Papers by Ajoy Chakrabarti
npj Vaccines, 2021
A new oral polio vaccine, nOPV2, has become the first vaccine to pursue a WHO Emergency Use Listi... more A new oral polio vaccine, nOPV2, has become the first vaccine to pursue a WHO Emergency Use Listing. Many lessons were learned as part of the accelerated development plan and submission, which have been categorized under the following sections: regulatory, clinical development, chemistry manufacturing and controls, and post-deployment monitoring. Efforts were made to adapt findings from these studies to COVID-19 vaccine candidates. Specific concepts for accelerating COVID-19 vaccine development across multiple functional domains were also included. The goals of this effort were twofold: (1) to help familiarize vaccine developers with the EUL process; and (2) to provide general guidance for faster development and preparations for launch during the COVID-19 pandemic.
npj Vaccines, 2020
In 2018, the Bill and Melinda Gates Foundation convened over thirty subject matter experts in cli... more In 2018, the Bill and Melinda Gates Foundation convened over thirty subject matter experts in clinical development, manufacturing, and regulatory assessment to determine how the development and approval of medical countermeasures could be accelerated in the event of Disease X. Disease X is the result of a presently unknown pathogen with epidemic or pandemic potential. A key opportunity to accelerate the scientific assessment and regulatory approval of medical countermeasures exists within efficient navigation of facilitated regulatory pathways. It was identified that not all stakeholders will be able to skillfully navigate the facilitated pathways offered by the various regulatory agencies during a public health emergency. To democratize this knowledge, we have written an overview of the facilitated approaches which have been developed and refined by Stringent Regulatory Authorities and the World Health Organization for the primary assessment of medical products. We discuss the cond...
The Lancet Infectious Diseases, 2020
WHO has listed several priority diseases with epidemic potential for which there are no, or insuf... more WHO has listed several priority diseases with epidemic potential for which there are no, or insufficient, medical countermeasures. In response, the Bill & Melinda Gates Foundation (with support from PricewaterhouseCoopers) coordinated subject matter experts to create a preparedness plan for Disease X. Disease X is caused by Pathogen X, an infectious agent that is not currently known to cause human disease, but an aetiologic agent of a future outbreak with epidemic or pandemic potential. We have identified crucial areas for acceleration in medical countermeasure product development and international coordination. We have also reviewed novel platforms and process improvements related to manufacturing, which could revolutionise the response to the next pandemic. Finally, we created several coordination and engagement guides. These guides range from the rational design of an intervention target product profile, to the key facets of vaccine and therapeutic development, to accelerated manufacturing and regulatory mechanisms. In this Personal View, we provide a high-level summary of the outcomes of the medical countermeasure development workstream, intended for a broad audience including academia, medical countermeasure developers, and multilateral coordinating bodies. We hope that they might find this piece useful in prioritising strategic investments and efforts to accelerate medical countermeasure development. We observed that in-depth analyses of clinical trial design, chemistry, manufacturing and control activities, and accelerated regulatory pathways are necessary for shortening the timelines for the product development of medical countermeasures. We intend to cover these topics in future publications.
Use of transmembrane pH gradients and A 23187 to produce dense iron liposomes
Biochemical Society Transactions, 1990
Journal of Molecular Evolution, 1994
Catalyzed polymerization reactions represent a primary anabolic activity of all cells. It can be ... more Catalyzed polymerization reactions represent a primary anabolic activity of all cells. It can be assumed that early cells carded out such reactions, in which macromolecular catalysts were encapsulated within some type of boundary membrane. In the experiments described here, we show that a templateindependent RNA polymerase (polynucleotide phosphorylase) can be encapsulated in dimyristoyl phosphatidylcholine vesicles without substrate. When the substrate adenosine diphosphate (ADP) was provided externally, long-chain RNA polymers were synthesized within the vesicles. Substrate flux was maximized by maintaining the vesicles at the phase transition temperature of the component lipid. A protease was introduced externally as an additional control. Free enzyme was inactivated under identical conditions. RNA products were visualized in situ by ethidium bromide fluorescence. The products were harvested from the liposomes, radiolabeled, and analyzed by polyacrylamide gel electrophoresis. Encapsulated catalysts represent a model for primitive cellular systems in which an RNA polymerase was entrapped within a protected microenvironment.
