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Advanced therapy medicinal products (ATMPs), such as somatic cell-therapy medicinal products or tissue-engineered products for human use, offer new and potentially curative opportunities to treat yet untreatable diseases or disorders. For cell-therapy medicinal products (CBMPs), multiple stability and quality challenges exist and relate to the cellular composition and unstable nature of these parenteral preparations. It is the aim of this review to discuss open questions and problems associated with the development, manufacturing and testing of CBMPs from a pharmaceutical drug product perspective. This includes safety, storage and handling, particulates, the choice of container closure systems and integrity. Analytical methods commonly used to evaluate the quality of the final CBMP to ensure patient's safety will be discussed. Particulate contamination in final products deserve special attention since CBMPs cannot be sterile filtered. Visible and sub-visible particles may represent environmental contaminations or may form during storage. They may be introduced from processing materials such as single use product contact materials, ancillary materials, or any components such as primary packaging used for the final product. Currently available analytical methods for detecting particulates may not be easily applicable to CBMPs due to their inherent particulate nature and appearance.

For injectable biopharmaceuticals, the subcutaneous route of administration is increasingly preferred over intravenous administration. However, one of the challenges in the development of subcutaneously administered biopharmaceuticals is a reduced bioavailability, which is difficult to predict. Since animal models do not reliably reflect bioavailability in patients, in vitro models could help to develop drug candidates. The purpose of this study was to evaluate a versatile set of in vitro tools for their suitability to predict bioavailability of biopharmaceuticals after subcutaneous administration.We examined seven commercially available biopharmaceuticals using three instruments, i.e., the Subcutaneous Injection Site Simulator (Scissor), the Osmomat 050, and a dialysis system using three artificial extracellular matrices, two dissolution apparatuses, i.e., the USP4 and the USP7, and two evaluation tools, i.e., the affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS) and the Developability Index (DI). Results were evaluated for their usefulness to predict the bioavailability and other pharmacokinetic parameters in humans using the Pearson correlation.None of the tested instruments and methods could reliably approximate bioavailability. Only pressure values derived with the Osmomat 050 instrument correlated with CNo single in vitro method confidently predicted the bioavailability in humans. We only found a correlation to maximum plasma concentration values for one of the tested approaches. However, a more focused evaluation would be necessary to confirm our findings and test combinations of orthogonal methods that may improve the confidence of such a prediction.

Therapeutic breakthroughs in a number of retinal degenerative diseases have come about through the development of biotherapeutics administered directly into the eye. As a consequence of their use, we have gained more insight into the immune privileged status of the eye and the various considerations that development, manufacturing, and use of these drugs require. It has been observed that therapeutic proteins injected into the vitreous can elicit an immune response resulting in the production of anti-drug antibodies (ADAs) which can have clinical consequences. This review includes discussion of the anatomy, physiology, and specific area of the eye that are targeted for drug administration. The various immunologic mechanisms involved in the immune responses to intraocularly administered protein are discussed. This review entails discussion on chemistry, manufacturing, and control (CMC) and formulation-related issues that may influence the risk of immunogenicity. Based on the available immunogenicity profile of the marketed intraocular drugs and their reported adverse events, the animal models and the translational gap from animals to human are discussed. Thus, the objective of this review article is to assess the factors that influence immunogenicity in relation to intraocular administration and the steps taken for mitigating immunogenicity risks.

Cell therapy products represent an exciting new class of medicinal products, which must be parenterally administered. Thus, compliance with parenteral preparation guidelines is required. One requirement for parenteral products is the characterization of particle contaminations. As cell-based products are turbid suspensions, containing particles, the cells, characterization and control of foreign particle impurities remain a challenge. Within this study, we evaluated a flow imaging microscopy method for the detection and characterization of subvisible particle contaminations in cell-based products. We found that flow imaging microscopy is a potential method where subvisible particle contaminations can be differentiated from the cells in cell therapy products.

A toxicological evaluation to determine the product specific permitted daily exposure (PDE) value is an accepted method to determine a safe limit for the carry-over of product residues in multipurpose manufacturing facilities. The PDE calculation for intravitreal (IVT) injection of small and large molecular weight (MW) drugs follows the guiding principles set for systemic administration. However, there are specific differences with respect to the volume administered with IVT administration, pharmacokinetic and pharmacodynamics (PK-PD) parameters and potential for toxicity. In this publication, we have proposed a method to derive PDEIVT in the presence of IVT dose. In the absence of an IVT dose we have a proposed default extrapolationof the systemic PDE for intravenous (IV) administration to the PDEIVT dose by applying a factor of 500 based on comparison of the volume of vitreous humour with the plasma volume, as well as provided examples for PK-PD and toxicity considerations.

