Continuous Monitoring of IgG using Immobilized Fluorescent Reporters

In the manufacture of therapeutic monoclonal antibodies (mAbs), the clarified cell culture fluid is typically loaded onto an initial protein A affinity capture column. Imperfect mass transfer and loading to maximum capacity can risk antibody breakthrough and loss of valuable product, but conservative underloading wastes expensive protein A resin. In addition, the effects of column fouling and ligand degradation require the frequent optimization of IgG loading to avoid wastage. Therefore, continuous real-time monitoring of IgG flowthrough is of great interest. We previously developed a fluorescence-based monitoring technology that allows mix-and-read mAb detection in cell culture fluid. Here we report the use of reporters immobilized on CNBr-activated Sepharose 4B resin for continuous detection of IgG in column breakthrough. The column effluent is continuously contacted with immobilized fluorescein-labeled Fc-binding ligands to produce an immediately detectable change in fluorescence intensity. The technology allows rapid and reliable monitoring of IgG in a flowing stream of clarified cell culture fluid emerging from a Protein A column, without prior sample preparation. We observed a significant change in fluorescence intensity at 0.5 g/L human IgG, sufficient to detect a 5% breakthrough of a 10 g/L load, within 2 minutes at a flow rate of 0.5 mL/min.

Establishing a surrogate model for inactivation of enveloped viruses to screen viral clearance conditions during biotherapeutics process development

Viral surrogates to screen for virus inactivation (VI) can be a faster, cheaper and safer alternative to third-party testing of pathogenic BSL2 (Biosafety Level 2) model viruses. Although the bacteriophage surrogate, Ø6, has been used to assess low pH BSL2 VI, it has not been used for evaluation of detergent-mediated VI. Furthermore, Ø6 is typically assayed through host cell infectivity which introduces the risk of cross-contaminating other cell lines in the facility. To circumvent contamination, we developed an in-house RT-qPCR (reverse transcriptase quantitative polymerase chain reaction) assay for selective detection of active Ø6 from a population of live and dead phage. The RT-qPCR assay was used to evaluate Ø6 inactivation in cell culture fluid of monoclonal antibody and fusion protein. Complementary Ø6 infectivity was also conducted at a third-party testing facility. The Ø6 RT-qPCR and infectivity data was modeled against VI of three BSL2 viruses, X- MuLV, A- MuLV and HSV-1 in corresponding therapeutics. Both Ø6 methods demonstrate that any VI agent showing Ø6 clearance of ≥ 2.5 logs would demonstrate complete BSL2 VI of ≥ 4.0 logs. Compared to BSL2 virus testing, this in-house Ø6 RT-qPCR tool can screen VI agents at 5% the cost and a turnaround time of 2-3 days versus 4-7 months.

Cell culture model vaccine trial against equine influenza

The main goal of our study was to develop a cell culture based vaccine model for equine influenza virus and within the purpose, a total of 161 equine nasal swabs were collected to detect the equine influenza virus and 15 (9.3%) samples were tested as positive with haemagglutination test (HA assay). From these positive swabs, equine influenza virus (EIV) was inoculated in Madin-Darby Canine Kidney (MDCK) cell line. The infected cell-culture fluid was inactivated with 2-Bromoethylamine Hydrobromide and mixed with MONTANIDE ISA 206 oil-based adjuvant (acid) at ratio 1: 1. The purity, toxicity, viscosity, stability, and activity of the newly prepared vaccine model was analyzed. According to our experimental results, the vaccinated horse developed an antibody titer against equine influenza 1:64-1: 128 at 30 days after the first injection, and the titer was increased at 1: 128-1: 256 at 60 days after the first injection and gradually decreased to 1: 16-1:32 at 180 days. These results showed that the vaccine model is active for 6 months. Адууны томуу өвчний эсийн өсгөвөрт вакцины загвар бэлтгэн туршсан дүн Бидний судалгааны ажлын гол зорилго нь адууны томуу өвчний эсийн өсгөвөрт вакцины загвар гарган авах бөгөөд зорилгын хүрээнд адууны томуу өвчний нутгийн үүсгэгчийг илрүүлэхээр нийт 161 адууны хамрын арчдас цуглуулж, цус наалдуулах урвалаар шалгахад 15(9.3%) дээж эерэг дүн үзүүлсэн. Эдгээр эерэг дүн үзүүлсэн арчдаснаас MDCK дамжмал эсийн өсгөвөрт халдвар хийв. Хураан авсан эмгэгт шингэнийг 2-Bromoethylamine Hydrobromide бодисоор идэвхгүйжүүлээд, MONTANIDE ISA 206 тосон суурьт адьювант (хүчлүүр) бодистой 1:1 харьцаатай хольж вакцины загварыг бэлтгэсэн. Бэлтгэсэн вакцины загварын ариун чанар, хорон чанар, зуурамтгай байдал, тогтвортой байдал болон идэвхит чанарыг шалгалаа. Бидний хийсэн туршилтын дүнгээс үзвэл вакцин таригдсан адууны анхны тарилтын дараа 30 хоногтоо 1.64-1:128 таньцтай дархлаа тогтсон бөгөөд 60 дахь хоногтоо 1:128-1:256 таньцтай болж хадгалагдан тэр нь аажмаар буурч 180 хоногтоо 1:16-1:32 таньцтай болсон байна. Үр дүнгээс харахад бидний бэлтгэсэн вакцины загвар нь 6 сарын хугацаанд хамгаалах идэвхитэй байна. Түлхүүр үг: Томуу, вирус, MDCK эс , вакцин

Profiling Active Enzymes for Polysorbate Degradation in Biotherapeutics by Activity-Based Protein Profiling

Polysorbate is widely used to maintain stability of biotherapeutic proteins in formulation development. Degradation of polysorbate can lead to particle formation in drug products, which is a major quality concern and potential patient risk factor. Enzymatic activity from residual enzymes such as lipases and esterases can cause polysorbate degradation. Their high activity, even at low concentration, constitutes a major analytical challenge. In this study, we evaluated and optimized the activity-based protein profiling (ABPP) approach to identify active enzymes responsible for polysorbate degradation. Using chemical probes to enrich active serine hydrolases, more than 80 proteins were identified in harvested cell culture fluid (HCCF) from monoclonal antibodies (mAb) production. A total of 8 known lipases were identified by ABPP, while only 5 lipases were identified by a traditional abundance-based proteomics (TABP) approach. Interestingly, phospholipase B-like 2 (PLBL2), a well-known problematic HCP was not found to be active in process-intermediates from two different mAbs. In a proof-of-concept study, phospholipase A2 group VII (PLA2G7) and sialic acid acetylesterase (SIAE) were identified by ABPP as possible root causes of polysorbate-80 degradation. The established ABBP approach can fill the gap between lipase abundance and activity, which enables more meaningful polysorbate degradation investigations for biotherapeutic development.

AN EFFICIENCY COMPARISON OF CLARIFYING FILTERS DURIG THE HARVEST OF CHO CELL CULTURE FLUID

The turbidity of the culture fluid (CF) and the volume of clarified CF obtained by using nine different filters with the same area (25 cm2) and their combinations were compared.