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Regulatory and manufacturing requirements exist to perform product-specific microbial retention testing on sterilizing filters. The implementation of a Quality by Design approach to sterilizing filtration supports a paradigm that would obviate the need for product-specific testing for early-stage products that do not have the quantity of material required to perform such testing easily and efficiently. Process and product parameters were varied to determine their effect on microbial retention to define a design space. To minimize the burden of filter validation retention studies for early stage (Phase 1) manufacturing, it is recommended that manufacturers perform a risk assessment to confirm their product and process conditions are within the established design space. For later stage product development prior to marketing authorization, product specific filter validation testing is expected.

In hospitals, often drug products in intravenous (IV) bags are transported via pneumatic tube systems (PTS). The goal of this study was to evaluate the effects of such transportation of protein products on particle formation in polyvinyl chloride (PVC) and polyolefin (PO) IV bags, containing either IV saline or dextrose. We studied intravenous immunoglobulin (IVIG) and a monoclonal antibody (mAb). Particles were quantified with flow imaging, light obscuration and nanoparticle tracking analysis. PTS transportation of IVIG caused large increases in protein particle concentrations, with much greater increases observed in saline than in dextrose. The increases were greater in IV solutions in PO than those in PVC bags. With the mAb, PTS transportation in saline caused increases in protein particle levels in PO bags, but not in PVC bags. Transportation in dextrose did not result in significant increases in mAb particle concentrations in IV bags made of either material. Overall, the results document that the PTS transportation can result in large increases in protein particles and that magnitude of these increases depends the protein itself, the bag material and the IV solution. The main conclusion is that protein products in IV solutions should not be transported in hospital PTS.

The control strategy is a critical aspect of vaccine drug development. This chapter introduces concepts of control strategy and how the holistic control strategy can be applied for a multicomponent suspension vaccine drug product. Various concepts and hypothetical case studies are utilized to help the readers to get familiarity with the topic.

One of the major product quality challenges for injectable biologics is controlling the amount of protein aggregates and particles present in the final drug product. This article focuses on particles in the submicron range (2 μm). A cross-industry collaboration was undertaken to address some of the analytical gaps in measuring submicron particles (SMPs), developing best practices, and surveying the concentration of these particles present in 52 unique clinical and commercial protein therapeutics covering 62 dosage forms. Measured particle concentrations spanned a range of 4 orders of magnitude for nanoparticle tracking analysis and 3 orders of magnitude for resonant mass measurement. The particle concentrations determined by the 2 techniques differed significantly for both control and actual product. In addition, results suggest that these techniques exhibit higher variability compared to well-established subvisible particle characterization techniques (e.g., flow-imaging or light obscuration). Therefore, in their current states, nanoparticle tracking analysis and resonant mass measurement-based techniques can be used during product and process characterization, contributing information on the nature and propensity for formation of submicron particles and what is normal for the product, but may not be suitable for release or quality control testing. Evaluating the level of SMPs to which humans have been routinely exposed during the administration of several commercial and late-phase clinical products adds critical knowledge to our understanding of SMP levels that may be considered acceptable from a safety point of view. This article also discusses dependence of submicron particle size and concentration on the dosage form attributes such as physical state, primary packaging, dose strength, etc. To the best of our knowledge, this is the largest study ever conducted to characterize SMPs in late-phase and commercial products.

Particulate matter present in drug products intended for parenteral administration to patients is typically monitored and controlled in the finished drug product to minimize potential risks to patients. In contrast to particulates found in drug products, the current study evaluated particulates representative of materials and operations typically used in the dose preparation and administration of drug products. A comprehensive assessment of intrinsic and extrinsic sources of subvisible and submicron particulates arising from materials associated with subcutaneous and intravenous dose preparation and administration was conducted. In particular, particles arising from disposable syringes, commercial sterile diluents, and intravenous supplies were quantitated using established methods for subvisible (light obscuration, flow imaging) and submicron particles (resistive pulse sensing). Each of these sources contributed varying amounts of particulates; therefore, owing to sources from materials required for administration, it is inadequate to assume that the total particulate load delivered to patients arises solely from the drug product. Careful consideration of the administration method and supplies used can improve the predictability of particulate levels present in dose preparations or administration volumes.

