Surfactant Impact on Interfacial Protein Aggregation and Utilization of Surface Tension to Predict Surfactant Requirements for Biological Formulations.

Biological drug products are formulated with excipients to maintain stability over the shelf life of the product. Surfactants are added to the drug product to stabilize air-water interfaces known to induce protein aggregation. Early formulation development is focused on maintaining protein conformation and colloidal stability over the course of the drug product shelf life but rarely considers stability through dose preparation and administration. Specifically, intravenous (IV) bag preparation exposes the therapeutic protein to a different solution environment concurrently diluting the stabilizing excipients that had been added to the drug product formulation. Mixing in IV bags can generate dynamic changes in the air-water interfacial area known to cause protein aggregation if not sufficiently protected. Therefore, understanding the surfactant requirements for drug product end-to-end stability in early formulation development provides critical information for a right-first-time approach to drug product formulation and robust clinical preparation. The goal of these studies was to understand if interfacial properties of proteins could predict surfactant formulation requirements for end-to-end stability. Specifically, the interfacial properties of five proteins were measured in 0.9% saline and 5% dextrose. Furthermore, shaking studies were conducted to identify the minimum surfactant concentration required to prevent subvisible and visible particle formulation in each diluent. The impact of surfactant type and concentration on particle generation and size was explored. A mathematical model was generated to predict the minimum surfactant concentration required to prevent interface-driven aggregation in each diluent based on the change in surface pressure upon exposure of the protein to the interface. The model was tested under typical IV-preparation conditions with experimental output closely matching the model prediction. By employing this model and better understanding the role of surfactants in interfacial stability, drug product development can generate robust end-to-end large molecule formulations across shelf life, dose preparation, and administration.

Residue-Specific Impact of EDTA and Methionine on Protein Oxidation in Biotherapeutics Formulations Using an Integrated Biotherapeutics Drug Product Development Workflow.

The rational design and selection of formulation composition to meet molecule-specific and product-specific needs are critical for biotherapeutics development to ensure physical and chemical stability. This work, based on three antibody-based (mAb) proteins (mAbA, mAbB, and mAbC), evaluates residue-specific impact of EDTA and methionine on protein oxidation, using an integrated biotherapeutics drug product development workflow. This workflow includes statistical experimental design, high-throughput experimental automation and execution, structure-based in silico modeling, inferential statistical analysis, and enhanced interactive data visualization of large datasets. This oxidation study evaluates the impact of formulation parameters including pH, protein concentration, and the presence of polysorbate 80 on the oxidation of specific conserved and variable residues of mAbs A, B, and C in the presence of stressors (iron, peroxide) and/or protectants (EDTA, L-methionine). Residue-specific analysis by automated high-throughput peptide mapping demonstrates differential residue-specific effects of EDTA and methionine in protecting against oxidation, highlighting the need for molecule-specific and product-specific selection of these excipients during formulation development. Computational modeling based on a homology model and the two-shell water coordination method (WCN) was employed to gain mechanistic understanding of residue-specific oxidation susceptibility of methionine residues. The computational determinants of local solvent exposure of methionine residues showed good correlation of WCN with experimentally determined oxidation for corresponding residues. The rapid generation of high-resolution data, statistical data analysis and interactive visualization of the high-throughput residue-level data containing ∼200 unique formulations facilitate residue-specific, molecule-specific and product-specific oxidation (global and local) assessment for oxidation protectants during early development for mAbs and related mAb-based modalities.

The effect of spray drying on the compaction properties of hypromellose acetate succinate.

BACKGROUND: The aim of this study was to evaluate the compaction behavior of a model two-component amorphous spray-dried dispersion system compared with the unprocessed excipients, using simulated rotary tablet press production conditions. METHOD: In this study, the stabilizing polymer, hypromellose acetate succinate (HPMCAS), was solubilized and spray dried with and without sodium lauryl sulfate (SLS). The impact of compression force and speed on the tabletting process was quantified by means of tablet tensile strength, compaction energy, and Heckel analysis. RESULTS: Addition of the surfactant SLS, spray dried or as a physical mix, reduced the tablet strength. However, a lesser impact on the unprocessed excipients was observed in comparison with the spray-dried excipients. In the presence of 1% (w/w) SLS, tablets displayed a tendency to cap when compressed at higher speeds, supported by high elastic energy values indicating high uniaxial stress upon decompression. In the presence of 3% (w/w) SLS, tablets could not be produced at high speeds. Heckel analysis revealed a greater strain rate sensitivity of HPMCAS when spray dried in the presence of surfactant. Exposure of samples to a range of relative humidities before compaction had no effect on tablet strength. CONCLUSION: This study has shown that spray drying of HPMCAS in the presence of a surfactant affects the compressibility of the material, resulting in decreased tablet strength, increased elastic deformation, and capping.

