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High concentration protein solutions are generally produced by spin column concentration (SCC) during early development and by tangential flow filtration (TFF) during later stages, when greater quantities of protein become available. This is based on the assumption that the protein generated by the SCC process would be fairly similar to the TFF process material. In this study, we report the case of high concentration solutions of an Fc fusion protein produced by the two processes using the same upstream drug substance (DS) with very different storage stability. The TFF and SCC batches were characterized for aggregation, viscosity, and hydrodynamic radius before and after storage at different temperatures (5°C, 25 °C, and 40 °C). Aggregation and viscosity of the solutions processed by TFF were higher than those processed by SCC upon storage at 25 °C and 40 °C for three months. Differential scanning fluorimetry (DSF) revealed differences in initial protein conformation. Upon exposure to shear stress, protein solutions showed conformational instability and increased aggregation upon storage at 35 °C. In addition, protein solution showed higher aggregation upon shearing under mixed (downstream purification process and final formulation) buffer conditions – which are more likely to be encountered during the TFF, but not SCC, process. These results were further confirmed in an independent experiment by Fourier transform-infrared (FT-IR) spectroscopy and aggregation analysis. Taken together, these data indicate that shearing the protein in intermediate, unstable buffer conditions can lead to conformational perturbation during TFF processing, which led to higher rate of aggregation and viscosity upon storage. This study highlights the importance of testing shear stress sensitivity in the transitional buffer states of the TFF process early in development to de-risk process related product instability.

Formation of isoaspartate (IsoAsp) from spontaneous asparagine (Asn) deamidation or aspartate (Asp) isomerization is one of the most common non-enzymatic pathways of chemical degradation of protein and peptide pharmaceuticals. Rapid quantitation of IsoAsp formation can enable rank-ordering of potential drug candidates, mutants, and formulations as well as support shelf life prediction and stability requirements. A coupled enzymatic fluorescence-based IsoAsp assay (CEFIA) was developed as a high-throughput method for quantitation of IsoAsp in peptides and proteins. In this note, application of this method to two therapeutic candidate proteins with distinct structural scaffolds is described. In addition, the results obtained with this method are compared to those from conventional assays.

An efficient route to two functionalized 2-aryl-5H-chromeno[2,3-b]pyridines (azaxanthenes) is reported. The addition of lithiated 2,6-dichloropyridine to salicylaldehyde followed by cyclization was a key process improvement identified for the formation of the azaxanthene core. Further elaboration of 2-chloro-5H-chromeno[2,3-b]pyridin-5-ol at the 5 position was accomplished via Lewis acid-catalyzed coupling with commercially available ((1-methoxy-2-methylprop-1-en-1-yl)oxy)trimethylsilane. A partial classical resolution coupled with a preparative chiral supercritical fluid chromatography (SFC) separation was used to isolate the desired enantiomer of the azaxanthene carboxylic acid that is a common intermediate for both compounds 1 and 2. Suzuki–Miyaura cross-coupling with appropriately substituted boronic acids, followed by condensation with 2-amino-1,3,4-thiadiazole, provided the target compounds with an overall yield of approximately 10%. The use of stable, amorphous materials to support clinical compari...

This study aimed in understanding the degradation behaviors of an IgG 1 subtype therapeutic monoclonal antibody A (mAb-A) associated with pH and buffer species. The information obtained in this study can augment conventional, stability-based screening paradigms by providing the direction necessary for efficient experimental design. Differential scanning calorimetry (DSC) was used for studying conformational stability. Dynamic light scattering (DLS) was utilized to generate B 22*, a modified second virial coefficient for the character of protein-protein interaction. Size-exclusion chromatography (SEC) and hydrophobic interaction chromatography (HIC) were employed to separate degradation products. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used for determining the molecular size and liquid chromatography mass spectrometry (LC-MS) were used for identifying the sequence of the separated fragments. The results showed that both pH and buffer species played the roles in controlling the degradation behaviors of mAb-A, but the pH was more significant. In particular, pH 4.5 induced additional thermal transition peaks occurring at a low temperature compared with pH 6.5. A continual temperature-stress study illustrated that the additional thermal transition peaks related to the least stable structure and a greater fragmentation. Although mAb-A showed the comparable conformational structures and an identical amount of aggregates at time zero between the different types of buffer species at pH 6.5, the aggregation formation rate showed a buffer species-dependent discrepancy over a temperature-stress period. It was found that the levels of aggregations associated with the magnitudes of protein-protein interaction forces.