Trumenba (MenB-FHbp; bivalent rLP2086), the first meningococcal serogroup B vaccine approved in the United States and subsequently approved in Europe, Canada, and Australia, is well-characterized. Pfizer devised a control strategy approach by using a simplified control strategy wheel for Trumenba based on International Council for Harmonisation (ICH) Q8 (R2), Q9, Q10, and Q11 guidelines, which provide complementary guidance on pharmaceutical development, quality risk management, quality systems, and development and manufacture of drug substances, respectively. These guidelines ensure product quality using a scientific and risk-based approach. Trumenba contains two factor H binding proteins (FHbps), one from each of the two FHbp subfamilies (A and B), adsorbed onto aluminum phosphate. Trumenba manufacturing processes are complicated by the recombinant protein expression of Subfamily A and B proteins and the nature of the drug product (suspension in syringes); the latter also introduces challenges in controlling product critical quality attributes during the development process. In such complex systems, the control strategy is critical to ensuring consistent desired product quality; it also supports the regulatory requirement of continued improvement through continuous process verification and aids regulatory filing. This article describes Pfizer's approach toward robust control strategy development, built on product and process understanding, and links control strategy to regulatory document sections and flow of controls. Specifically, an approach is presented on product quality attribute criticality determination based on safety and efficacy and on an understanding of process parameter criticality. This was achieved by studying the impact of the approach on product quality attributes to define process parameter and in-process controls. This approach is further explained through Trumenba case studies, highlighting specific quality attributes and the associated controls implemented, and provides a holistic view of controls employed for both drug substance and drug product.

A case study is presented for a vaccine drug product (DP) that showed variable re-dispersion times between syringes within a given DP lot and between different DP lots when shipped from the manufacturing site to the receiving site. A simulated shipping study was designed to understand the effect of individual shipping stresses on re-dispersion time and product quality. Shipping stresses simulating shock/drop, aircraft, and truck vibrations were applied separately to 3 syringe orientations, namely tip up, tip down, and tip horizontal (TH). Results from the simulated shipping study showed that shock/drop reduced re-dispersion time while truck and aircraft vibrations increased re-dispersion time in the tip down orientation. The dissimilar effects of different shipping stresses on re-dispersion resulted in the observed intra and inter DP lot variability in re-dispersion time. Shipping stresses did not impact re-dispersion in the TH or tip up orientation. No vaccine product quality attributes or physical properties were affected by shipping stresses. Actual shipping results correlated well with simulated shipping data. Because re-dispersion time was influenced mainly by shipping stress and syringe orientation, the mitigation measure to reduce end-user re-dispersion time was to implement the TH orientation for DP syringes during shipment and storage.

Protein drug substance is typically frozen to enable manufacturing flexibility through prolonging the shelf life of drug substance and providing better biochemical stability. The process of freezing and thawing of bulk protein solutions poses several challenges. It is important to understand the process and define mitigation strategies to address these challenges. Just not the process but the choice of storage container can have an impact on stability and downstream operation of formulation during drug product process. Therefore, understanding the options currently available in terms of drug substance storage containers and how to balance the need based on specific scenarios is critical. Storage temperature is as important as the container the drug substance is stored at. Furthermore, shipping drug substance in frozen state requires thorough understanding of logistics and dependence of shipping temperature on stability. This chapter fundamentally looks at the freezing and thawing process, discusses phenomenon of cryoconcentration in various containers and mitigation strategies, consequence of not choosing appropriate storage temperature, and provides considerations for storage and shipping of drug substance.

Ex situ and in situ techniques were used to characterize the suspended phase over time for a vaccine drug product supplied in syringes. Micro-computed tomography was used to characterize the suspended sediment in situ in the syringe, while traditional techniques such as particle size distribution, charge (zeta potential), settling rate, and front-faced fluorescence were used to characterize the suspension ex situ. In addition, analytical chemical measurements were conducted in parallel during the course of the study. The ex situ and in situ techniques together with the chemical analyses provided different sets of data, but all leading to the same conclusion that the older, hard to re-disperse vaccine product syringes were similar in product quality attributes (both physical and chemical) to the freshly made, easy to re-disperse syringes. Longer re-dispersion time with age was not a result of any altered physical or chemical attributes of the product but simply because of the distance travelled by the sediment from the neck of the syringe barrel deeper into the bore of the syringe over time under the influence of gravity in the tip down orientation, making it harder for the continuous external phase to access the sediment in the bore and enable easy re-dispersion.

