| Abstract | Cilj istraživanja
Cilj rada je dati sveobuhvatnu analizu karakterizacije i kontrole agregatnih onečišćenja u monoklonskim protutijelima, radi osiguravanja odgovarajuće kakvoće i učinkovitosti te smanjivanja njihove imunogenosti.
Hipoteze istraživanja su:
- nastanak agregatnih onečišćenja u monoklonskim protutijelima se može smanjiti, ali ne i u potpunosti izbjeći;
- razumijevanje mehanizama agregacije i primjena najsuvremenijih metoda karakterizacije i kvantifikacije agregata u monoklonskim protutijelima osigurava odgovarajuću kontrolu kakvoće i smanjuje mogućnost nastanka agregata tijekom proizvodnje i čuvanja lijeka;
- agregacija proteina između referentnog i biosličnog lijeka je usporediva.
Materijal i metode
Literatura je pretraživana prema temi i predmetu istraživanja, autorima i časopisu, od općih prema specijaliziranim člancima, na temu razvoja i proizvodnje monoklonskih protutijela, nastanka agregatnih onečišćenja i njihovog imunogenog potencijala te načina kako izbjeći, odnosno minimizirati agregaciju proteina. Pri pretraživanju literature su traženi odgovori na specifična pitanja vezana uz problematiku ovog specijalističkog rada, u cilju potkrepe predloženih hipoteza.
Pretražene su bibliografska baza podataka (PubMed) i baza podataka s cjelovitim tekstom (Science Direct), kojima je pristupano elektroničkim putem preko umreženog računala koje ima
online pristup spomenutim bazama. Regulatorne smjernice i pravni dokumenti relevantni za problematiku ovog rada su istraženi pristupom mrežnih stranicama i bazama regulatornih Agencija (EMA, FDA, HALMED), Europske komisije te Europske (Ph. Eur.) i Američke farmakopeje (USP). Na temelju pretraživanih izvora su izvedeni vlastiti zaključci o predmetnoj problematici.
Rezultati
Agregacija proteina je neizbježan događaj karakterističan za gotovo sve faze u proizvodnji proteina, transport, čuvanje i primjenu lijeka te predstavlja velik izazov u proizvodnji monoklonskih protutijela. Proteinska agregacija se uglavnom može predvidjeti i donekle prevenirati još tijekom razvoja formulacije i proizvodnje monoklonskih protutijela. Agregati različitih proteina pokazuju veći imunogeni potencijal u odnosu na pojedinačne podjedinice (monomere), a pretpostavlja se da je razlog tomu razlika u veličini i vrsti agregata, njihovoj konformaciji te morfologiji. Razumijevanje mehanizama agregacije i razvoj prikladnih strategija njezine minimizacije su zadnjih desetljeća postali imperativ u razvoju proteinskih lijekova, u cilju osiguravanja njihove kliničke učinkovitosti. Zasad nije u potpunosti jasno imaju li sve vrste agregata jednak imunogeni potencijal te se ne može unaprijed znati koja je agregatna onečišćenja potrebno kontrolirati u lijeku. Prema posljednje važećim smjernicama i preporukama regulatornih agencija, svojstva svih agregata koji se mogu detektirati trebaju biti odgovarajuće karakterizirana, a sadržaj kontroliran i rutinski nadziran u svim biološkim lijekovima. Na temelju dosadašnjeg iskustva, zaključeno je kako se kvantifikacija proteinskih agregata treba provesti na temelju njihove veličine te se uobičajeno istovremeno koriste barem dvije ortogonalne metode temeljene na različitim principima rada. Usporednim ispitivanjima na razini kakvoće između biosličnih i referentnih lijekova je utvrđeno kako imaju slične profile onečišćenja, obzirom na sadržaj agregatnih onečišćenja.
Zaključak
Obzirom na strukturne i ostale karakteristike proteina, tijekom proizvodnje, čuvanja, transporta i primjene većine monoklonskih protutijela često dolazi do agregacije proteina. Ovisno o svojstvima, agregatna onečišćenja mogu značajno utjecati na kakvoću, djelotvornost i sigurnost monoklonskih protutijela, posebice u vidu izazivanja pojačane imunogenosti, što može dovesti do smanjenog djelovanja lijeka i značajnih nuspojava kod pacijenata. Nastanak agregatnih onečišćenja u monoklonskim protutijelima se može smanjiti, ali ne i u potpunosti izbjeći.