Polysorbate Degradation and Particle Formation in a High Concentration mAb: Formulation Strategies to Minimize Effect of Enzymatic Polysorbate Degradation.

Polysorbate (PS) 20 and 80 are the most common surfactants in monoclonal antibody (mAb) drug product (DP) formulations. Residual host cell proteins (HCP) present at extremely low concentrations in DP formulations can maintain enough enzymatic activity to degrade PS surfactants. Over time, the hydrolysis of surfactant causes the accumulation of minimally soluble free fatty acids resulting in precipitation and formation of subvisible and visible particulates. This manuscript summarizes the investigation of a batch of high concentration (>100 mg/mL) mAb DP where subvisible particles formed abruptly after prolonged storage at 5C°. The work also summarizes the effectiveness of different strategies for managing host cell proteins and fatty acid particles. The concentration and fatty acid composition of polysorbates were found to be significant factors in particle development. Solubilizers and alternative surfactants were all shown to be effective means of preventing particle formation. Lipase inhibitors proved to be a simple means to identify the problem but are more difficult to utilize as a solution.

Spectral Markers for T Cell Death and Apoptosis-A Pilot Study on Cell Therapy Drug Product Characterization Using Raman Spectroscopy.

Application of Raman spectroscopy as a T cell characterization tool supporting cell therapy drug product development has been evaluated. Statistically significant correlations between a set of Raman signals and established flow cytometry markers associated with apoptosis of T cells detected during drug product cryopreservation are presented in this study. Our study results demonstrate the potential of Raman spectroscopy for label-free measurements of T cell characteristics relevant to cell therapy product design and process control.

Product-Specific Impact of Viscosity Modulating Formulation Excipients During Ultra-High Concentration Biotherapeutics Drug Product Development.

Developing ultra-high concentration biotherapeutics drug products can be challenging due to increased viscosity, processing, and stability issues. Excipients used to alleviate these concerns are traditionally evaluated at lower protein concentrations. This study investigates whether classically known modulators of stability and viscosity at low (<50 mg/mL) to high (>50 - 150 mg/mL) protein concentrations are beneficial in ultra-high (>150 mg/mL) concentration protein formulations and drug products. This study evaluates the effect of arginine monohydrochloride, proline, and lysine monohydrochloride on viscosity and concentratability at different high and ultra-high protein concentrations using a monoclonal antibody, mAbN, formulation as a candidate protein system. The effect of excipients on the viscosity and concentratability (rate and extent) was different at high versus ultra-high protein concentrations. These results highlight that classical excipients in literature known to modulate protein interactions at low protein concentrations and reduce viscosity at high protein concentrations may need to be evaluated at target protein concentrations in a product-specific manner while developing ultra-high concentration biologics drug products.

Toward Biotherapeutics Formulation Composition Engineering using Site-Identification by Ligand Competitive Saturation (SILCS).

Formulation of protein-based therapeutics employ advanced formulation and analytical technologies for screening various parameters such as buffer, pH, and excipients. At a molecular level, physico-chemical properties of a protein formulation depend on self-interaction between protein molecules, protein-solvent and protein-excipient interactions. This work describes a novel in silico approach, SILCS-Biologics, for structure-based modeling of protein formulations. SILCS Biologics is based on the Site-Identification by Ligand Competitive Saturation (SILCS) technology and enables modeling of interactions among different components of a formulation at an atomistic level while accounting for protein flexibility. It predicts potential hotspot regions on the protein surface for protein-protein and protein-excipient interactions. Here we apply SILCS-Biologics on a Fab domain of a monoclonal antibody (mAbN) to model Fab-Fab interactions and interactions with three amino acid excipients, namely, arginine HCl, proline and lysine HCl. Experiments on 100 mg/ml formulations of mAbN showed that arginine increased, lysine reduced, and proline did not impact viscosity. We use SILCS-Biologics modeling to explore a structure-based hypothesis for the viscosity modulating effect of these excipients. Current efforts are aimed at further validation of this novel computational framework and expanding the scope to model full mAb and other protein therapeutics.

