Assessing Aluminum Vaccine Adjuvant Filling, Sedimentation, and Resuspension in Sealed Vials using Water Proton NMR

Abstract

The filling level, sedimentation, and resuspension of aluminum adjuvants (alum) in sealed vials can be quantitatively assessed in situ using the water proton NMR (wNMR) technology. wNMR demonstrates high sensitivity and high throughput capacity (10-40 sec per vial). wNMR makes it possible to quantify alum filling and suspension in every vial in a batch before product release by vaccine makers and before injection by end-users.

Introduction

Aluminum salts are the most widely used vaccine adjuvants, appearing in over twenty marketed vaccine products.1 Aluminum salts are covalent hydroxocomplexes between Al(III) and anions, such as OH-, PO4 3- and SO4 2-. These complexes form insoluble nanometersized primary particles which then agglomerate into irregular-shaped micrometer-sized particles.2 Aluminum-adjuvanted vaccines are formulated as aqueous suspensions. Aluminum salt particles, with or without adsorbed antigens, are heavier than water and thereby tend to sediment. This tendency to sediment creates potential quality problems for aluminum-adjuvanted vaccines, both before and after product release.

Sedimentation of the suspended particles during the fill-finish step of manufacturing may cause uneven filling of vials (here, the vials refer to any form of containers). Uneven filling, if severe enough, may lead to mis-dosing. One such example is Novomix 30®, where a manufacturing error in 2013 caused up to 50% over- or under-filling of the vial with insulin, i.e., insulin concentration deviates from the specified value by up to ±50%.3 A patient collapsed in a hypoglycemic coma state after taking this product in September, 2013.4 One month later, 33 batches of Novomix 30® were recalled.3 Like aluminum-adjuvanted vaccines, Novomix 30® is an aqueous suspension containing insoluble insulin particles. In general, suspensions have more complex flow properties than solutions, making uneven filling of the vials more likely. For some vaccines, 50% over-filling of aluminum salts will exceed the regulatory approved limit of the aluminum content - 0.85 mg of Al(III) per dose in US and 1.25 mg Al(III) per dose in Europe.5 Under-filling, on the other hand, might result in subpar efficacy of vaccines.

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After product release, aluminum-adjuvanted vaccines sediment to the bottom of the vial during transportation and storage. Package inserts typically instruct rigorous shaking immediately before use. Incomplete resuspension may lead to product recall. For example, in April 2010, the World Health Organization recommended recall and destruction of all lots of SHAN5 vaccine that contained particles that were not fully re-suspended by shaking.6 Shipping stresses may make re-suspension difficult, with significant vial-to-vial variability.7 Pre-release uneven filling and post-release shipping stress may cause defects at the drug product (DP) level that are not detectable by prefilling testing of the drug substance (DS), no matter how extensive the testing is. Such DP-level defects may display vial-to-vial variability and thereby pose stiff challenges to quality control (QC); unless every vial in a batch is quantitatively inspected, serious DP-level defects may escape detection.

In the context of aluminum-adjuvanted vaccines, current analytical technologies include atomic adsorption spectroscopy (AAS)8 and 27Al NMR spectroscopy9 to assess aluminum filling level, UV absorption spectroscopy,7 optical scanning analysis,10 microCT,11 and visual observation (the WHO shake test)12 to assess aluminum particle sedimentation and resuspension. All these technologies, except microCT and visual observations, are ex situ, i.e., they require taking the DS out of the vial for analysis, thereby compromising DP integrity. MicroCT and visual inspection are in situ methods and do not compromise DP integrity. However, microCT involves ionizing radiation, expensive equipment and complex and time-consuming procedures. Visual observation is subjective and might be difficult to execute, depending on vial size and labelling (a vial containing 0.5 mL whitish suspension in a sealed and labeled vial is challenging for visual inspection).

When an analytical procedure compromises DP integrity, it can only be applied to a small percentage of the fi nished vials in each batch (statistical sampling). This feature makes it hard to catch pre-release DP defects caused by manufacturing errors if the defect rate is low. For example, in the aforementioned Novomix 30® case, the defect rate was 0.14%.3 Also, the complexity and cost of current analytical procedures restrict their use to highly trained personnel at the manufacturing plant. This feature precludes their implementation at the point-of-care by end-users to detect post-release defects caused by shipping stresses.

