Dr. DeFilippi has over 37 years of experience in both basic and applied biochemistry
(biochemical research and enzymology) and biotechnology. He is very successful in
finding flaws in published and spoken materials, which has helped him both as a consultant
and as an expert witness. His greatest strengths are in the fields of physical biochemistry,
protein biochemistry, protein characterization, protein modification, protein stabilization
and protein structure. He is adept at using many chromatography applications, as
he has employed adsorption chromatography (column and TLC), as well as ion exchange
and gel permeation chromatography, for small molecule and protein isolation, enzyme
purification, and protein purification. A protein concentration process, usually
via membranes and pressure filtration, followed the purifications. Some proteins
/ enzymes that he purified and studied were hemoglobin (haemoglobin), methemoglobin,
methemoglobin reductase, cytochrome b5 reductase, cytochrome b5, superoxide dismutase,
IgG, glutathione peroxidase, glucoamylase, cytochrome P-450 and other oxidoreductases,
and numerous other proteins / enzymes, including erythrocyte (red blood cell) proteins.
He has applied these strengths in the use of immobilized enzymes to make value
added products. He is very familiar with HPLC (high-performance liquid chromatography)
and associated detectors. The prosthetic group of Cytochrome a was isolated from
cytochrome oxidase, derivatized via an original methylation process and the structure
characterized in this manner. He is very strong in analytical biochemistry and has
experience in managing an analytical biochemistry laboratory where such techniques
as UV/Vis spectrophotometry, GC/MS, Ion chromatography and the like were in common
usage. He studied free radical biochemistry and showed its negative effects on various
blood proteins and related it to peroxidase (HRP). He has had success in applied
enzymology (biochemical process development), especially with immobilized enzymes
(isomerase, esterase and lipase [lipid modifying] enzymes).
BIOCHEMISTRY AND BIOTECHNOLOGY
BIOCHEMISTRY AND BIOTECHNOLOGY
WE DELIVER EXPERT CONSULTING, LABORATORY, EXPERT WITNESS AND PATENT INFRINGEMENT
SERVICES IN BIOPROCESSING, BIOCATALYSIS, BIOTRANSFORMATIONS, ENZYMOLOGY & PROTEINS
and BIOCHEMISTRY, especially as applied to chiral transformations, by the application
of immobilized enzymes and immobilized cells,
"FROM MICROGRAMS TO METRIC TONS"™
BIOCATALYSIS, BIOPROCESSING &
BIOPROCESSING USING IMMOBILIZED ENZYMES AND WHOLE CELLS;
BIOCHEMISTRY & ENZYMOLOGY
Biocatalysis, bioprocessing (biocatalytic processing) and biotransformations are
all similar processes that involve the use of enzymes (derived from mammalian or
microbial sources and often immobilized) and/or microorganisms (enzymes still contained
within microorganisms, and often immobilized as whole cells) to perform chemical
conversions. The enzymes are not reagents, but rather catalysis, or more precisely,
biocatalysts, that perform highly specific biotransformations. Like all catalysts
they are not consumed in the reaction but are re-used over and over again. However,
like all catalysts, there are stability issues that must be addressed for the biocatalytic
process to be commercially viable.
Many synthetic organic chemists would like enzymes to be simple off-the-shelf catalysts
that can be used painlessly to perform desired organic transformations.
Unfortunately, the age of enzymes as simple reagents has not yet arrived although
tremendous progress is constantly being made!
So, although not yet an off-the-shelf technology, applied biotransformations and
biocatalysis are becoming more and more prevalent in the synthesis of fine chemicals
and pharmaceuticals as an efficient replacement for certain key reaction steps. Arguably,
the most important are in pharmaceutical applications. There, the use of enzymes
to catalyze stereospecific and regiospecific organic reactions is incredibly valuable
due to potentially high degrees of selectivity and yield effected by enzymes.
Another tremendous benefit is that the formation of side products can be significantly
FORTUNATELY YOU DO NOT HAVE TO BE AN EXPERT!
We can help you to develop screening tools to select the right enzymes and conditions
for your diverse applications. This can often be performed at the microgram level,
or sometimes even lower. We then work with you to take a laboratory-scale discovery
and develop a viable commercial product or process. We have successfully done this
on the metric ton scale. This often involves the use of immobilized enzymes and
microorganisms. Results are efficiently achieved by working with your process engineers,
biochemists, analytical chemists, and microbiologists, as appropriate.
We also have significant experience in taking a laboratory-scale process and successfully
developing it into a commercial process.
Some notable successes are where we have:
...worked directly with Molecular Biologists at the discovery level to select enzymes
appropriate for performing the biotransformation of interest.
...shepherded projects from discovery through early development.
...developed proprietary conditions for high yield production of pharmaceutical intermediates.
For example, we have developed, and carried out to completion, approaches for preparation
and application of immobilized PLE (pig liver esterase; Chirazyme ®) on the metric
ton scale, for the production of Emtriva™ (Coviracil®, Emtricitabine, (2R,5S)-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine).
Emtricitabine is the (-) enantiomer of a thio analogue of cytidine, which differs
from other cytidine analogues in that it has a fluorine in the 5-position, and functions
as a potent anti-AIDS drug marketed by Gilead Sciences.
...immobilized in a catalytically active state microbial esterases, such as Amano
pseudomonas sp lipase. They have been used in stereo- and regioselective biocatalytic
processes to produce a number of different pharmaceutical intermediates.
...significantly improved time space yield (productivity) of commercial biocatalysts
for continuous and semi-batch immobilized enzymatic process applications. For example,
for an historic reference, we developed key components of the world's first commodity-scale
immobilized enzyme process, glucose isomerase (Ketomaxâ ) for production of high
fructose corn syrup,
...successfully scaled up organic transformations employing immobilized microorganisms,
whole cells & enzymes (biocatalysts derived from mesophilic, thermophilic and hyperthermophilic
microorganisms), including enantioselective and regioselective transformations, for
continuous and semi-batch processes,
... developed, In conjunction with the Degussa AG Project House, conditions for biocatalytic
production of enantiomerically pure amino acids using a single construct,
...developed immobilized enzyme supports for unique applications,
...developed chemical stabilization method for commercial immobilized enzymes,
...developed, optimized and applied enzyme immobilization chemistry,
...developed analytical procedures under cGMP auspices for the accurate and reproducible
monitoring of immobilized enzyme processes, including the use of a pH stat for temporally
following esterase activity,
...optimized enzyme purification techniques,
...performed process optimizations, especially DOE (Statistical Design of Experiments)
and physical biochemistry approaches for product yield increase,
...performed enzymatic conversions / transformations in non-aqueous solvents.
Applied chemistry: proteins, carbohydrates and natural products
Performed chemical modification of peptide and enzymatic antimicrobials for use in
Optimized conjugate production (activated carbohydrate and peptide conjugation) for
Enzyme characterization (mammalian and bacterial) as well as
Small molecule isolation, characterization and derivatization.
Separations science and analytical chemistry
Developed separations technology for purification and characterization of numerous
enzymes, other proteins, peptides and natural products, used in commercial applications,
Applied quantitative ion chromatography (IC) of small molecules,
Accurate detection and quantitation of enzymes and proteins
Other areas of technical expertise mostly relating to biologically catalyzed conversions
(Bioprocessing & Biotransformation) and analysis, include:
stereospecific and stereoselective biological conversions and product isolation;
reclamation and bio-conversion of materials;
physical biochemistry, especially as they relate to protein, enzyme and peptide (mammalian
and microbial) production, selection, purification, characterization and chemical
protein biochemistry and enzymology including enzyme kinetics, assay and ligand-ligand