Comparison of 4% articaine with 1:100,000 epinephrine and 2% lidocaine with 1:100,000 epinephrine when used as a supplemental anesthetic.

A randomized, double-blind trial was conducted to compare the efficacy of 4% articaine with 1:100,000 epinephrine and 2% lidocaine with 1:100,000 epinephrine when used as a supplemental anesthetic. Forty-eight patients with irreversible pulpitis requiring supplemental buccal infiltration for endodontic therapy were given either 4% articaine with 1:100,000 epinephrine or 2% lidocaine with 1:100,000 epinephrine in a double-blind manner. A standard VAS pain scale was used to evaluate the patient's response to pain after a supplemental injection. The mean VAS score after supplemental anesthesia was 15.28 for 4% articaine with 1:100,000 epinephrine and 19.70 for 2% lidocaine with 1:00,000 epinephrine. The mean percentage change in VAS score was 70.5 and 62.2% for articaine and lidocaine, respectively. There was no statistically significant difference in the VAS pain score between 4% articaine with 1:00,000 epinephrine and 2% lidocaine with 1:00,000 epinephrine as a supplemental anesthetic.

Comparison of cost of immune globulin intravenous therapy to conventional immunosuppressive therapy in treating patients with autoimmune mucocutaneous blistering diseases.

Autoimmune mucocutaneous blistering diseases (AMBD) are a group of potentially fatal diseases that affect the skin and mucous membranes. AMBD have different target antigens as well as variable clinical presentation, course, and prognosis. The mainstay of conventional immunosuppressive therapy (CIST) for AMBD is long-term high-dose systemic corticosteroids and immunosuppressive agents. Such therapy has proven effective in many patients; however, in some patients, the disease continues to progress with significant sequelae such as blindness, loss of voice, anal, and vaginal stenosis which causes poor quality of life. Furthermore, the CIST may have some serious side effects including opportunistic infections which may cause death. Immune globulin intravenous (IGIV) therapy has been reportedly used in the management of patients with AMBD refractory to CIST. IGIV has shown to be more clinically beneficial than CIST by bringing about long-term clinical remission and less recurrence. The high cost of the IGIV is of concern to patients, physicians, and insurance companies. In this report, we compare the cost of IGIV to that of CIST in treating a cohort of 15 mucous membrane pemphigoid (MMP), 10 ocular cicatricial pemphigoid (OCP), 15 bullous pemphigoid (BP), and 32 pemphigus vulgaris (PV) patients. In each cohort of patients, CIST had significant side effects, many of which were hazardous and required prolonged and frequent hospitalizations. Some of these side effects were severe enough to require discontinuation of the treatment. We consider the total cost of CIST to be the actual cost of the drug, plus the cost of management of the side effects produced by CIST. In the same patient cohort, no significant side effects to IGIV were observed. None of the IGIV treated patients required physician visits, laboratory tests, or hospitalizations specifically related to IGIV therapy. Hence, the total cost of the IGIV therapy is the actual cost of the IGIV only. The mean total cost of treatment of IGIV therapy is statistically significantly less than that of CIST during the entire course of the disease and on an annual basis. In conclusion, IGIV therapy is a safe, clinically beneficial, and a cost effective alternative treatment in patients with AMBD, non-responsive to CIST.

In vitro hemolysis: guidance for the pharmaceutical scientist.

Pharmaceutical excipients are commonly incorporated into parenteral formulations to increase solubility and stability of active pharmaceutical ingredients. The biocompatibility of these excipients is an important consideration during formulation development. Despite the importance of hemolytic potential of parenteral formulations, there is considerable contradictory information in the literature related to the hemolytic potential of various excipients. The hemolytic potential of various formulation vehicles in dog, rabbit, and human blood by means of an in vitro hemolysis assay is compared. The selected formulation vehicles are found in currently marketed drug products. The guidance presented here considers formulations with a hemolysis value of <10% to be nonhemolytic while values > 25% to be at risk for hemolysis.