To catch DP-defects, whether they occurred pre- or post-release, in situ analytical technologies that maintain DP integrity are needed. Here, we report a technology that can quantitatively assess aluminum adjuvant filling level, sedimentation, and resuspension in sealed and labelled vials. It builds on the water proton NMR (wNMR) technology we previously presented in this journal.13

Suspended, Sedimented, and Resuspended Aluminum Adjuvants

Aqueous suspensions of two aluminum vaccine adjuvants – aluminum oxyhydroxide [Alhydrogel®85] and aluminum phosphate [Adju-Phos®] (both manufactured by Brenntag Biosector and distributed by Sergeant Adjuvants)—were prepared in sealed vials (4 mL total volume). The original commercial suspensions were diluted using water to give a concentration range of 0.1 to 5 mg/mL (here, weight refers to that of elemental (Al(III)). The goal is to determine the response of wNMR to aluminum salt fi lling concentration; the sensitivity of such responses forms the basis to detect potential fi lling errors during manufacturing. The same samples were also used to verify the sensitivity of wNMR towards aluminum salt sedimentation and resuspension. To this end, prior to the analysis, samples were allowed to sediment in an undisturbed environment for 2-3 weeks at 4°C. The fully sedimented aluminum salts were resuspended by vigorous shaking. wNMR data were collected on the fully sedimented and resuspended samples. Figure 1 shows photos of suspended and sedimented aluminum adjuvants in sealed vials.

wNMR measurements were performed in sealed and labelled vials, which were loaded into the NMR instrument using a plastic sample holder (Figure 2). The same parameters and experimental settings were used to measure suspended, sedimented, and resuspended samples. The water proton transverse relaxation rate, R2(1H2O), was measured using standard CPMG pulse sequence14 with 2 transients. The instrument was a low-field (0.56 T, 24 MHz) wide-bore (26 mm) benchtop NMR instrument. Duration of a single measurement for the fully suspended samples, depending on the aluminum adjuvant concentration, varied from 10 to 40 seconds. Duration of a single measurement of the fully sedimented samples within all concentration ranges was 40 seconds. For each vial, the measurement was repeated thrice. Alhydrogel® and AdjuPhos® were stored at 4°C respectively for 21 and 14 days for sedimentation of the aluminum salt to evaluate whether storage time has impact on the extent of resuspension.

Water Proton NMR Sensitivity toward Filling, Sedimentation and Resuspension of Aluminum Adjuvants

For both Alhydrogel® and Adju-Phos®, in the fully suspended states, wNMR demonstrated high sensitivity towards the changes in the concentration of aluminum salt. As seen from Figure 3, for both adjuvants, the dependences of R2(1H2O) vs. Al(III) concentration were perfectly linear with extremely high response eff ectiveness of 1-2 s-1 per mg/mL (cf. with < 0.1 s-1 per mg/mL, e.g., proteins).13

Figure 3 also shows about 70% larger response eff ectiveness observed for Adju-Phos® compared to Alhydrogel®. Alhydrogel® and AdjuPhos® differ in charge, exchangeable protons per Al(III), and proton exchange rates, which may all contribute to the observed diff erences in response. For example, Alhydrogel® particles are positively charged, while Adju-Phos® particles are negatively charged.15 Negatively charged Adju-Phos® particles likely exchange proton with water molecules more efficiently than positively charged Alhydrogel®, resulting in a higher water proton relaxation rate R2(1H2O). Also, differences in R2(1H2O) values may in part be the result of differences in average hydrodynamic diameters between Adju-Phos® particles and Alhydrogel® particles.16

Once fully sedimented, both Alhydrogel® and Adju-Phos®, demonstrated a sharp drop in the observed R2(1H2O) values within the whole range of the concentrations under study (Figure 4). In fact, R2(1H2O) in sedimented samples displayed little change over the experimental concentration range for both adjuvants. And, as seen from Figure 4, for both adjuvants, resuspension of the aluminum salt particles brings the R2(1H2O) values back to the levels observed for the original suspensions. This forms the basis for using R2(1H2O) to evaluate the extent and uniformity of vial filling in each batch.