The Biophorum Development Group (BPDG) is an industry-wide consortium enabling networking and sharing of best practices for the development of biopharmaceuticals. To gain a better understanding of current industry approaches for establishing biopharmaceutical drug product (DP) robustness, the BPDG-Formulation Point Share group conducted an intercompany collaboration exercise, which included a bench-marking survey and extensive group discussions around the scope, design, and execution of robustness studies. The results of this industry collaboration revealed several key common themes: (1) overall DP robustness is defined by both the formulation and the manufacturing process robustness; (2) robustness integrates the principles of quality by design (QbD); (3) DP robustness is an important factor in setting critical quality attribute control strategies and commercial specifications; (4) most companies employ robustness studies, along with prior knowledge, risk assessments, and statistics, to develop the DP design space; (5) studies are tailored to commercial development needs and the practices of each company. Three case studies further illustrate how a robustness study design for a biopharmaceutical DP balances experimental complexity, statistical power, scientific understanding, and risk assessment to provide the desired product and process knowledge. The BPDG-Formulation Point Share discusses identified industry challenges with regard to biopharmaceutical DP robustness and presents some recommendations for best practices.

Since the introduction of Herceptin and Rituximab in 1986, therapeutic antibodies have gained tremendous momentum in the treatment of broad range of several diseases such as cancer and inflammation. Selection of the clinical candidate mAb usually starts with large-scale in vitro screening and profiling of multiple mAbs to identify candidates that show high in vitro or in vivo activity, and thus it is necessarily to identify and eliminate potentially unstable mAbs during the lead selection process. Antibodies undergo a variety of degradation reactions that may result in compromised bioactivity and safety profile. The nonenzymatic post-translational modification of both deamidation of asparagine and isomerization of aspartate residues is one of the major chemical reactions occurring in proteins during production and storage resulting in formation of protein variants that may affect the quality, safety, and functionality of the therapeutic proteins. Current methods (HPLC and liquid chromatography and mass spectrometry) for monitoring isoaspartate (isoAsp) formation are time consuming, require specialized equipment and trained personnel, and are not amenable to high-throughput scaling. We have developed a robust, homogenous, high-throughput formatted, and sensitive assay to accurately monitor the formation of isoAsp under several conditions, such as new formulations, storage periods, and temperature.

Solution pH and ionic strength (I) have complex effects on protein stability. We developed an experimental approach based on exploitation of the zwitterionic characteristic of amino acid molecules to probe the relative contribution from each. A variety of types of amino acid solutions were adopted to investigate the effects of pH and I in a manner that allows independent evaluation of each factor. The same effect could not be achieved using conventional buffer solutions. Size-exclusion chromatography, capillary differential scanning calorimetry, and fluorescence spectroscopy were utilized to probe the protein aggregation and conformation. The results suggested that, in addition to pH, solution ionic strength as a function of ionization state of the amino acid molecules and the ions introduced by pH adjustment played an important role in the aggregation and conformation of the protein studied. This experimental approach offers a useful tool to aid fundamental understanding of the relative effects of solution pH and ionic strength on protein stability.

