34028) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). == Cell lifestyle == Therapeutic antibody-producing CHO cells were developed and cultured using the platform of the Molecular Biotechnology Laboratory (MY Lab) at Suranaree University of Technology (Thailand), which is based on CHO-S system, using DHFR and puromycin as selection markers (Thermo Fisher Scientific, Waltham, MA, USA, catalog # A1155701). set of samples of polyclones. The results indicated that while all predicted % cell viability were very similar to the actual value at RSEP value of 6.7 andR2of 0.8908, the predicted productivity from 14 of 18 samples were closed to the reference measurements at RSEP value of 22.4 andR2of 0.8522. These results indicated that UV-Vis combined with PLS has potential to be used for monitoring antibody titer, VCD, and % cell viability for both online and off-line therapeutic production process. == Graphical abstract == The process of multivariate analysis and partial least squares regression of UV-Vis spectrum for the determination of CHO cell densities and antibody titers obtained from small volume of cell culture supernatant samples. Keywords:Multivariate data analysis, Partial least squares regression, Viable cell density, Therapeutic antibody titer, CHO, UV-Vis spectroscopy == Introduction == Since the production of therapeutic antibodies involves the use of living cells, the manufacturing process is much more complicated than generating small molecule drugs, which requires chemical reactions [1]. Multiple bioprocess parameters such as viable cell density (VCD), antibody titer, pH, temperature, %CO2, and level of metabolites need to be tightly monitored in order to obtain maximum yield and to ensure consistency of product quality [2]. These bioprocess parameters are necessary to monitor the performance of cell lines both during the process development and routine manufacturing [3]. Among these parameters, VCD and antibody titers are the two key parameters for determining optimal condition for cell line cultivation during bioprocess development [4]. However, monitoring of these parameters need specific methods and instruments, which are expensive, laborious [5], require substantial volume of samples [6], or sophisticated instruments in case of real-time measurement [7,8]. For example, the determination of antibody titers by ELISA [9] involves several reagents and a microplate reader, and the determination of %viability by trypan blue staining requires a cell T-5224 counter T-5224 or inverted microscope as well as experienced personnel [10]. Recently, multivariate data analysis (MVDA) such as principal component analysis T-5224 (PCA) and partial least squares (PLS), in conjunction with spectroscopic spectra, has been considered to be a powerful tool in the process analytical technology (PAT) or quality by design (QbD) of biopharmaceutical processes [11,12]. The most popular spectroscopies combined with MVDA in PAT tool are Raman spectroscopy [13,14], near-infrared spectroscopy (NIR) [15], and Fourier-transform infrared spectroscopy (FTIR) [16]. This is because these spectroscopic methods can be used to identify chemical functional groups [17]. Ultraviolet-Visible (UV-Vis) spectra provide less information when compared with other spectroscopy techniques as the bands are less specific and T-5224 have high chances of spectral overlapping [17]. However, this method is more attractive in terms of ease of handling and cost, which is more suitable for lab-scale or manufacturing of therapeutic antibody in low-to-middle-income countries. There has been a previous report on successful application of this technique to monitor glutamine and glucose concentrations in BHK-21 cell cultures [18]. In this study, the potential of monitoring VCD and antibody titers from lab-scale Chinese hamster ovary (CHO) cell cultures by applying UV-Vis spectra in combination with MVDA and PLS calibration model was demonstrated. The data was obtained from an off-line UV-Vis spectrum of samples from lab-scale CHO cell culture supernatants. == Materials and methods == == Reagents and materials == Dynamis (catalog no. A2661501), CD FortiCHO (catalog no. A1148301), and anti-clumping agent (catalog no. 0010057AE) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). For ELISA method, Invitrogen Nunc MaxiSorp flat-bottom 96-well plates (catalog no. 44-2404-21) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Protein A (catalog no. GSZ02201) was purchased from GenScript (Piscataway, NJ, USA). Peroxidase AffiniPure F(ab)2fragment goat anti-human IgG (H + L) HRP (catalog no. 109-036-088) T-5224 was purchased from Jackson Immuno Research Laboratories (West Grove, PA, USA). ABTS (Diammonium 2,2-azinobis[3-ethyl-2,3-dihydrobenzothiazole-6-sulfonate]) was ordered from VWR (Tuas, Singapore). TMB (3,3,5,5-tetramethylbenzidine) solution (catalog no. 34028) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). == Cell culture == Therapeutic antibody-producing CHO cells were developed and cultured using the platform of the Molecular Biotechnology Laboratory (MY Lab) at Suranaree KLHL11 antibody University of Technology (Thailand), which is based on CHO-S system, using DHFR and puromycin as selection markers (Thermo Fisher Scientific, Waltham, MA, USA, catalog # A1155701). Four different single clones were cultured separately in 125-mL shaker flasks with Dynamis, supplemented with 8 mM ofl-glutamine and anti-clumping.
IGF Receptors