Of note, our results demonstrate high sensitivity of wNMR to aluminum adjuvant concentration in the fully suspended state. Insets in Figure 3 show the response effectiveness in the very low concentration range, from 0.0 to 0.1 mg/mL of Al(III). The aluminum concentration range used in this work (0.1 to 5 mg/mL) likely encompasses any realistic adjuvant concentration variation caused by filling errors. Therefore, the resulting R2(1H2O) response effectiveness could be used to estimate the wNMR sensitivity to potential filling errors in vaccine DPs containing aluminum oxyhydroxide (such as Alhydrogel®) or aluminum phosphate (such as Adju-Phos®). Table 1 shows the estimated R2(1H2O) changes in response to various levels of fi lling errors for two vaccine products, Pediarix® containing 1.70 mg/ mL of Al(III) in the form of aluminum oxyhydroxide, and TDVAXTM containing 1.06 mg/mL of Al (III) in the form of aluminum phosphate.

Thus, the high sensitivity of wNMR as shown in Figure 3 supports its application as a noninvasive analytical tool for accurate quantification of the filling level, sedimentation, and resuspension of aluminumadjuvanted vaccines.

Comparison of wNMR with Other Techniques

Compared with other analytical techniques, such as atomic adsorption spectroscopy, 27Al NMR spectroscopy, UV and fluorescence spectroscopy, and optical scanning analysis, the foremost advantage of wNMR is that it is an in situ technology, capable of quantitative inspection of sealed vials. As opposed to other techniques, wNMR does not require opening the sealed vial, and thus does not compromise the DP integrity. Secondary advantages including simplicity, affordability, high throughput potential and the ability to quantify, make wNMR more preferable than micro-CT or visual inspection for in situ inspection. These advantages arise because wNMR detects not the DS, but water, which is by far the most abundant component of all aluminum-adjuvanted vaccines.

The disadvantage of wNMR is that it is an indirect technology; it does not monitor aluminum adjuvants directly. To convert R2(1H2O) of a vial into absolute Al(III) content, the vaccine maker needs to establish a calibration curve for each product, as shown in Figure 3. wNMR is more suited to detect inconsistency, change and anomaly at the DP level rather than ascertain the absolute chemical composition of the aluminum adjuvants at the DS level; the latter can be accomplished by invasive analytics before the fill finish step of vaccine manufacturing.

Conclusion

wNMR is a noninvasive analytic based on parameters of the water proton NMR signal, such as the transverse relaxation rate, R2(1H2O). For aluminum adjuvants, wNMR can assess filling, sedimentation and resuspension of aluminum adjuvants in sealed vials in an in situ fashion.

As such, it opens the possibility to quantitatively inspect every vial in a batch of vaccines both at the point-of-release by manufacturers and the point-of-care by end-users.

Acknowledgement

Work at the University of Maryland was supported in part by the FDA through the Maryland Center of Excellence in Regulatory Science and Innovation (1U01FD005946)

Author Biographies

Marc Taraban, PhD, is assistant professor (research) in the Department of Pharmaceutical Sciences at the University of Maryland School of Pharmacy. He was actively involved in research in the areas of structural biology, biophysics of enzymatic reactions and structure-properties relationships in soft matter biomaterials. Currently, he is exploring dynamic NMR applications to study biologics and nanoparticle products.

Christopher Fox, PhD, is the Vice President of Formulations at IDRI. He has developed multiple vaccine adjuvant formulations for a variety of infectious disease indications, including cGMP manufacturing, clinical testing, and technology transfer to developing country manufacturers. He currently leads eff orts to develop a thermostable tuberculosis vaccine as well as scale up adjuvant manufacturing capacity for pandemic influenza preparedness.

Yihua Bruce Yu, PhD, is professor of pharmaceutical sciences at the University of Maryland School of Pharmacy and the director of its Bioand Nano-Technology Center. He has conducted research on protein biophysics, biomaterials and imaging agents. His current research is on analytical technologies for complex drugs, including biologics and nanomedicines.

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