Bulk production of the antiviral lectin griffithsin

Fuqua, Joshua L.; Hamorsky, Krystal; Khalsa, Guruatma; Matoba, Nobuyuki; Palmer, Kenneth E.

Being cognizant that problems in method scalability and technology transfer are inevitable when changing from a research to manufacturing-orientated setting, we optimized purification processes for GRFT at pilot production scale, in the Kentucky Bioprocessing, Inc. (Owensboro, KY, USA) facility where cGMP manufacturing will occur, to enable seamless scale-up of the process. Pilot scale production assessing feasibility and limitations of processes is important to understand yield and properly plan scale-up studies. Process optimization is therefore a necessary initial step in bulk production prior to developing the documentation and analytics necessary for proper cGMP production. cGMP requires the development of validated product release assays that prove acceptability of critical product parameters of identity, purity, potency and safety. Quality is established through char-acterization and stability testing with quality parameters established through repeated measurements on multiple batches. The current release criteria for GRFT include visual appearance, molecular weight determined by mass spectrometry, pH determination of the solution, protein concentration determined by OD 280 , purity determined by SDS-PAGE and size-exclusion highperformance liquid chromatography (SEC-HPLC), potency determined by a gp120 ELISA, safety determined by endotoxin levels and bioburden and residual TMV determined by infectivity tittering in a local lesion host (N. tabacum cv Glurk). Decisions regarding acceptable bioburden and endotoxin levels are informed by dosage form, dosage regime and route of administration. FDA guidance states that the calculation methods described in United States Pharmacopeia monograph 85 (USP <85>) or Association for the Advancement of Medical Instrumentation (AAMI) standards are appropriate for establishing the endotoxin limit for a product (US Department of Health and Human Services Food and Drug Administration, 2012). Unfortunately, there are no specific benchmark standards or guidances for bioburden for rectal product administration that are provided by any regulatory agency (although a USP chapter is proposed at this issue). USP guidelines in Chapter 1111, 'Microbiological Attributes of Nonsterile Pharmaceutical Products', do not provide specific guidance for topical preparations, but do specify that the 'significance of microorganisms in nonsterile pharmaceutical products should be evaluated in terms of the use of the product, the nature of the product, and the potential hazard to the user' (United States Pharmacopeia Convention, 2013) . It is helpful to note that the maximum total aerobic microbial count (TAMC) for a rectally administered product specified in both the World Health Organization's International Pharmacopoeia as well as the European Pharmacopoeia is 10 3 CFU/G, CFU/ML; the maximum total combined yeasts and moulds count (TYMC) is 10 2 CFU/G, CFU/ ML (European Directorate for the Quality of Medicines & Healthcare, 2015) (World Health Organization, 2014). The rectum is a notably nonsterile biological compartment. Testing is therefore included in the rectal GRFT CofA for information only and is likely not an actual acceptance criterion. Table 2 details the current quality profile for GRFT bulk API and will be used for the release of GRFT manufactured under cGMP. Stability testing includes some methods not required for release but used for information only. Common methods include OD 280 and SEC-HPLC and potency