A monoclonal antibody (mAb) product development case study is presented to address some of the issues faced during developing a pre-filled syringe (PFS) product for a biotherapeutic. In particular, issues involving incompatibility with silicone oil and a stability-based approach for selection of PFS barrel and tip cap components have been discussed. Silicone spiking studies followed by exposure to agitation stress or accelerated temperature conditions were used to check for incompatibilities of the mAb with silicone oil, a necessary product contact material in PFS. In addition, screening studies to compare various closure materials as well as syringe barrel processing methods were used to select the optimum closure materials as well as the correct syringe processing method. Results indicate that the model mAb formulation used was sensitive to high levels of silicone oil especially under accelerated temperature conditions resulting in formation of protein–silicone particles in the solution for samples that were spiked with the silicone oil. Agitation stress did not have any significant impact on the quality attributes tested. Samples stored in syringe barrels that were processed with sprayed-on silicone had higher levels of subvisible particles as compared to those that were processed with the baked-on process. The tip cap comparability study resulted in one tip cap material having superior compatibility among the three that were tested. The quality attribute that was most impacted by the tip cap materials was mAb oxidation. An approach for evaluation of primary packaging components during the development of pre-filled syringe presentations for biotechnology-based compounds has been highlighted.

Freezing of biologic drug substance at large scale is an important unit operation that enables manufacturing flexibility and increased use-period for the material. Stability of the biologic in frozen solutions is associated with a number of issues including potentially destabilizing pH changes. The pH changes arise from temperature-associated change in the pK(a)s, solubility limitations, eutectic crystallization, and cryoconcentration. The pH changes for most of the common protein formulation buffers in the frozen state have not been systematically measured. Sodium phosphate buffer, a well-studied system, shows the greatest change in pH when going from +25 to -30 degrees C. Among the other buffers, histidine hydrochloride, sodium acetate, histidine acetate, citrate, and succinate, less than 1 pH unit change (increase) was observed over the temperature range from +25 to -30 degrees C, whereas Tris-hydrochloride had an approximately 1.2 pH unit increase. In general, a steady increase in pH was observed for all these buffers once cooled below 0 degrees C. A formulated IgG2 monoclonal antibody in histidine buffer with added trehalose showed the same pH behavior as the buffer itself. This antibody in various formulations was subject to freeze/thaw cycling representing a wide process (phase transition) time range, reflective of practical situations. Measurement of soluble aggregates after repeated freeze-thaw cycles shows that the change in pH was not a factor for aggregate formation in this case, which instead is governed by the presence or absence of noncrystallizing cryoprotective excipients. In the absence of a cryoprotectant, longer phase transition times lead to higher aggregation.

Dendrimers have emerged as promising multifunctional nanomaterials for drug delivery due to their well-defined size and tailorability. We compare two schemes to obtain methylprednisolone (MP)-polyamidoamine dendrimer (PAMAM-G4-OH) conjugate. Glutaric acid (GA) was used as a spacer to facilitate the conjugation. In scheme A, PAMAM-G4-OH was first coupled to GA and then further conjugated with MP to obtain PAMAM-G4-GA-MP conjugates. This scheme yields a lower conjugation ratio of MP, presumably because of lower reactivity and steric hindrance for the steroid at the crowded dendrimer periphery. In scheme B, this steric hindrance was overcome by first preparing the MP-GA conjugate, which was then coupled to the PAMAM-G4-OH dendrimer. The (1)H NMR spectrum of the conjugate from scheme B indicates a conjugation of 12 molecules of MP with the dendrimer, corresponding to a payload of 32 wt %. In addition, conjugates were further fluorescent-labeled with fluoroisothiocynate (FITC) to evaluate the dynamics of cellular entry. Flow cytometry and UV/visible spectroscopic analysis showed that the conjugate is rapidly taken up inside the cell. Fluorescence and confocal microscopy images on A549 human lung epithelial carcinoma cells treated with conjugates show that the conjugate is mostly localized in cytosol. MP-GA-dendrimer conjugate showed comparable pharmacological activity to free MP, as measured by inhibition of prostaglandin secretion. These conjugates can potentially be further conjugated with a targeting moiety to deliver the drugs to specific cells in vivo.