Cancer gene therapy using tumor suppressor genes is considered to be an attractive approach for arresting cell growth and inducing apoptosis. Programmed cell death 4 (Pdcd4) is a tumor suppressor gene, which prevents tumorigenesis and tumor progression. To address the issue of whether expression of PDCD4 protein induces apoptosis in cancerous cells, the Pdcd4 gene was delivered using folate-PEG-baculovirus. Folate-PEG-baculovirus containing Pdcd4 gene (F-P-Bac-Pdcd4) was constructed by attachment of F-PEG to the baculovirus surface using chemical modification. The F-P-Bac-Pdcd4 showed enhanced transduction efficiency, efficiently expressed PDCD4 protein, and induced apoptosis in human epidermal carcinoma (KB) cells as compared with an unmodified baculovirus. In a tumor xenograft study, injection of F-P-Bac-Pdcd4 into tumors established from the KB cell line by subcutaneous implantation significantly suppressed tumor growth and induced apoptosis. Thus, this study shows a new baculovirus-mediated tumor suppressor gene delivery system for cancer therapy.

Biodegradable nano-polymeric carrier composed of polycaprolactone (PCL) and polyethylenimine (PEI) (BNPP) was successfully synthesized for the delivery of sh/siRNA in lung cancer cells. BNPP efficiently and safely delivered siRNA in lung cancer cells. BNPP-delivered Akt1 siRNA silenced Akt1 protein, and reduced the cancer cell survival, proliferation, malignancy and metastasis.

Gene therapy is a powerful approach in the treatment of a wide range of both inherited and acquired diseases. Nonviral delivery systems have been proposed as safer alternatives to viral vectors because they avoid the inherent immunogenicity and production problems that are seen when viral systems are used. Many cationic polymers, including high-molecular-weight polyethylenimine (PEI) have been widely studied as gene-delivery carriers, both, in vitro and in vivo. However, interest has recently developed in degradable polymeric systems. The advantage of degradable polymer is its low in-vivo cytotoxicity, which is a result of its easy elimination from the cells and body. Degradable polymer also enhances transfection of DNA or small interfering RNA (siRNA) for efficient gene expression or silencing, respectively. This review paper summarizes and discusses the recent advances with degradable PEIs, such as cross-linked and grafted PEIs for DNA and siRNA delivery.

Baculovirus, containing an appropriate eukaryotic promoter, is considered an attractive approach for an efficient and safe gene delivery vehicle. However, the drawbacks of baculovirus, such as the lack of specificity and the inactivation of baculovirus by the complement system in human serum, negatively affect efficient gene delivery. Therefore, a hybrid system utilizing the positive aspects of both viral and non-viral vector systems would be useful to overcome the obstacles of either system alone. In this study, we constructed a hybrid system composed of baculovirus (B) and galactosylated polyethylenimine (GP)/DNA complexes through electrostatic interaction. The resulting GP/B hybrid had suitable physicochemical properties and low cytotoxicity for use in gene therapy. Furthermore, the GP/B significantly enhanced transduction efficiency and showed good cell-specificity compared to either viral or non-viral vector systems. These results suggest that the GP/B hybrid system can be used in gene therapy to enhance transduction efficiency and hepatocyte specificity.

Background Polyethylenimine (PEI) vectors are widely used in gene delivery because of their high transfection efficiency owing to a unique proton sponge effect. An increase in molecular weight increases transfection efficiency, but simultaneously results in increased toxicity. Therefore, the design and synthesis of new degradable gene delivery carriers having high transfection efficiencies and reduced cytotoxicity are necessary. Methods In the present study degradable poly(ester amines) (PEAs) based on glycerol dimethacrylate (GDM) and low molecular weight branched polyethylenimine (LMW-PEI) were synthesized in anhydrous methanol at 60 °C following Michael addition reaction. The transfection efficiencies of the synthesized PEA/DNA complexes were evaluated using three different cell lines (HeLa, HepG2 and 293T cells) in vitro. Results PEAs with zeta potential in the range of 30–55 mV (at physiological pH) condensed plasmid DNA into nanosized particles (