Razumijevanje mehanizama agregacije i razvijanje strategija smanjivanja nastanka agregatnih onečišćenja su bitni za minimiziranje nastanka imunogenog učinka monoklonskih protutijela. Isto tako, primjena sofisticiranih i specifičnih ortogonalnih analitičkih metoda za karakterizaciju i kvantifikaciju agregatnih onečišćenja te njihov daljnji razvoj osiguravaju odgovarajuću kontrolu kakvoće i smanjuje mogućnost nastanka agregata tijekom proizvodnje i čuvanja lijeka. Utvrđeno je kako su referentni biološki lijekovi i njihove bioslične inačice usporedivi obzirom na čistoću i profil onečišćenja (obzirom na fragmente, procesna onečišćenja i neglikozilirane forme), stupanj agregacije te vrstu agregata i ostalih molekula veće molekulske mase. |
| Abstract (english) | Objectives
The objective of this work is to provide the comprehensive analysis of the characterization and quality control of the aggregate impurities in monoclonal antibodies (mABs). The aim is to ensure that mABs and other biotherapeutics are of adequate safety, quality and efficacy, with the reduced immunogenic potential as much as possible.
Hypotheses are the following:
- the formation of aggregate impurities in mABs can be decreased, but not completely avoided;
- understanding of the mechanisms of aggregation and the use of the sophisticated methods of characterization and quantification of aggregates in mABs ensure an adequate control of aggregation and reduce the possibility for the formation of aggregates during the production and storage of the drug products;
- protein aggregation between the reference drug product and the biosimilar is comparable.
Material and methods
Through this work, the importance of an adequate characterization and control of the aggregate impurities in monoclonal antibodies in a timely manner has been emphasized, in order to ensure their safety, quality and efficacy. This is especially important from the aspect of the mABs immunogenicity and its consequent possible decrease. The mechanisms of protein aggregation and the specific types of aggregates that can occur considering the protein properties and the drug product storage conditions have been described. There is a special emphasis on the
possible enhanced immune response of the patients on the drug product due to the presence of aggregate impurities.
Results
Protein aggregation is an inevitable event specific for almost all protein manufacturing stages, as well as its transport, storage and drug administration. It represents a huge challenge in the production of monoclonal antibodies. Protein aggregation is mostly predictable and in some way it can be prevented during the development of the formulation and the production of mABs. Aggregates from different proteins show a higher immunogenic potential compared to the monomers and it is assumed that the main reason for this is the difference in size and type of aggregates, their conformation and morphology. Understanding of the mechanisms of aggregation and the strategy development for its minimization have become imperative in the development of protein drug products in recent decades, all with the aim to ensure their clinical efficacy. So far, it is not entirely clear whether all types of aggregates have the same immunogenic potential and it is not possible to know which aggregate impurities should be controled in the drug product in advance. According to the recent guidelines and the recommendations from the regulatory bodies, the properties of all detectable aggregates should be properly characterized and their content routinely monitored in mABs. Based on the previous experience, it was assumed that the quantification of protein aggregates should be carried based on their size, at least with two orthogonal methods based on different principles. From the comparative studies regarding quality between biosimilars and the reference drug products it has been concluded that impurity profiles are similar, also considering the content of protein aggregates.
Conclusion
Considering the structural and other characteristics of proteins, protein aggregation occurs often during the production of the most of monoclonal antibodies, their storage, transport and administration. Depending on their properties, aggregates could significantly affect the quality, safety and efficacy of mABs, especially considering the immunogenic potential that could lead to decreased drug activity, as well as significant side effects in patients. The formation of protein aggregates in mABs can be reduced but not completely avoided. Understanding of the mechanisms of aggregation and strategy development for reduction of aggregates formation are essential for minimization of the immunogenic effects of mABs. Likewise, the application of sophisticated and specific orthogonal analytical methods for the characterization and quantification of aggregates and their further development ensure adequate quality control and reduce the possibility of aggregates formation during the production and storage of biological drugs. It has been established that the reference biological drug products and their biosimilar versions are comparable considering the purity and impurity profile (with regard to fragments, impurities and non-glycosylated forms), the level of aggregation, as well as the type of aggregates and other molecules of higher molecular weight. |