Biochemistry, 1994
has shown that basic amino acids and peptides, in which the C-terminal carboxyl groups have been ... more has shown that basic amino acids and peptides, in which the C-terminal carboxyl groups have been modified to form amides or methyl esters, can be rapidly and efficiently accumulated into large unilamellar vesicle (LUV) systems in response to transmembrane pH gradients (ApH, inside acidic). In this work, the ability of small (di and tri) peptides, composed exclusively of basic (lysine) and hydrophobic (tryptophan) amino acids, to accumulate into LUV systems in response to ApH has been investigated. In the case of the dipeptides Trp-Lys-amide and Lys-Trp-amide, remarkable differences in the rate constants associated with net transport were observed. In EPC:cholesterol LUV systems exhibiting a ApH of 3 units (pHi = 4.0; pH, = 7.0). for example, the rate constant for the uptake of Lys-Trp-amide is some 5 X 10' faster than for Trp-Lys-amide. Activation energies associated with the uptake also varied from 24 (Lys-Trp) to 29 kcal/mol (Trp-Lys). Related effects were observed for the tripeptides composed of one lysine and two tryptophan residues; however, the differences in rate constants were less sensitive to amino acid sequence. It is concluded that different charge distributions in short peptides of identical amino acid composition can strongly influence the ability of these groups to associate with and permeate across lipid bilayers. These observations may have relevance to the ability of basic peptides, such as signal sequences and peptide hormones, to translocate across biological membranes. '~istestarcbwassupportbdbytbeMcdicrlResearchCouncil(MRC) of Canada. A.C. is grateful to the University of British Columbia for a University Graduate Fellowship and to tbe Science Council of British Columbia for a GREAT Award. l Author to whom correspondence should be addressed.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1992
Previous work (Veim and Cullis (tqgO) Bioehim, Biophys. Asia 1023, 109-115) has shown that Ca 2÷ ... more Previous work (Veim and Cullis (tqgO) Bioehim, Biophys. Asia 1023, 109-115) has shown that Ca 2÷ can be accumulated into large unilamellar vesicles (LUVs) in the presence of a transmcmbran¢ pH gradient (inside acidic) and the Ca"+-ionuphore A23187. Here, ~ht" ability of A23!87 to mediate the uptake of i J on and barium into LUVs has been investigated. It is shown that under approprime conditions of temlxraturc and A23187 coneemration, iron (in the form of Fe 2 +) can be accumulated into EPC and DSPC/cholcstcrol (55:45; tool/reel} LUVs with an acidic interior. This uptake is dependent on the internal buffer concentratinn, with maximum levels of uptake in the range of 300 nmol of cation per ~.mol lipid, The DSPC-chnlgstcrol LUV s/gems exhibit superior retention properties compared to the F.PC systems, It is demonstrated that Ba 2+ can also he loaded by similar methods. It is also shown that the maximally loaded F¢ ~+-and BaZ+-containin8 LUVs exhibit increased densities, This is expressed by enhanced gravimctric properties, as an increased proportion of the loaded LUVs ~n be pcRetcd by low speed centrifugation, and by anhaneed electron densities, in that the BaZ+-Ioaded systems can be directly visualized cmpi~:¢ing cryo-electron mieroscnpy.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1992
Permeability coefficients I'or amino acid classes, including neutral, polar, hydrophobic, and cha... more Permeability coefficients I'or amino acid classes, including neutral, polar, hydrophobic, and charged species, were measured and compared with values for other ionic solutes such as phosphate. The rates of efflux of glycine, lysine, phenylalanine, serine and tryptophan were determined after they were passively entrapped in large unilamellar vesicles (LUVs) composed of egg phosphatidylcholine (EPC) or dimyristoylphosphati0ylcholine (DMPC). The following permeability coefficients were obtained for: glycine, 5.7.10 t2 cm s-t (EPC), 2.0.10-tt cm s-' (DMPC); serine, 5.5.10-J2 cm s-i (EPC), 1.6.10-II cm s i (DMPC); lysine, 5.1.1()-12 cm s-I (EPC), 1.9.10-II cm s-~ (DMPC); tryptophan, 4.1.10-"D cm s '~ (EPC); and phenylalaninc, 2.5.10-"~ cm s-' (EPC). Decreasing lipid chain length increased permeability slightly, while variations in pH had only minor effects on the permeability coefficients of the amino acids tested. Phosphate permeability was i,~ the range of 10-~2-10-~-~ cm s-m depending on the pH of the medium. The values for the polar and charged amino acids were surprisingly similar to those previously measured for monovalent cations such as sodium and potassium, which are in the range of 10-~2-10-L~ cm s-m depending on conditions and the lipid species used. This observation suggests that the permeation rates for the neutral, polar and charged amino acids are controlled by bilayer fluctuations and transient defects, rather than partition coefficients and Born energy barriers. The re,,;ults are relevant to the permeation of certain peptides into lipid bilayers during protein translocation and membrane biogenesis.
