport to review paperwork. Such a delay is not uncommon; however, during this period the container was held outside in cold weather conditions, and the temperature in the container dropped to 1 °C for up to 5 hours (Figure 4). The cold-chain data for this product indicated it was not stable upon freeze/thaw at temperatures ranging from -20 °C to ambient; however, no data were available at 1 °C. The regulators were informed and the clinical trial was put on hold until the quality of the material was reviewed.
An urgent excursion study on the material was performed, with a representative sample being taken from the batch and held in a programmed thermal cycling chamber at +5 °C for 4 hours, followed by 1 °C for 5, 10 and 15 hours. Stability tests were performed on an unexposed control sample and on the exposed samples, with the resulting data confirming that the product quality was unaffected following this level of exposure. The data were used to provide scientifically sound justification that product quality had not been affected and the clinical trials were continued.
Excursion studies for new products are not mandatory. Studies that include mapping of the intended shipment routes, however, can be used to perform risk analyses and identify any high-risk excursions in advance. Conducting a small-scale study to identify the impact of most likely shipping events upfront allows drug developers to be proactive, rather than reactive, when an unexpected temperature excursion occurs.
Stability studies are an integral part of drug development with stringent timelines for analytical testing. However, it is not uncommon that atypical or unexpected stability data may arise that cause severe impact on the drug development program. It is crucial, therefore, for drug developers to take rapid and effective action that minimizes the risk to their timelines and satisfies regulatory authorities. As discussed in this article, it is recommended that potential risks to the biopharmaceutical due to temperature excursions or agitation during shipment are identified in early development rather than late-phase stages. Furthermore, it is vital to develop appropriate stability-indicating analytical methods to be able to identify degradants that present potential risk to patient safety.
ALL FIGURES ARE COURTESY OF THE AUTHOR.
About the Author
Stella-Christiana Chotou is team leader, stability services, both at SGS Life Science Services.
Article DetailsBioPharm International
Vol. 28, No. 9
Citation: When referring to this article, please cite it as S.-C. Chotou, "Stability Testing in Biopharma," BioPharm International28 (9) 2015.