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To be administered to patients, therapeutic monoclonal antibodies must have very high purity, with process related impurities like host-cell proteins (HCPs) and DNA reduced to <100ppm and <10ppb respectively relative to desired product. Traditionally, Protein-A chromatography as a capture step has been the work horse for clearing a large proportion of these impurities. However, remaining levels of process and product related impurities still present significant challenges on the development of polishing steps further downstream. In this study, we have incorporated high throughput screening to evaluate three areas of separation a) Harvest treatment b) Protein-A Chromatography and c) Low pH Viral Inactivation. Precipitation with low pH treatment of cell culture harvest resulted in selective removal of impurities while manipulating the pH of wash buffers used in Protein-A chromatography and incorporating wash additives that disrupt various modes of protein-protein interaction resulted in further and more pronounced reduction in impurity levels. In addition, our study also demonstrate that optimizing the neutralization pH post Protein-A elution can result in selective removal of impurities. When applied over multiple mAbs, this optimization method proved to be very robust and the strategy provides a new and improved purification process that reduces process related impurities like HCPs and DNA to drug substance specifications with just one chromatography column and open avenues for significant decrease in operating costs in monoclonal antibody purification.

Ultra­high expression from a single­ or double­vector system >> Antibody expression 5–7 times higher than with CMV­derived promoters >> Excellent functionality shown in mammalian cell culture >> Overview Many biomedically relevant proteins require something beyond standard expression in E. coli.Mammalian expression systems offer the advantages of proper folding and post­translational modifications not possible in prokaryotic cells. Recombinant antibodies are increasingly important subjects for research, and mammalian expression systems are popular platforms for these studies. Being able to rely on vectors that produce high expression of functional antibodies is a critical piece of the puzzle. TAR­Tat transactivation for ultra­high protein expression in mammalian cells The pHEK293 Ultra Expression Vector systems exploit the HIV­1 TAR­Tat transcriptional activation mechanism to express proteins of interest at ultra­high levels in human HEK 293 cells. These systems are capable of providing protein expression up to 10 times higher than what is typically obtained with vectors containing CMV­derived promoters. Please visit our overview page to learn more about this transcriptional activation mechanism. The pHEK293 Ultra Expression system is available in two formats. The single­vector format (Cat. # 3390) allows for high expression of your protein of interest with the convenience of single­plasmid transfection. The double­ vector system (Cat. # 3392) includes a separate Enhancer Vector. While experiments with this system will require cotransfection of cells with both plasmids, they will give you the ability to optimize expression levels by varying the ratio of the two vectors. Quantification of highly expressed monoclonal antibodies The double­vector system (pHEK293 Ultra Expression Vector II, Cat. # 3392) was used to produce anti­human CD3 IgG monoclonal antibody clone OKT3. The pHEK293 Ultra Expression Vector II system was compared to a negative control and a vector with a CMV­derived promoter (pBApo­CMV DNA). Protein expression was examined in adherent and suspension cells transfected as shown in the table below. Cells were cotransfected with IgG heavy chain (HC) and light chain (LC) expression constructs. Culture samples taken at 2, 5, 7, and 9 days after transfection showed high expression for all of the pHEK293 vector conditions; expression levels were 5­ to 7­fold higher than the CMV vector.

Purification of therapeutic monoclonal antibodies usually involves a protein A affinity capture step. Because column breakthrough of antibody in complex, UV-absorbing culture fluid cannot be readily detected in real time, processes are designed so conservatively that column capacity is usually underutilized, wasting adsorbent and reducing productivity. We have developed a fluorescence-based monitoring technology which allows real-time mAb monitoring and used it to detect IgG in column breakthrough. The column effluent was continuously contacted with soluble fluorescein-labeled Fc-binding ligands to produce an immediately-detectable shift in both fluorescence polarization and intensity. To extend the upper limit of inlet flow rate, a 14:1 split-ratio flow splitter was tested with an inlet flow of 15 mL/min (0.9 L/h), producing a sampling stream at 1 mL/min while still enabling continuous detection functionality. We observed significant shifts in fluorescence intensity in CHO cell culture fluid spiked with human IgG, and detected 0.02-0.1 g/L human IgG in protein A column breakthrough at a flow velocity of 80 cm/h. The increase in fluorescence intensity upon 0.1% breakthrough of an 1 g/L feed was used to trigger column switching using Python-enabled two-way communication with the standard Unicorn OPC process control protocol. The technology allows rapid, continuous and reliable monitoring of IgG in a flowing process stream, without elaborate sample preparation.

Cutinase, a member of /-fold hydrolase family possess potentially diverse applications in several industrial processes and products. The present work aims towards thermo-stabilization of cutinase from novel source Fusarium sp. ICT SAC1 via non-covalent interaction with polysaccharides. Although all six polysac-charides chosen for study enhanced the thermal stability, pectin was found to be most promising. The interaction protocol for cutinase with pectin was optimized sequentially with respect to the ratio of enzyme to pectin, solution pH, and buffer strength. Cutinase-pectin conjugate under optimized conditions (1:12, pH-6.5, 50 mM) showed enhanced thermal stability as evident from lower inactivation rate constant, higher half-life and D-value within the 40–55 • C. A slender rise in K m and V max values and enhanced thermodynamic parameters of cutinase-pectin conjugate were observed after non-covalent interaction. Entropy values were 1.5-fold higher for cutinase-pectin conjugate at each temperature suggesting an upsurge in number of protein molecules in a transition activated state. Positive values of entropy for both forms of cutinase suggested a rise in disordered conformation. Noticeable conforma-tional changes in cutinase after conjugation with pectin were confirmed by FTIR as well as fluorescence emission spectra. An increment in helix to turn conversion was observed in complexed cutinase vis-à-vis free cutinase.