A sensitive and effective method has been developed for the rapid determination of polysorbate-80 content in therapeutic monoclonal antibody (mAb) products. The method is based on the detection of the fluorescence emission of 4,4 0 -dianilino-1,1 0 -binaphthyl-5,5 0 disulfonic acid dipotassium salt (bis-ANS) enhanced by the presence of polysorbate-80. The developed method includes two approaches. One requires removal of the mAb from solution prior to analysis, while the other requires only simple sample dilution. The limits of detection and quantitation, calculated from the calibration curve generated in the absence of mAb-A, were 1.5 and 4.7 parts per million, respectively. Given the comparable linear range and linearity of the linear line between the solutions, with or without mAb, the limit of detection and quantitation is assumed to be similar. The dilution method is not only fast and simple in terms of sample preparation, but it is also particularly useful for analyzing the level of polysorbate-80 contained in highly concentrated mAb products. However, given that this method does require availability of polysorbate-80-free materials of mAb for preparation of calibration standards, the protein removal method may be useful for the cases where appropriate protein materials for standard preparation are limited or unavailable.

A high-throughput thermal-scanning method to rank-order formulation conditions for therapeutic proteins is described. Apparent transition temperatures for unfolding and aggregation of four different proteins are determined using the dyes SYPRO Orange and thioflavin T (ThT) under a variety of buffer conditions. The results indicate that the ThT-based thermal scanning method offers several advantages over the previously described SYPRO Orange-based thermal scanning and allows rapid rank ordering of solution conditions relevant toward long-term storage of therapeutic molecules. The method is also amenable to high protein concentration and does not require sample dilution or extensive preparation. Additionally, this parallel use of SYPRO Orange and ThT can be readily applied to the screening of mutants for their unfolding and aggregation propensities.

Micro-flow imaging (MFI) has been used for formulation development for analyzing sub-visible particles. Archimedes, a novel technique for analyzing sub-micron particles, has been considered as an orthogonal method to currently existing techniques. This study utilized these two techniques to investigate the effectiveness of polysorbate (PS-80) in mitigating the particle formation of a therapeutic protein formulation stored in silicone oil-coated pre-filled syringes. The results indicated that PS-80 prevented the formation of both protein and silicone oil particles. In the case of protein particles, PS-80 might involve in the interactions with the hydrophobic patches of protein, air bubbles, and the stressed surfaces of silicone oil-coated pre-filled syringes. Such interactions played a role in mitigating the formation of protein particles. Subsequently, quartz crystal microbalance with dissipation (QCM-D) was utilized to characterize the interactions associated with silicone oil, protein, and PS-80 in the solutions. Based on QCM-D results, we proposed that PS-80 likely formed a layer on the interior surfaces of syringes. As a result, the adsorbed PS-80 might block the leakage of silicone oil from the surfaces to solution so that the silicone oil particles were mitigated at the presence of PS-80. Overall, this study demonstrated the necessary of utilizing these three techniques cooperatively in order to better understand the interfacial role of PS-80 in mitigating the formation of protein and silicone oil particles.

