Role of Cytokines and Growth Factors in the Manufacturing of iPSC-Derived Allogeneic Cell Therapy Products.

Cytokines and other growth factors are essential for cell expansion, health, function, and immune stimulation. Stem cells have the additional reliance on these factors to direct differentiation to the appropriate terminal cell type. Successful manufacturing of allogeneic cell therapies from induced pluripotent stem cells (iPSCs) requires close attention to the selection and control of cytokines and factors used throughout the manufacturing process, as well as after administration to the patient. This paper employs iPSC-derived natural killer cell/T cell therapeutics to illustrate the use of cytokines, growth factors, and transcription factors at different stages of the manufacturing process, ranging from the generation of iPSCs to controlling of iPSC differentiation into immune-effector cells through the support of cell therapy after patient administration.

Virus filtration of high-concentration monoclonal antibody solutions.

The ability to process high-concentration monoclonal antibody solutions (> 10 g/L) through small-pore membranes typically used for virus removal can improve current antibody purification processes by eliminating the need for feed stream dilution, and by reducing filter area, cycle-time, and costs. In this work, we present the screening of virus filters of varying configurations and materials of construction using MAb solutions with a concentration range of 4-20 g/L. For our MAbs of interest-two different humanized IgG1s-flux decay was not observed up to a filter loading of 200 L/m(2) with a regenerated cellulose hollow fiber virus removal filter. In contrast, PVDF and PES flat sheet disc membranes were plugged by solutions of these same MAbs with concentrations >4 g/L well before 50 L/m(2). These results were obtained with purified feed streams containing <2% aggregates, as measured by size exclusion chromatography, where the majority of the aggregate likely was composed of dimers. Differences in filtration flux performance between the two MAbs under similar operating conditions indicate the sensitivity of the system to small differences in protein structure, presumably due to the impact of these differences on nonspecific interactions between the protein and the membrane; these differences cannot be anticipated based on protein pI alone. Virus clearance data with two model viruses (XMuLV and MMV) confirm the ability of hollow fiber membranes with 19 +/- 2 nm pore size to achieve at least 3-4 LRV, independent of MAb concentration, over the range examined.

Effect of electrostatic interactions on binding and retention of DNA oligomers to PNA liposomes assessed by FRET measurements.

A FRET-based method is used to observe the desorption of di-alkyl peptide nucleic acid amphiphiles (PNAA) from liposomes occurring on binding of complementary DNA oligomers. PNA liposomes were prepared containing fluorescein-labeled PNAA and rhodamine-labeled dipalmitoylphosphoethanolamine (DPPE). These liposomes showed efficient energy transfer from the fluorescein to rhodamine, with an average donor-to-acceptor distance of 5.91nm. In low-ionic-strength buffer (50mM Tris-HCl, pH 8.0), the FRET signal was maintained in the presence of a stoichiometric amount of 10- and 20-mers DNA complements, but the signal attenuated for 40-mer complements, indicating that DNA first binds the PNAA before the PNAA/DNA duplex desorbs from the lipid bilayer. The FRET signal was maintained in the presence of 10-, 20-, 40-, and 60-mer DNA in high ionic-strength buffer, showing that the driving force for the desorption is electrostatic repulsion between the bound DNA oligomer and the liposome surface. This conclusion is corroborated by comparison of the PNA/DNA binding energy, the energy of adsorption of the di-alkyl PNAA to the lipid bilayer, and a calculation of the DNA/lipid bilayer electrostatic repulsion using the linearized Poisson-Boltzmann equation.

Sequence-specific binding of DNA to liposomes containing di-alkyl peptide nucleic acid (PNA) amphiphiles.

We present a method to covalently attach peptide nucleic acid (PNA) to liposomes by conjugation of PNA peptide to charged amino acids and synthetic di-alkyl lipids ("PNA amphiphile," PNAA) followed by co-extrusion with disteroylphosphatidylcholine (DSPC) and cholesterol. Attachment of four Glu residues and two ethylene oxide spacers to the PNAA was required to confer proper hydration for extrusion and presentation for DNA hybridization. The extent of DNA oligomer binding to 10-mer PNAA liposomes was assessed using capillary zone electrophoresis. Nearly all PNAs on the liposome surface are complexed with a stoichiometric amount of complementary DNA 10-mers after 3-h incubation in pH 8.0 Tris buffer. No binding to PNAA liposomes was observed using DNA 10-mers with a single mismatch. Longer DNA showed a greatly attenuated binding efficiency, likely because of electrostatic repulsion between the PNAA liposome double layer and the DNA backbone. Langmuir isotherms of PNAA:DSPC:chol monolayers indicate miscibility of these components at the compositions used for liposome preparation. PNAA liposomes preserve the high sequence-selectivity of PNAs and emerge as a useful sequence tag for highly sensitive bioanalytical devices.

Thermodynamic and structural characterization of amino acid-linked dialkyl lipids.

Using differential scanning calorimetry (DSC), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR), we determined some thermodynamic and structural parameters for a series of amino acid-linked dialkyl lipids containing a glutamic acid-succinate headgroup and di-alkyl chains: C12, C14, C16 and C18 in CHES buffer, pH 10. Upon heating, DSC shows that the C12, C14 and annealed C16 lipids undergo a single transition which XRD shows is from a lamellar, chain ordered subgel phase to a fluid phase. This single transition splits into two transitions for C18, and FTIR shows that the upper main transition is predominantly the melting of the hydrocarbon chains whereas the lower transition involves changes in the headgroup ordering as well as changes in the lateral packing of the chains. For short incubation times at low temperature, the C16 lipid appears to behave like the C18 lipid, but appropriate annealing at low temperatures indicates that its true equilibrium behavior is like the shorter chain lipids. XRD shows that the C12 lipid readily converts into a highly ordered subgel phase upon cooling and suggests a model with untilted, interdigitated chains and an area of 77.2A(2)/4 chains, with a distorted orthorhombic unit subcell, a=9.0A, b=4.3A and beta=92.7 degrees . As the chain length n increases, subgel formation is slowed, but untilted, interdigitated chains prevail.