Highly branched, functionalized polymers have potential to act as efficient drug carrier systems. Dendrimers are ideal candidates among model hyperbranched polymers because of their well-defined structure and high density of functional groups. Using ibuprofen as a model drug, we studied the interaction between the drug and Polyamidoamine (PAMAM) dendrimers (generations 3 and 4 with --NH2 functionality) and Perstrop Polyol (generation 5, hyperbranched polyester with --OH functionality). FTIR and NMR studies suggest that ibuprofen predominantly forms a complex with PAMAM dendrimers because of the ionic interaction between the --NH2 end groups and the carboxyl group of ibuprofen. On an average, up to 78 molecules of ibuprofen could be incorporated into one molecule of PAMAM-G4-NH2 with 64 end groups. This complex is stable in deionized water and methanol. The in vitro release of ibuprofen from drug-dendrimer complex is appreciably slower compared to pure ibuprofen. The complexed drug enters A549 cells much more rapidly than pure drug suggesting that dendrimers may be able to carry the complexed drug inside cells efficiently. Hyperbranched Polyol (with 128 --OH end groups) appears to encapsulate approximately 24 drug molecules. Perhaps the lack of strong interactions between the --OH end groups and the drugs prevents complex formation.

Chitosan is an important biomaterial used widely in medical applications. One of the key concerns about its use is the fragile nature of chitosan films. By comparing the component molecular interactions using FTIR, this study attempts to understand how the ductility of chitosan can be improved by blending and copolymerizing with poly(ethylene glycol) (PEG). An improvement in ductility was obtained for all compositions of blend as manifested by a decrease in modulus and an increase in strain at break. For comparable PEG composition (∼30%), the properties of the solution-cast blend were better than those of the grafted copolymer. Therefore, blending may be a more efficient way to improve ductility of chitosan. FTIR characterization of the materials revealed subtle decreases in molecular interactions upon annealing the partially miscible blend. These may not be apparent in DSC or X-ray diffraction, yet they play a key role in the mechanical behavior. It appears that in the case of the graft copolymer the imp...

Anti-nicotine vaccines aim to prevent nicotine entering the brain, and thus reduce or eliminate the reward that drives nicotine addiction. Those tested in humans to date have failed to improve quit rates over placebo, possibly because antibody (Ab) responses were insufficient to sequester enough nicotine in the blood in the majority of subjects. We have previously shown in mice that the carrier, hapten and linker used in the nicotine conjugate antigen each influence the function (nicotine-binding capacity) of the Ab induced. Herein we have evaluated immunogenicity in mice of 27 lots of NIC7–CRM, a conjugate of 5-aminoethoxy-nicotine (Hapten 7) and a mutant nontoxic form of diphtheria toxin (CRM197), that differed in three antigen attributes, namely hapten load (number of haptens conjugated to each molecule of CRM197), degree of conjugate aggregation and presence of adducts (small molecules attached to CRM197 via a covalent bond during the conjugation process). A range of functional responses (reduced nicotine in the brain of immunized animals relative to non-immunized controls) were obtained with the different conjugates, which were adjuvanted with aluminum hydroxide and CpG TLR9 agonist. Trends for better functional responses in mice were obtained with conjugates having a hapten load of 11 to 18, a low level of high molecular mass species (HMMS) (i.e., not aggregated) and a low level of adducts and a more limited testing in cynomolgus monkeys confirmed these results. Thus hapten load, conjugate aggregation and presence of adducts are key antigen attributes that can influence Ab function induced by NIC7–CRM.

To investigate the cause of unexpected and erratic increase in aggregation during long-term storage of an IgG2 monoclonal antibody in a trehalose formulation at -20°C.Frozen matrix was sampled, stored frozen at various temperatures and analyzed by SEC over time.Aggregation increased with time at -20°C but not at -40°C or -10°C. The cause of the instability was the crystallization of freeze-concentrated trehalose from the frozen solute when the storage temperature exceeds the glass transition temperature of the matrix (-29°C). Crystallization at -20°C deprives the protein of the cryoprotectant, leading to a slow increase in aggregation. Storage at -10°C also leads to crystallization of trehalose but no increase in aggregation. It is hypothesized that significantly higher mobility in the matrix at -10°C allows protein molecules that are unfolded at the ice interface on freezing to refold back before significant aggregation can occur. In contrast, lack of mobility at -40°C prevents crystallization, refolding, and aggregation.Aggregation in the frozen state when stored above the glass transition temperature is a consequence of balance between rate of crystallization leading to loss of cryoprotectant, rate of aggregation of the unfolded protein molecules, and rate of refolding that prevents aggregation.