Saccharides, including sucrose, trehalose, mannitol, and sorbitol, are commonly employed as stabilizers, cryoprotectants, and/or tonicity adjusters in protein formulations. During the thawing of a protein-containing formulated bulk drug substance conducted prior to a drug product (DP) filling operation, a white, crystalline precipitate was observed. In addition, upon thawing, vial breakage was observed for filled DP that had been previously frozen at −40 °C. To investigate the causes of both phenomena, the freeze/thaw behavior of the formulation components was studied. Multiple physical characterization techniques, including differential scanning calorimetry (DSC), electrical resistance measurements, thermomechanical analysis (TMA), and powder X-ray diffraction (PXRD), were utilized to characterize the formulations. The PXRD pattern of precipitate collected from thawed bulk was consistent with that of a mannitol control. An exothermic transition observed by DSC, a sharp increase in electrical resistance detected via resistivity measurements, and the onset of volumetric expansion of the frozen matrix evident in the TMA curve offer evidence that the frozen mannitol solution undergoes transitions at or near the vial breakage temperature (−22 to −23 °C) observed during warming. In addition, osmolality measurements taken from fractionated aliquots of frozen samples indicated that non-uniform concentration gradients contributed to precipitation of mannitol observed at larger scales. Small-scale laboratory experiments (i.e., 10–125 mL) failed to adequately predict behavior at larger scale (i.e., in 1 L and 2 L bottles). Upon linking the detrimental behavior to the freeze/thaw properties of the tonicity adjustor, mannitol, alternative saccharide excipients, including sorbitol, sucrose, and trehalose, were evaluated at isotonic concentrations over a temperature range of −80 to 25 °C using physical-chemical techniques and visual observation. Neither precipitation nor vial breakage was observed for the alternate saccharides. Recommendations for saccharide selection are provided based on storage conditions and scale considerations for liquid biopharmaceutical formulations. LAY ABSTRACT: Saccharides, including sucrose, trehalose, mannitol, and sorbitol, are commonly employed as stabilizers, cryoprotectants and/or tonicity adjusters in protein formulations. During thawing of formulated bulk drug substance, a white, crystalline precipitate was observed. In addition, upon thawing, vial breakage was observed for filled drug product that had been previously frozen at −40 °C. To investigate the causes of both phenomena, multiple physical characterization techniques were utilized to characterize the formulations. The powder X-ray diffraction pattern of precipitate collected from thawed bulk was consistent with that of a mannitol control. Upon linking the detrimental behavior to the freeze/thaw properties of the tonicity adjustor, mannitol, alternative saccharide excipients, including sorbitol, sucrose, and trehalose, were evaluated at isotonic concentrations over a temperature range of −80 to 25 °C using physico-chemical techniques and visual observation. Neither precipitation nor vial breakage was observed for the alternate saccharides. Recommendations for saccharide selection are given based on storage conditions and scale considerations for liquid biopharmaceutical formulations.

PEGylated poly(carboxylic acid)s, PEG-b-PCAs, were evaluated as additives for solubilized oral formulations of weakly acidic compounds. Micelles of poly(ethylene glycol)-block-poly(acrylic acid), PEG-b-PAA, and poly(ethylene glycol)-block-poly(methacrylic acid), PEG-b-PMAA, were prepared. Fluorescence spectroscopy and dynamic light scattering revealed that both polymers assemble into nanoscopic structures (200 nm) in acidic media and exhibit pH-sensitive colloidal phase behavior. Using a solvent evaporation technique, the block copolymers and corresponding PCA homopolymers were incorporated into PEG3350-based solid dispersions. The kinetic solubility profile of a BMS compound, BMS-A (Seq ~ 12.5 μg/mL at pH 1.1) in 0.1 N HCl was monitored as a function of polymer composition. While BMS-A precipitated rapidly in 0.1 N HCl in the absence of PEG-b-PCAs, a supersaturated level of ca. 400 μg/mL was maintained for variable lengths of time in the presence of PEG-b-PCAs. Although the kinetic solubility of BMS-A was also enhanced in the presence of the PCA homopolymers, the relative magnitude and duration of supersaturation as a function of polymer composition suggests that micellar solubilization, rather than specific interaction, contributes to enhanced solubility of BMS-A in 0.1 N HCl. Under acidic conditions, pH-responsive PEG-b-PCAs may offer the kinetic supersaturation necessary to minimize precipitation of compounds which have limited solubility in acidic milieu.

To support dose reduction, low dose of a monoclonal antibody (mAb) was required to be administered via IV infusion at a concentration of 0.1 mg/mL. To achieve the target protein concentration, the infusion solution was prepared by diluting the drug product containing 10-mg/mL mAb with normal saline, a 0.9% sodium chloride injection solution. However, particles were observed in the diluted solution. Particle formation must be avoided to administer the low dose using the existing drug product. To mitigate the particle formation, an unconventional compounding approach was used. With this approach, a stabilizing vehicle containing polysorbate-80 was added to saline before drug-product dilution to maintain suitable surfactant level to prevent precipitation of the mAb. In this way, use of the stabilizing vehicle to support low doses ensured suitable quality across a wider range of mAb concentrations, thereby allowing additional flexibility to the clinical trial. Such an approach may be useful for broader application in early-stage clinical trials where there is an uncertainty regarding doses or the need to revise to lower doses based on clinical observations or other drivers.