Applied Biochemistry and Biotechnology, 1990
Glucose isomerase was immobilized by itself and coimmobilized with cellulase and fl-glucosidase u... more Glucose isomerase was immobilized by itself and coimmobilized with cellulase and fl-glucosidase using a polyurethane foam (Hypol | FHP 2002). Approximately 50% of the enzyme added was immobilized. The immobilized enzyme was active at pH values as low as 6.8. When immobilized alone, the Km for Mg 2 § increased by 5.5fold and the Km for fructose increased 62%. The half-life of the immobilized glucose isomerase was approximately 160 h of continuous hydrolysis, with a substantial (about 35-40%) amount of activity remaining even after 1000 h. When all three enzymes were immobilized together, the system was found capable of functioning at pH 7.0 to produce fructose from both soluble and insoluble cellulose substrates. At this pH, the glucose:fructose ratio was 70:30. The advantageous properties of the foam as a support for enzyme immobilization and the efficiency of the one-step conversion process outlined combine to make this system appear valuable for use in high fructose syrup production.
Applied Biochemistry and Biotechnology, 1988
Cellulase was covalently immobilized using a hydrophilic polyurethane foam (HypoI| 2002). Compare... more Cellulase was covalently immobilized using a hydrophilic polyurethane foam (HypoI| 2002). Compared to the free enzyme, immobilized cellulase showed a dramatic decrease (7.5-fold) in the Michaelis constant for carboxymethylcellulose. The immobilized enzyme also had a broader and more basic pH optimum (pH 5.5-6.0), a greater stability under heat-denaturing or liquid nitrogen-freezing conditions, and was relatively more efficient in utilizing insoluble cellulose substrates. High molecular weight compounds (Blue Dextran) could move throughout the foam matrix, indicating permeability to insoluble celluloses; activity could be further improved 2.4-fold after powdering foams under liquid nitrogen. The improved kinetic and stability features of the immobilized cellulase combined with advantageous properties of the polyurethane foam (resistance to enzymatic degradation, plasticity of shape and size) suggest that this mechanism of cellulase immobilization has high potential for application in the industrial degradation of celluloses.
Immobilization of amyloglucosidase using two forms of polyurethane polymer
Applied Biochemistry and Biotechnology, 1990
Amyloglucosidase was covalently immobilized using two hydrophilic prepolymers: Hypol FHP 2002 (cr... more Amyloglucosidase was covalently immobilized using two hydrophilic prepolymers: Hypol FHP 2002 (creates foams) and Hypol FHP 8190H (creates gels). The foamable prepolymer was superior as a support for enzyme immobilization. The percent activity immobilized in the polyurethane foams was 25 +/- 1.5%. Large substrates (greater than 200,000 daltons in mol wt) were hydrolyzed as effectively as smaller ones by the immobilized enzyme. The Km value of the foam-immobilized enzyme increased from 0.76 mg/mL (free) to 0.86 mg/mL (immobilized), whereas the Vmax dropped from 90.9 (free) to 12.4 nmol glucose/min/mL (immobilized). The long-term (2 mo) storage stability of amyloglucosidase was enhanced by immobilization in foams (70% activity retained; free enzyme only retained 50%). Immobilization also improved the enzyme stability to various denaturing agents (sodium chloride, urea, and ethanol). The immobilized enzyme exhibited increased stability compared to the free enzyme at high temperatures (95 degrees C). Both glycogen and starch could be utilized by the immobilized enzyme, indicating that this technique could prove useful for starch hydrolysis.
Antimicrobial Agents and Chemotherapy, 2013
Development of anthrax countermeasures that may be used concomitantly in a postexposure setting r... more Development of anthrax countermeasures that may be used concomitantly in a postexposure setting requires an understanding of the interaction between these products. Anthrax immune globulin intravenous (AIGIV) is a candidate immunotherapeutic that contains neutralizing antibodies against protective antigen (PA), a component of anthrax toxins. We evaluated the interaction between AIGIV and BioThrax (anthrax vaccine adsorbed) in rabbits. While pharmacokinetics of AIGIV were not altered by vaccination, the vaccine-induced immune response was abrogated in AIGIV-treated animals.