Efficient delivery of small interfering RNA (siRNA) or small hairpin RNA (shRNA) is a critical concern in RNA interference (RNAi) studies. In the present study, we evaluated biodegradable poly(beta-amino ester) (PAE) carrier composed of low molecular weight polyethylenimine and poly(ethylene glycol) for si/shRNA delivery in lung cancer cells. PAE carrier successfully delivered EGFP (enhanced green fluorescence protein) siRNA (siGFP) and silenced EGFP expression. The silencing achieved with PAE carrier was found to be nearly 1.5 times superior and safer than standard PEI25K. Also, our PAE carrier exhibited superior Akt1 shRNA delivery (shAkt) and thereby silenced oncoprotein Akt1 efficiently. PAE-shAkt mediated Akt1 knock-down hindered cancer cell growth in Akt1 specific manner. Superior shAkt delivery and low cytotoxicity of PAE carrier promoted Akt1 knock-down specific apoptosis, while low delivery efficiency and high cytotoxicity of PEI25K carrier mainly exhibited undesirable necrosis. Moreover, basic cancer properties like cell proliferation, malignancy and metastasis were reduced more efficiently using PAE-shAkt system. These findings demonstrated the potential of PAE as an alternative to PEI25K in si/shRNA-based RNAi studies.

Application of combinatorial chemistry and high throughput screening for the synthesis and evaluation of mini-library of novel biodegradable poly (beta-amino ester)s (PAE)s composed of gamma-aminopropyl-triethoxysilane (APES) and poly (ethylene glycol) diacrylate (PEGDA) for gene delivery efficiency and safety in 293T and HeLa cells in the presence of and absence of serum.PAEs were synthesized at different mole ratios of APES and PEGDA by Michael addition reaction and synthesis was confirmed by 1H nuclear magnetic resonance (1H-NMR). Ninety six ratios of polyplexes were evaluated for luciferase and MTS assay in 293T and HeLa cells in the presence of and absence of serum. Relationship between transfection efficiency and DNA binding ability of PAEs was studied by gel electrophoresis. Particle sizes and molecular weight of selected PAEs were measured by dynamic light scattering and gel permeation chromatography multi-angle light scattering, respectively.1H-NMR confirmed the synthesis of PAEs. In both cell lines, transfection efficiency and cell viability were increased for PAEs obtained from R106 (0.7:1, APES:PEGDA) to R121 (6:1, APES:PEGDA) with a marginal increase in APES concentration. Transfection pattern was uniform in the absence of and presence of serum. In both cell lines, PAE obtained from R121 demonstrated high transfection efficiency and low cytotoxicity as compared to polyethylenimine (25 KDa) and Lipofectamine. PAE obtained from R121 showed good DNA binding and condensation with average particle sizes of 133 nm.Addition of PEGDA over APES resulted in a novel PAE which has high safety and transfection efficiency. Transfection and cytotoxicity are very sensitive to monomer ratios and mainly governed by concentration of amine monomer.

Folate conjugated poly(ester amine) (PEA) was prepared by reaction of folic acid with PEAs based on polycaprolactone (PCL) and low molecular weight polyethylenimine (LMW-PEI) with PEG as a linker. This novel gene carrier showed excellent physicochemical properties and relatively low cytotoxicity compared with PEI 25K. It showed excellent transfection efficiency through folate receptor mediated endocytosis.

To circumvent carrier related obstacles, we developed a biodegradable, folate conjugated poly (ester amine) (FP-PEA) that mediates high level folate receptor (FR) mediated endocytosis in vitro as well as in vivo. We report the efficacy of a therapeutic strategy that combines the potency of FP-PEA based on polycaprolactone (PCL) and low molecular weight polyethylenimine (LMW-PEI) with the tumor targeting potential of receptor mediated endocytosis. When tested on cells in culture, FP-PEA was found to retain high affinity for FR-positive cells compared with PEA without folate moiety (P-PEA). The FR specific activity of FP-PEA was drastically decreased in the presence of an excess free folic acid and very less significant transfection was detected against FR-negative cells. FP-PEA showed marked anti-tumor activity against FR-positive human KB tumors in nude mice with no evidence of toxicity during and after therapy using TAM67 gene. Furthermore, the therapeutic effect occurred in the apparent absence of weight loss or noticeable tumor apoptosis. In contrast, no significant anti-tumor activity was observed in P-PEA treated mice which were co dosed with an excess of FR, thus demonstrating the target specific gene delivery. Furthermore, anti-tumor activity with PEA without folic acid moiety (P-PEA) proved not to be effective against xenograft mice model with KB cells when administered at the same dose to that of FP-PEA. Taken together, these results indicate that FP-PEA is highly effective gene carrier capable of producing therapeutic benefit in xenograft mice model without any sign of toxicity.