BMS-779788 contains a reactive tertiary hydroxyl attached to a weakly basic imidazole ring. Propensity of the carbinol toward dehydration to yield the corresponding alkene, BMS-779788-ALK, was evaluated. Elevated levels of BMS-779788-ALK were observed in excipient compatibility samples. Stability studies revealed that BMS-779788 degrades to BMS-779788-ALK in capsules and tablets prepared by both dry and wet granulation processes. An acid-catalyzed dehydration mechanism, in which the heterocyclic core contributes resonance stability to the cationic intermediate via charge transfer to the imidazole ring, was proposed. Therefore, neutralization via a buffered (pH 7.0) granulating solution was used to mitigate dehydration. Solution studies revealed degradation of BMS-779788 to BMS-779788-ALK over the pH range of 1-7.5. Reversibility was confirmed by initiating reactions with BMS-779788-ALK over the same pH range. Accordingly, a simple reversible scheme can be used to describe reactions initiated with either BMS-779788 or BMS-779788-ALK. To eliminate potential for charge delocalization across the heterocycle and probe the degradation mechanism, the imidazole ring of BMS-779788 was methylated (BMS-779788-Me). The propensity for acid-catalyzed dehydration was then evaluated. The acid stability of BMS-779788-Me confirmed that the heterocyclic core contributes to reactivity liability of the tertiary hydroxyl.

Amphiphilic block copolymers (ABCs) have been used extensively in pharmaceutical applications ranging from sustained-release technologies to gene delivery. The utility of ABCs for delivery of therapeutic agents results from their unique chemical composition, which is characterized by a hydrophilic block that is chemically tethered to a hydrophobic block. In aqueous solution, polymeric micelles are formed via the association of ABCs into nanoscopic core/shell structures at or above the critical micelle concentration. Upon micellization, the hydrophobic core regions serve as reservoirs for hydrophobic drugs, which may be loaded by chemical, physical, or electrostatic means, depending on the specific functionalities of the core-forming block and the solubilizate. Although the Pluronics, composed of poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide), are the most widely studied ABC system, copolymers containing poly(L-amino acid) and poly(ester) hydrophobic blocks have also shown great promise in delivery applications. Because each ABC has unique advantages with respect to drug delivery, it may be possible to choose appropriate block copolymers for specific purposes, such as prolonging circulation time, introduction of targeting moieties, and modification of the drug-release profile. ABCs have been used for numerous pharmaceutical applications including drug solubilization/stabilization, alteration of the pharmacokinetic profile of encapsulated substances, and suppression of multidrug resistance. The purpose of this minireview is to provide a concise, yet detailed, introduction to the use of ABCs and polymeric micelles as delivery agents as well as to highlight current and past work in this area.

We systematically altered the chemical structure of the core-forming poly(L-amino acid) block of an amphiphilic diblock copolymer series based on poly(ethylene oxide)-block-poly(N-hexyl-L-aspartamide), PEO-b-p(N-HA), acyl esters by varying the length of the attached acyl side chain. Drug-loaded micelles were prepared in good yield by a modified solvent evaporation procedure. In addition, the relative aggregation state and hemolytic activity of encapsulated amphotericin B (AmB) were analyzed by absorption spectroscopy. The length of the attached acyl side chain in PEO-b-p(N-HA) acyl ester micelles modulates the relative aggregation state of encapsulated AmB. Furthermore, acyl chain length appears to have a profound influence on the time-dependent hemolytic profile of encapsulated AmB toward bovine erythrocytes. For all acyl conjugate micelle-AmB formulations, the onset of hemolysis is delayed relative to free AmB. Particularly in the case of stearate ester micelles, the incomplete and gradual build-up of hemolysis might reflect the sustained release of drug over a period of 24 h. Based on the corresponding absorption spectrum, we speculate that encapsulated AmB may interact strongly with stearate side chains, resulting in sustained release. Via chemical manipulation of the core-forming region, it may be possible to fine-tune the release of encapsulated AmB from PEO-b-p(N-HA)-acyl ester micelles.