Antimicrobial Agents and Chemotherapy, 2013
Bacillus anthracis toxins can be neutralized by antibodies against protective antigen (PA), a com... more Bacillus anthracis toxins can be neutralized by antibodies against protective antigen (PA), a component of anthrax toxins. Anthrivig (human anthrax immunoglobulin), also known as AIGIV, derived from plasma of humans immunized with BioThrax (anthrax vaccine adsorbed), is under development for the treatment of toxemia following exposure to anthrax spores. The pharmacokinetics (PK) of AIGIV was assessed in naive animals and healthy human volunteers, and the efficacy of AIGIV was assessed in animals exposed via inhalation to aerosolized B. anthracis spores. In the clinical study, safety, tolerability, and PK were evaluated in three dose cohorts (3.5, 7.1, and 14.2 mg/kg of body weight of anti-PA IgG) with 30 volunteers per cohort. The elimination half-life of AIGIV in rabbits, nonhuman primates (NHPs), and humans following intravenous infusion was estimated to be approximately 4, 12, and 24 days, respectively, and dose proportionality was observed. In a time-based treatment study, AIGIV...
Biophysical Journal, 1992
The uptake of derivatives of lysine and a pentapeptide (ala-met-leu-trp-ala) into large unilamell... more The uptake of derivatives of lysine and a pentapeptide (ala-met-leu-trp-ala) into large unilamellar vesicle (LUV) systems in response to transmembrane pH gradients has been examined. In these derivatives, the C-terminal carboxyl functions have been converted to methyl esters or amides. It is shown that the presence of a pH gradient (interior acidic) results in the rapid and efficient accumulation of these weak base amino acid and peptide derivatives into LUVs in a manner consistent with permeation of the neutral (deprotonated) form. It is suggested that this property may have general implications for mechanisms of transbilayer translocation of peptides, such as signal sequences, which exhibit weak base characteristics.
Accumulation d'acides amines et de peptides dans des liposomes
Cette invention concerne des compositions de liposomes ayant un gradient de concentration qui cha... more Cette invention concerne des compositions de liposomes ayant un gradient de concentration qui charge dans des liposomes, des acides amines et des peptides presentant de faibles caracteristiques acides ou basiques. Plus specifiquement, on peut, a l'aide des procedes de cette invention, charger dans des liposomes, des acides amines ou des peptides a substitution de la terminaison C. Les liposomes sont, de preference, de grandes vesicules unilamellaires. On forme le gradient de concentration en encapsulant un premier milieu dans les liposomes, ledit milieu ayant une premiere concentration d'une ou plusieurs especes chargees, puis on met en suspension les liposomes dans un deuxieme milieu ayant une deuxieme concentration d'une ou de plusieurs especes chargees, comme par exemple un gradient de pH. L'invention concerne egalement des preparations pharmaceutiques comprenant de tels acides amines ou peptides a substitution de la terminaison C qui ont ete charges dans les lipo...
~-Glucosidase was covalently immobilized alone and coimmobilized with cellulase using a hydrophil... more ~-Glucosidase was covalently immobilized alone and coimmobilized with cellulase using a hydrophilic polyurethane foam (Hypol | FHP 2002). Immobilization improved the functional properties of the enzymes. When immobiliTed alone, the Km for cellobiose of fl-glucosidase was decreased by 33% and the pH optimum shifted to a slightly more basic value, compared to the free enzyme. ImmobiliTed/~-glucosidase was extremely stable (95% of activity remained after 1000 h of continuous use). CoimraobfliTation of cellulase and/5-glucosidase produced a cellulose-hydrolyzing complex with a 2.5-fotd greater rate of glucose production for soluble cellulose and a four-fold greater increase for insoluble cellulose, compared to immobiliTed cellulase alone. The imrno-biliTed enzymes showed a broader acceptance of various types of insoluble cellulose substrates than did the free enzymes and showed a long-term (at least 24 h) linear rate of glucose production from microcrystalline cellulose. The pH optimum for the coimrnobiliTed enzymes was 6.0. This method for enzyme immobilization is fast, irreversible, and does not require harsh conditions. The enhanced glucose yields obtained indicate that this method may prove useful for commercial cellulose hydrolysis.
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Papers by Ajoy Chakrabarti