Lyophilization of polyethylene glycol mixtures.

Lyophilization of cosolvent systems may be a beneficial way of enhancing both physical and chemical stability of a drug product. The objective of this research is to establish whether cosolvent systems commonly used in the formulation of poorly water-soluble drugs can be successfully lyophilized. Polyethylene glycol (PEG) 400 was selected because it is widely used and can be easily frozen. The addition of PEG 400 to commonly used bulking agents, such as mannitol, sucrose, or polyvinylpyrrolidone, caused a significant change in the thermal properties of the bulking agents as observed by modulated differential scanning calorimetry. In addition, PEG 8000 was evaluated as a bulking agent because it also can function as a cosolvent in solution and forms an acceptable cake after lyophilization. Addition of PEG 400 to PEG 8000 caused negligible changes in the thermogram of this bulking agent. Surprisingly, the combination of PEG 8000 and PEG 400 forms a solid lyophilized cake. The current system can be best described as the lyophilization of a miscible solution of PEG 8000 and PEG 400 resulting in a lyophile that has a crystalline structure of PEG 8000 which is able to support PEG 400.

Cell association of liposomes with high fluid anionic phospholipid content is mediated specifically by LDL and its receptor, LDLr.

We have sought to confirm indications in our recent studies suggesting that association of liposomes composed of 75-100 mol % egg phosphatidylglycerol (ePG), a fluid anionic phospholipid, with cells is mediated by low density lipoprotein (LDL) and the classical LDL receptor (LDLr). In the present study, binding of liposomes composed of 75-100 mol % ePG to CV1-P cells, either in serum-supplemented medium or in defined medium supplemented with LDL, is blocked by the presence of either of two monoclonal antibodies. The first is immunoglobulin (Ig)G C7, an antibody specific for LDLr. The second is IgG 5E11, an antibody specific for domain 3441-3569 of apolipoprotein B100. CHOldlA7, a cell line known to lack the LDLr and previously shown by us to associate minimally with 75-100 mol % ePG liposomes, was transfected with the human LDLr. The transfected cells bound 75-100 mol % ePG liposomes at high levels, and this binding was blocked by IgG C7. Previously, we have shown that serum, but not LDL or high density lipoprotein, induces association of 25-50 mol % ePG liposomes with both CV1-P and CHO wild type cells, but not CHOldlA7. In the present study, IgG C7 does not block this interaction, and transfected CHOldlA7 cells do not show this interaction. Hence, this form of liposome binding appears not to involve LDL or LDLr, but requires a receptor, currently unknown, and a serum component other than LDL or high density lipoprotein. The unknown receptor, in addition to LDLr, is missing from CHOldlA7.

Cost of intravenous immunoglobulin therapy versus conventional immunosuppressive therapy in patients with mucous membrane pemphigoid: a preliminary study.

BACKGROUND: Intravenous immunoglobulin (IVIG) is an expensive biologic agent used to treat patients with mucous membrane pemphigoid (MMP) nonresponsive to conventional immunosuppressive therapy (CIST). The high cost of IVIG is of concern to healthcare providers and insurance companies. OBJECTIVE: To compare the cost of IVIG with that of CIST in treating a cohort of 15 patients with severe and extensive MMP. METHODS: Fifteen patients with biopsy-proven MMP nonresponsive to CIST were subsequently treated with IVIG and demonstrated a positive clinical response. This was a comparative, retrospective study; the mean total duration of the observation period was 8.4 years. A comparison of the cost of IVIG with that of CIST during the study period and the annual cost was performed. The cost of CIST was defined as the actual cost of the drug plus the cost of management of the multiple adverse effects, including hospitalizations, produced by CIST. In the same patient cohort, no significant adverse effects to IVIG were observed and no hospitalizations were required. Hence, the cost of IVIG therapy is simply the actual cost of the IVIG. RESULTS: In this cohort of patients, CIST had significant adverse effects, many of which were hazardous and required prolonged and frequent hospitalizations. The mean total cost using IVIG therapy was significantly less than that of CIST during the entire course of the disease (p < 0.001) and on an annual basis (p < 0.05). CONCLUSIONS: IVIG therapy is a safe, clinically beneficial, and less-expensive alternative treatment in patients with progressive MMP that is nonresponsive to CIST.