Alexey L. Margolin

Crystalline monoclonal antibodies for subcutaneous delivery

Therapeutic applications for mAbs have increased dramatically in recent years, but the large quantities required for clinical efficacy have limited the options that might be used for administration and thus have placed certain limitations on the use of these agents. We present an approach that allows for s.c. delivery of a small volume of a highly concentrated form of mAbs. Batch crystallization of three Ab-based therapeutics, rituximab, trastuzumab, and infliximab, provided products in high yield, with no detectable alteration to these proteins and with full retention of their biological activity in vitro. Administration s.c. of a crystalline preparation resulted in a remarkably long pharmacokinetic serum profile and a dose-dependent inhibition of tumor growth in nude mice bearing BT-474 xenografts (human breast cancer cells) in vivo. Overall, this approach of generating high-concentration, low-viscosity crystalline preparations of therapeutic Abs should lead to improved ease of administration and patient compliance, thus providing new opportunities for the biotechnology industry.

Protein crystals for the delivery of biopharmaceuticals

The year 2002 marked the 20th anniversary of the first successful product of modern biotechnology, the regulatory approval of recombinant insulin for biopharmaceutical applications. Insulin is also the first crystalline protein to be approved for therapeutic use. Over the past two decades, almost 150 biopharmaceuticals have gained marketing authorisation; however, insulin remains the only crystalline protein on the market. Significant research and development efforts have focused on the engineering of protein molecules, efficacy testing, model development, and protein production and characterisation. These advances have dramatically boosted the therapeutic applications of proteins, which now include treatments against acute conditions, such as cancer, cardiovascular disease and viral disease, and chronic conditions, such as diabetes, growth hormone deficiency, haemophilia, arthritis, psoriasis and Crohn’s disease. Despite these successes, many challenges normally associated with biopharmaceuticals, such as poor stability and limited delivery options, remain. Protein crystals have shown significant benefits in the delivery of biopharmaceuticals to achieve high concentration, low viscosity formulation and controlled release protein delivery. This review will discuss challenges related to the broader utilisation of protein crystals in biopharmaceutical applications, as well as recent advances and valuable new directions that protein crystallisation-based technologies present.

Stereoselective oligomerizations catalyzed by lipases in organic solvents

Abstract Lipases have been found to act as stereoselective catalysts in polycondensation reactions between racemic diesters and achiral diols (or vice versa) in organic solvents. Optically active trimers andpentamers have been formed as a result.

Novel Long-Acting Crystal Formulation of Human Growth Hormone

PurposeThe aim of the study is to solve a significant challenge of extending the half-life of therapeutic proteins using crystalline biopharmaceuticals and without redesigning the molecules.MethodsCrystals of recombinant human growth hormone were coated with a monomolecular layer of positively charged poly(arginine). The pharmacokinetics and pharmacodynamics of this poly(arginine)-coated human growth hormone crystalline formulation were determined in hypophysectomized rats and monkeys.ResultsHere we have demonstrated for the first time that crystals of human growth hormone coated with positively charged poly(arginine) allowed for in vivo pharmacokinetic release profiles of over several days in animal models. The efficacy of this crystalline formulation injected subcutaneously once a week was found to be equivalent to seven daily soluble injections in the standard weight gain assay using the hypophysectomized rat model and in measurement of serum insulin-like growth factor in monkeys. The nonviscous nature of the suspension facilitated easy administration through a fine, 30-gauge needle and should provide for improved patient convenience and compliance.ConclusionsThe approach described here offers an exciting possibility of being broadly applicable to other therapeutic proteins.

Cofactor-Bound Cross-Linked Enzyme Crystals (CLEC) of Alcohol Dehydrogenase

Horse liver alcohol dehydrogenase was crystallized in the presence of its cofactor. The cross-linked enzyme crystals (CLECs) produced showed good catalytic activity without the addition of extra cofactor (see scheme). The enantioselectivity and stereochemical preference of the CLEC were the same as with the soluble enzyme, and both cofactor and enzyme were considerably more stable in CLEC form. Cofactor regeneration studies on the reduction of cinnemaldehyde indicate the potential for a high level of catalyst productivity.

