Characterisation of protein stability in rod-insert vaginal rings

A major goal in vaccine development is elimination of the 'cold chain', the transport and storage system for maintenance and distribution of the vaccine product. This is particularly pertinent to liquid formulation of vaccines. We have previously described the rod-insert vaginal ring (RiR) device, comprising an elastomeric body into which are inserted lyophilised, rod-shaped, solid drug dosage forms, and having potential for sustained mucosal delivery of biomacromolecules, such as HIV envelope protein-based vaccine candidates. Given the solid, lyophilised nature of these insert dosage forms, we hypothesised that antigen stability may be significantly increased compared with more conventional solubilised vaginal gel format. In this study, we prepared and tested vaginal ring devices fitted with lyophilised rod inserts containing the model antigen bovine serum albumin (BSA). Both the RiRs and the gels that were freeze-dried to prepare the inserts were evaluated for BSA stability using PAGE, turbidimetry, microbial load, MALDI-TOF and qualitative precipitate solubility measurements. When stored at 4°C, but not when stored at 40°C/75% RH, the RiR formulation offered protection against structural and conformational changes to BSA. The insert also retained matrix integrity and release characteristics. The results demonstrate that lypophilised gels can provide relative protection against degradation at lower temperatures compared to semi-solid gels. The major mechanism of degradation at 40°C/75% RH was shown to be protein aggregation. Finally, in a preliminary study, we found that addition of trehalose to the formulation significantly reduces the rate of BSA degradation compared to the original formulation when stored at 40°C/75% RH. Establishing the mechanism of degradation, and finding that degradation is decelerated in the presence of trehalose, will help inform further development of RiRs specifically and polymer based freeze-dried systems in general.

The Romanian Orthodox Church

At first glance, the nationalist ideology of the French Revolution seems to have had little impact on the Orthodox Church in Romanian-speaking territories. Romanians were the predominant inhabitants of the principalities of Wallachia and Moldavia and the neighboring territories of Transylvania (including Crişana, Maramureş and Banat), Bukovina, Bessarabia, and Dobrudja. The majority of ethnic Romanians belonged to the Orthodox faith while their communities were at the intersection of geopo liti cal interests of the Rus sian, Ottoman, and Habsburg empires. In 1859 the Principalities of Wallachia and Moldavia (known as the Old Kingdom between 1866 and 1918) united into a single state under the rule of a local prince. The term "Romania" began to be used by the new state in its of cial documents in 1862. Two years later, the state supported the declaration of a Romanian autocephalous (in de pen dent) church that was recognized by the Ecumenical Patriarchate in 1885. As an integrative part of the Orthodox commonwealth, the church was situated between the competing jurisdictions of the Ecumenical Patriarchate and the Rus sian Orthodox Church, while its declaration of autocephaly followed a pattern in the spread of national churches in Southeastern Europe. From the Treaty of Kuchuk Kainardji of 1774 to the beginning of the Greek War for In de pen dence in 1821, the Romanian principalities were under the suzerainty of the Ottoman Empire, which had full control of their po liti cal and economic affairs. The sultan appointed princes, and the Porte determined their po liti cal and judicial status. The princes were drawn from the "Phanariots," and were directly appointed by the Porte from preponderantly Greek elite rather than the Romanian local elite, the boyars (boieri).1 In each principality, the church was headed by a metropolitan who was under the direct jurisdiction of the Ecumenical Patriarchate. That religion mattered to local population as a means of social cohesion was suggestively depicted by Anatole de Demidoff, an En glish traveler in the region in 1837. Arriving in Bucharest, the capital of Wallachia, he claimed that: I know of no city in Europe in which it is possible to find more agreeable society, or in which there is a better tone, united with the most charming gaiety⋯. Religion, which is here of the schismatic Greek creed, does not, properly speaking, hold any great empire over the minds of the Wallachian people, but they observe its outward forms, and particularly the austerities of fasting, with scrupulous exactitude. The people are seen to attend divine ser vice with every sign of respect, and the great number of churches existing in Wallachia, bear witness to the ardent zeal with which outward worship is honored.2 The Romanian Orthodox Church was a national institution, closely linked to social, economic, and po liti cal structures. In most cases, Orthodox hierarchs were appointed from the families of boyars, thus ensuring a close relationship with the state authorities and its policies. As one of the largest landowners in the principalities, the church had a prime role in administrating healthcare and education. Although the majority of the clergy was uneducated, it dispensed both ecclesiastical and civil justice and in many cases worked closely with boyars in local administration.3 The lower clergy not only contributed directly to the economy but also benefited from tax privileges. Some small villages had an unusually high proportion of clergy in comparison to the overall population. For example, in 1810, Stənisləveşti, a village in the south of Wallachia, was composed of eleven houses and had two priests, five deacons, and three cantors; similarly, the Frəsinet village of nineteen houses had two priests and five deacons.4 Although these cases were exceptional, they indicate both the economic value of being a member of the clergy and the wider canonical dimension of church jurisdiction. The special status of the clergy was reflected not only at lower but also at higher levels. Bishops and metropolitans engaged with state policy and in many cases opposition to the authorities led to the loss of a spiritual seat. The metropolitan of each principality worked with the prince and was president of the divan, the gathering of all boyars. He held the right to be the first person to comment on state policy and to make recommendations when the prince was absent. The metropolitan replaced the prince when the principality had no political ruler, such as in the cases of Metropolitan Veniamin Costachi of Moldavia in 1806 and Metropolitan Dositei Filitti of Wallachia, while the bishops of Buzəu and Argeş were members of the provisional government during the Rus sian occupation of the principalities in 1808. The higher clergy had both religious and political prerogatives in relation to foreign powers as evident in their heading of the boyars' delegation to peace negotiation between the Rus sian and Ottoman empires at Focşani in 1772 and addressing memoranda to the Austrian and Rus sian governments in 1802.5 The primary role of the church in the principalities of Moldavia and Wallachia was paralleled by the national mobilization of Orthodox communities in the neighboring territories that had Romanian inhabitants. Although throughout the region Orthodox communities were incorporated into church structures as part of the Habsburg, Austrian or Rus sian empires, the nineteenth century was characterized by the leadership's search for political autonomy and the building of a Romanian national identity. The Orthodox communities outside the Old Kingdom maintained relations with the faithful in principalities across the Carpathian Mountains and the Dniester River and sought support in their struggle for political and religious rights.