Efficient delivery of small interfering RNA (siRNA) remains a challenging task in RNA interference (RNAi) studies. In this study, we used chitosan-graft-polyethylenimine (CHI-g-PEI) copolymer composed of chitosan and low molecular weight polyethylenimine (PEI) for the delivery of siRNA. The CHI-g-PEI carrier formed stable complexes with siRNA with compact spherical morphology. CHI-g-PEI delivered EGFP siRNA (siGFP) silenced EGFP expression nearly 2.5 folds higher than PEI25K at 50 pM siGFP concentration. Cell viability was found to be 2 folds high with CHI-g-PEI carrier than PEI25K. Also, our CHI-g-PEI carrier efficiently delivered Akt1 siRNA (siAkt) and thereby silenced onco-protein Akt1. Silencing of this crucial cell survival protein significantly reduced the lung cancer cell survival and proliferation. Additionally, Akt1 protein knock-down decreased A549 cell malignancy and metastasis. These findings suggest that the CHI-g-PEI carrier efficiently and safely delivered siRNA. Moreover, CHI-g-PEI mediated Akt1 siRNA delivery may emerge as a viable approach for lung cancer treatment.

Gene transfer using non-viral vectors is a promising approach for the safe delivery of therapeutic genes. Among non-viral vectors, chitosans have been proposed as alternative, biocompatible cationic polymers for non-viral gene delivery. However, the low transfection efficiency and low specificity of chitosan needs to be addressed prior to clinical application. In this study, mannosylated chitosan-graft-polyethylenimine (Man-CHI-g-PEI) copolymer was prepared by thiourea reaction between the isothiocyanate group of mannopyranosylphenylisothiocyanate and the amine groups of chitosan-graft-PEI (CHI-g-PEI) for targeting into antigen presenting cells (APCs) having mannose receptors. The composition and molecular weight were characterized using (1)H NMR and GPC, respectively. The copolymer was complexed with plasmid DNA in various copolymer/DNA (N/P) charge ratios, and the complexes were characterized. Man-CHI-g-PEI showed good DNA binding ability and high protection of DNA from nuclease attack and had low cytotoxicity compared with PEI 25K. The transfection efficiency of Man-CHI-g-PEI/DNA complexes into the Raw 264.7 macrophage cell line, which has mannose receptors, was higher than CHI-g-PEI itself as well as PEI 25K, indicating Man-CHI-g-PEI can be used as an APCs' targeting gene delivery carrier.

Chitosan and chitosan derivatives have been proposed as alternative and biocompatible cationic polymers for non-viral gene delivery. However, the low transfection efficiency and low specificity of chitosan is an aspect of this approach that must be addressed prior to any clinical applications. In the present study a chitosan derivative, galactosylated poly(ethylene glycol)-chitosan-graft-polyethylenimine (Gal-PEG-CHI-g-PEI), was investigated as a potential hepatocyte-targeting gene carrier. The composition of Gal-PEG-CHI-g-PEI was characterized using (1)H nuclear magnetic resonance ((1)H NMR), and the particle size and zeta potential of Gal-PEG-CHI-g-PEI/DNA complexes were measured using dynamic light scattering (DLS). The Gal-PEG-CHI-g-PEI exhibited lower cytotoxicity compared to PEI 25K as a control. Likewise, Gal-PEG-CHI-g-PEI/DNA complexes showed good hepatocyte specificity. Furthermore, Gal-PEG-CHI-g-PEI/DNA complexes transfected liver cells more effectively than PEI 25K in vivo after intravenous (i.v.) administration. Together, these results suggest that Gal-PEG-CHI-g-PEI, which has improved transfection efficiency and hepatocyte specificity both in vitro and in vivo, may be useful for gene therapy.

Cationic polymers have been increasingly proposed as potential gene delivery vectors because of their versatility. In this paper, we focus on the characteristics of poly(ester amine)s (PEAs) as gene delivery carriers, degradation pattern as an essential parameter for reduced toxicity, classification based on its physicochemical properties followed by its success as efficient gene delivery carrier in vitro and in vivo. We also discuss the conjugation of ligands/charged groups to the side chain of the polyester in order to achieve receptor-mediated endocytosis as well as target-specific delivery of DNA. Capable of delivering exogenous genes to a cell nucleus, these cationic PEAs also serve as a valuable model to understand the important characteristics that render a polymer an effective gene carrier.

Bioreducible disulfide linkage-employing drug conjugate has already been approved for drug delivery application, and also has shown immense potential in gene and siRNA transfection. This paper will focus on the recent developments in bioreducible polymeric systems for gene and siRNA delivery application, and will discuss the advantages and challenges associated with reducible polymeric carriers.