Hyperoxaluria Is Reduced and Nephrocalcinosis Prevented with an Oxalate-Degrading Enzyme in Mice with Hyperoxaluria

Background/Aims: Hyperoxaluria is a major risk factor for recurrent urolithiasis and nephrocalcinosis. We tested an oral therapy with a crystalline, cross-linked formulation of oxalate-decarboxylase (OxDc-CLEC®) on the reduction of urinary oxalate and decrease in the severity of kidney injury in two models: AGT1 knockout mice (AGT1KO) in which hyperoxaluria is the result of an Agxt gene deficiency, and in AGT1KO mice challenged with ethylene glycol (EG). Methods: Four different doses of OxDc-CLEC mixed with the food, or placebo were given to AGT1KO mice (200 mg/day, n = 7) for 16 days and to EG-AGT1KO mice (5, 25, and 80 mg, n = 11) for 32 days. Results: Oral therapy with 200 mg OxDc-CLEC reduced both urinary (44%) and fecal oxalate (72%) in AGT1KO mice when compared to controls. Similarly, in EG-AGT1KO mice, each of the three doses of OxDc-CLEC produced a 30–50% reduction in hyperoxaluria. A sustained urinary oxalate reduction of 40% or more in the 80 mg group led to 100% animal survival and complete prevention of nephrocalcinosis and urolithiasis. Conclusion: These data suggest that oral therapy with OxDc-CLEC may reduce hyperoxaluria, prevent calcium oxalate nephrocalcinosis and urolithiasis, and can represent a realistic option for the treatment of human hyperoxaluria, independent of cause.

Enzyme-catalyzed acylation of castanospermine and 1-deoxynojirimycin

Abstract Several esters of the alkaloids deoxynojirimycin and castanospermine have been synthesized via subtilisin-catalyzed regioselective acylation in pyridine.

Candida rugosa lipase: Enantioselectivity enhancements in organic solvents

Abstract Chiral resolutions of carboxylic acids ( 1–3 ) and alcohol ( 4 ) were carried out through esterification or transesterification in organic solvents using cross-linked enzyme crystals (CLEC®) of Candida rugosa lipase (CRL). Comparison of these results with those of crude CRL reveal significant differences. As was seen in resolution through hydrolysis, 1 a marked improvement in enantioselectivity is realized with the CLEC. Additionally, the stability afforded the enzyme in CLEC form leads to a higher activity in organic solvent.

Synthesis of chiral building blocks for selective adenosine receptor agents. Lipase-catalyzed resolution of 2-benzylpropanol and 2-benzylpropionic acid.

Abstract Both enantiomers of 2-benzylpropanol and 2-benzylpropionic acid have been synthesized using a lipase-catalyzed resolution in an organic solvent and in water.

[20] Cross-linked enzyme crystals of lipases as catalysts for kinetic resolution of acids and alcohols

Publisher Summary The cross-linked crystalline form of a lipase is superior to the soluble lipase preparation under some specific circumstances. Situations where a CLEC (cross-linked enzyme crystals) lipase is preferred include (1) the highly pure catalyst gives higher enantioselectivity, (2) a recoverable form of the catalyst is needed, (3) high catalyst productivity is needed, and (4) organic-solvent-based reactions where high activity is needed. Preparation of Candida rugosa lipase and Pseudomonas cepacia lipase in the CLEC form involves crystallization to give platelike crystals about 30μm in length, followed by glutaraldehyde treatment of the protein crystals while suspended in the crystallization medium. Lipase CLECs, similar to other protein crystals, are macroporous, containing about 50% solvent by volume. The large pore diameter of CLECs has been shown to allow rapid diffusion of relatively large molecules into the protein crystal. These heterogeneous lipase catalysts are active as a suspension in water, in aqueous organic solvents mixtures, or in substantially water-free organic solvent. On completion of an ester synthesis or hydrolysis reaction, the heterogeneous catalyst can be recovered by filtration, to be stored or reused.