Derivatives of poly(ethylene oxide)-block-poly(beta-benzyl-aspartate), 12:25 have been prepared via aminolysis of the benzyl protecting group with 6-amino-1-hexanol, followed by subsequent acylation with acetic anhydride, hexanoic acid, lauric acid, or stearic acid. A series of amphiphilic diblock copolymers based on poly(ethylene oxide)-block-poly(N-hexyl-aspartamide)acyl conjugates with various acyl chain lengths have been prepared. The extent of esterification was determined by 1H-NMR. Aqueous micelle solutions were prepared by a dialysis method and the polymer series was characterized as a function of the acyl chain length. Transmission electron microscopy and dynamic light scattering revealed micelle-like structures of nanoscopic dimensions (< 100 nm). Environmentally sensitive fluorescent probes were loaded into the micelles in order to study the properties of the hydrophobic microdomain and to determine the critical micelle concentration (CMC). Steady-state fluorescence measurements indicated that the relative apparent core viscosity and polarity are modulated by the relative length of the attached acyl chains, as is the CMC. Increasing the acyl chain length results in a decreased CMC and a more viscous and less polar core region. Carefully chosen chemical moieties can be introduced in order to influence the properties of the poly(L-Asp) blocks of the micelles. As a result, the micellar properties can be altered via chemical modification in order to impact several key properties relevant to drug delivery.

Poly(vinyl alcohol) (PVA)-based formulations are used for pharmaceutical tablet coating with numerous advantages. Our objective is to study the stability of PVA-based coating films in the presence of acidic additives, alkaline additives, and various common impurities typically found in tablet formulations. Opadry® II 85F was used as the model PVA-based coating formulation. The additives and impurities were incorporated into the polymer suspension prior to film casting. Control and test films were analyzed before and after exposure to 40°C/75% relative humidity. Tests included film disintegration, size-exclusion chromatography, thermal analysis, and microscopy. Under stressed conditions, acidic additives (hydrochloric acid (HCl) and ammonium bisulfate (NH(4)HSO(4))) negatively impacted Opadry® II 85F film disintegration while NaOH, formaldehyde, and peroxide did not. Absence of PVA species from the disintegration media corresponded to an increase in crystallinity of PVA for reacted films containing HCl. Films with NH(4)HSO(4) exhibited slower rate of reactivity and less elevation in melting temperature with no clear change in melting enthalpy. Acidic additives posed greater risk of compromise in disintegration of PVA-based coatings than alkaline or common impurities. The mechanism of acid-induced reactivity due to the presence of acidic salts (HCl vs. NH(4)HSO(4)) may be different.

The core-forming blocks of amphiphilic diblock copolymers based on methoxypoly(ethylene oxide)-block-poly(L-aspartate), PEO-b-p(L-Asp), were derivatized to incorporate stearate side chains. The effects of stearate esterification were assessed in terms of micelle stability and amphotericin B (AmB) encapsulation/release. The level of stearate esterification modulates the relative self-aggregation state of encapsulated AmB as evidenced by absorption spectroscopy. When AmB is physically loaded into polymeric micelles, the onset of hemolytic activity toward bovine erythrocytes is delayed relative to that of the free drug. Furthermore, the extent of esterification (0, 46, or 91%) appears to have profound influence on the time-dependent hemolytic profile of AmB toward bovine erythrocytes. Particularly in the case of highly substituted stearate ester micelles, incomplete and gradual build-up of hemolysis was observed over a period of 24 h. On the basis of the corresponding absorption spectra, we speculate that encapsulated AmB may interact strongly with stearate side chains, resulting in sustained release. In a neutropenic murine model of disseminated candidiasis, kidney colony-forming unit determination revealed dose-dependent efficacy for the polymeric micelle/AmB formulation, which was not significantly different from that of Fungizone at doses of 0.2, 0.3, and 0.6 mg/kg (p = 0.7). Thus, AmB administered via a polymeric micelle formulation retained potent in vivo activity.

The original article to which this Erratum was published in Journal of Pharmaceutical Sciences 92(7) 2003, 1343–1355