Active pharmaceutical ingredient for biotechnology-based drugs, commonly known as drug substance (DS), is often stored frozen for longer shelf-life. Freezing DS enhances stability by slowing down reaction rates that lead to protein instability, minimizes the risk of microbial growth, and eliminates the risk of transport-related stress. High density polyethylene bottles are commonly used for storing monoclonal antibody DS due to good mechanical stress/strain resistant properties even at low temperatures. Despite the aforementioned advantages for frozen storage of DS, this is not devoid of risks. Proteins are known to undergo ice-water surface denaturation, cryoconcentration, and cold denaturation during freezing. A systematic investigation was performed to better understand the protein and solute distribution along with potential of aggregate formation during freeze and thaw process. A significant solute and protein concentration gradient was observed for both frozen and thawed DS bottles. In case of thawed DS, cryoconcentration was localized in the bottom layer and a linear increase in concentration as a function of liquid depth was observed. On the other hand, for frozen DS, a "bell shaped" cryoconcentration distribution was observed between the bottom layers and centre position. A cryoconcentration of almost three-fold was observed for frozen DS in the most concentrated part when freezing was conducted at -20 and -40 °C and 2.5-fold cryoconcentration was observed in the thawed DS before mixing. The information obtained in this study is critical to design freeze thaw experiments, storage condition determination, and process improvement in manufacturing environment.

Drug release from hyperbranched polymer-drug conjugates and the subsequent activity are influenced by the branching architecture and the linker. To gain an understanding of these effects, we used hyperbranched polyol and G4-OH polyamidoamine (PAMAM) dendrimer with methyl prednisolone (MP) as the model drug. The drug was conjugated to dendrimer or polyol using a glutaric acid (GA) or a succinic acid (SA) spacer. Drug payload was the highest with polyol, while in the case of dendrimer, a higher payload was achieved with the GA than the SA spacer. Cell uptake of the polymer conjugates in A549 lung epithelial cells was higher than that of the free drug, and the conjugates largely localized in the cytosol. The anti-inflammatory activity of polymer conjugated MP, as measured by inhibition of prostaglandin synthesis, was the highest for MP-SA-dendrimer conjugate, followed by MP-GA-polyol conjugate, and then MP-GA-dendrimer conjugate. This study suggests that the branching architecture and spacer influence the drug payload and pharmacological activity of a drug-nanopolymer conjugate, which may significantly influence the in vivo efficacy of these nanodevices. This has key implications in the eventual in vivo efficacy of these nanodevices.

Dendrimers are emerging as a relatively new class of polymeric biomaterials with applications in drug delivery, and imaging. Achieving a high drug payload in dendrimers, and understanding the therapeutic effect of the dendrimer-drug conjugates are receiving increasing attention. A high drug payload nanodevice was obtained by covalent conjugation of ibuprofen to a polyamidoamine (PAMAM-G4-OH) dendrimer. Using DCC as a coupling agent, 58 molecules of ibuprofen were covalently conjugated to one molecule of generation 4 PAMAM-OH dendrimer. Cellular entry of the fluoroisothiocynate (FITC)-labeled dendrimer-drug conjugate was evaluated in vitro by using human lung epithelial carcinoma A549 cells by flow cytometry, confocal microscopy and UV/Visible spectroscopy. The pharmacological activity of the dendrimer-ibuprofen conjugate was compared to pure ibuprofen at various time points by measuring the suppression of prostaglandin E2. Significant amounts of the conjugate entered the cells rapidly within 15 min. Suppression of prostaglandin was noted within 30 min for the dendrimer-drug conjugates versus 1 h for the free ibuprofen. The results suggest that dendrimers with high drug payload improve the drug's efficacy by enhanced cellular delivery, and may produce a rapid pharmacological response. These dendrimer-drug conjugates can potentially be further modified by attaching antibodies and ligands for targeted drug delivery.

To synthesize and evaluate hyperbranched polymer (HBP)-drug conjugates with high drug payload for enhanced cellular delivery.Polyol- and polyglycerol-ibuprofen conjugates with or without imaging agent fluorescein isothiocyanate (FITC) were synthesized using dicyclohexilcarbodiimide (DCC) as a coupling agent. Drug-polymer conjugates were characterized using 13C NMR, 1H NMR, and gel permeation chromatography (GPC). Stability of the drug-conjugates was studied using free drug release through a dialysis membrane. Cellular entry of FITC-labeled HBP conjugates was studied using fluorescence activated cell sorter (FACS), and cell supernatant was analyzed by UV-visible spectrophotometer. The intracellular localization of FITC-labeled conjugates in A549 lung epithelial cells was imaged using fluorescence microscopy. Anti-inflammatory activity of the HBP-ibuprofen conjugates was estimated in vitro by measuring the concentration of prostaglandin (PGE2) using an ELISA kit.The average number of ibuprofen molecules conjugated per molecule of HBP was estimated to be 50 for polyol and 53 for polyglycerol. The HBP-drug conjugates did not release the drug up to 72 h in methanol, indicating the presence of stable ester bonds. Both the polymer-drug conjugates entered the cells rapidly. The conjugates were localized in the cell cytosol as evidenced by fluorescence microscopy. Within 30 min, the HBP-drug conjugates showed rapid suppression of PGE2 synthesis, whereas free ibuprofen did not show any activity. At later times, the conjugates showed comparable activity.For the first time, we report HBP conjugates with a high drug payload. HBP-drug conjugates entered the cells rapidly and produced the desired pharmacological action. This study demonstrates that hyperbranched polyol and polyglycerol are promising nanovehicles for achieving enhanced cellular delivery of drugs.

Dendrimers and hyperbranched polymers are emerging as potentially ideal drug delivery vehicles because they provide a significant amount of tailorability and a large density of functional groups. This study explores the dynamics of cellular entry of dendrimers and hyperbranched polymers alone, and in the complexed form with ibuprofen, into A549 human lung epithelial carcinoma cells using UV/Vis spectroscopy, flow cytometry and fluorescence microscopy. Both dendrimers and hyperbranched polymers appear to enter these cells rapidly. The polyamidoamine (PAMAM) dendrimers, with NH2 and OH end functionalities appear to enter cells (in approx. 1 h) faster than the hyperbranched polyol (OH functionality) (in approx. 2 h). Cellular entry of PAMAM-NH2 was detected as early as 5 min. All branched polymers and their ibuprofen complexes entered A549 lung epithelial cells rapidly when compared to the pure drug. The drug payload was about 50% by weight in the complexes formed by PAMAM-NH2 dendrimers and was about 30% in the encapsulated form for Polyol-OH and PAMAM-OH. The complexation and encapsulation of ibuprofen with the polymers appear to facilitate rapid cellular entry of ibuprofen. The anti-inflammatory effect of the polymer-complexed drug was demonstrated by more rapid suppression of COX-2 mRNA levels than that achieved by the pure drug. This suggests that these dendritic polymers can act as efficient drug carriers, delivering high 'payloads' of drug even with complexation and encapsulation.

Chitosan is an important biomaterial used widely in medical applications. One of the key concerns about its use is the fragile nature of chitosan films. By comparing the component molecular interactions using FTIR, this study attempts to understand how the ductility of chitosan can be improved by blending and copolymerizing with poly(ethylene glycol) (PEG). An improvement in ductility was obtained for all compositions of blend as manifested by a decrease in modulus and an increase in strain at break. For comparable PEG composition (approximately 30%), the properties of the solution-cast blend were better than those of the grafted copolymer. Therefore, blending may be a more efficient way to improve ductility of chitosan. FTIR characterization of the materials revealed subtle decreases in molecular interactions upon annealing the partially miscible blend. These may not be apparent in DSC or X-ray diffraction, yet they play a key role in the mechanical behavior. It appears that in the case of the graft copolymer the improvement in the properties comes from suppression of the crystallinity of each component and not from component interactions. On the other hand, in the blend, the improvement appears to come predominantly from the "well-dispersed", "kinetically trapped" phase morphology and from the intermolecular interactions. Therefore, annealing the blend leads to decreased intermolecular interactions, phase coarsening, and deterioration in properties.