Bioengineering Techniques

Bioengineering requires a very broad and diverse set of skills to be effective. IBBR leverages a unique combination of skills, equipmment, and collaborators to achieve real results.

Monoclonal antibodies and mapping

We have recently established a multicolor Fluorescence Activated Cell Sorting (FACS) cell sorting platform to isolate monoclonal antibodies from antigen-specific memory B cell repertoires. This FACS-based single B cell sorting platform affords rapid antigen-specific monoclonal antibody cloning compared to conventional monoclonal antibody cloning techniques such as B cell hybridoma.

IBBR is fully equipped to map protein epitopes recognized by monoclonal antibodies (mAbs) using two independent techniques: 1) deuterium-exchange mass spectrometry (DXMS) and 2) X-ray crystallography.

DXMS is a powerful, moderate throughput method to map protein–protein, including antigen–antibody, interactions. Although DXMS does not provide the resolution of X-ray crystallography, this footprinting technique offers the advantage of speed. Complete data acquisition can be accomplished in several days, with another day or two required for full processing and interpretation. In addition, DXMS does not require crystallization of the target proteins, which is typically the bottleneck in X-ray crystallography. DXMS consumes only small quantities of sample (1–2 nmoles), and is tolerant of proteins that are not completely pure. Proteins and protein–protein complexes that are fairly large, including multimers, can be analyzed, with an upper-size limit of ~400–500 kDa.

X-ray crystallography is the gold standard for mapping epitopes recognized by mAbs. This method allows direct visualization of the interaction between the antigen and antibody at the atomic level, a resolution that is not attainable by DXMS or any other epitope mapping technique. Crystal diffraction data is collected in-house at IBBR using Rigaku image plate detectors and/or at a synchrotron radiation source to which IBBR has access. Structure determinations of antigen–mAb complexes are typically performed by molecular replacement using known mAb and antigen structures as search models.

B cell immunology and evolution

We have recently established a comprehensive platform to study B cell response using a well characterized soluble form of HIV-1 envelope glycoproteins, namely Env, as model antigen. With this platform, we coupled single B cell FACS sorting and cloning with next-generation sequencing (NGS) technology to investigate Env-specific memory B cell responses, illustrated in Fig. 1. This platform can also be adapted to investigate neutralizing antibody response evolution in other viral systems, such as ebola.

In brief, 1) we used single B cell FACS sorting and cloning technology to isolate HIV Env-specific memory B cells and recover the IgG heavy/light chain encoding gene; 2) we used NGS (deep sequencing) to reconstitute the sequences of total Ig cDNA, (antibodyome) using PBMC from the immunized animal as the source of the total B cell repertoire to prepare the total Ig cDNA library; and 3) we used the Ig sequences of the memory B cells of known function from 1) to query the total Ig sequences of the antibodyome with intensive depth and to reveal function-related genetic variants. A combination of single B cell sorting and NGS of the antibodyome affords comprehensive understanding of the immunogenetics of antibody induction and maturation.

Materials science

IBBR researchers are pioneering the application of advanced biological methods for materials fabrication with a particular focus on the biofabrication of the materials interface between biological and electronic systems. Specifically, their focus is on soft matter (e.g., biopolymeric systems and hydrogels) and they are making contributions in four major areas.

  • Self-assembly. Biological polymers (e.g., proteins and polysaccharides) and virus-based nanoparticles are being engineered to meet needs in sensing and energy applications.
  • Enzymatic assembly. Enzymes are being enlisted to tailor macromolecular structure and confer biological function to material systems.
  • Protein engineering. Genetic methods are enlisted to manipulate protein structure and function to facilitate assembly, couple function-conferring components (e.g., in vivo enzyme pathways), and permit molecular-based communication.
  • Synthetic biology. Engineered bacteria are being created to confer biotic functions to materials (e.g., chemically-based computations).

Polymer synthesis, design, and engineering

Unique expertise in the design and synthesis of multifunctional polyphosphazenes for life sciences applications. Experience in the synthesis of water-soluble polymers, biodegradable polyphosphazenes, polyionic immunoadjuvants, ionically cross-linked hydrogels, polymer modification reactions, reactive macromolecular precursors, environmentally responsive polyelectrolytes, multilayer macromolecular assemblies, sulfonated and fluorinated polymers. The laboratory equipment, such as MBraun glove box system, Parr 1L titanium benchtop pressure reactor, De Dietrich 50 Gallon glass lined reactor, fume hoods, allows to carry out a wide range of organic chemistry reactions including those requiring high temperature, high pressure, and anhydrous conditions.

Formulation chemistry and delivery

Expertise in preparation of hydrogel and polymer nanoparticulates, nano- and micro-encapsulation of therapeutic proteins and enzymes, controlled release formulations, microneedles and transdermal patches, formulations for intravenous and oral delivery, adjuvanted vaccine formulations, characterization of biophysical carrier properties, protein-polymer interactions, formulation stability in storage and physiological conditions, interaction, uptake and subcellular trafficking in cell culture and in vivo models, pharmacokinetics and biodistribution and laboratory animals, and effects..
Equipment for advanced characterization of proteins, biomacromolecules, interpolymer complexes, nanoparticles and microcapsules: Postnova AF2000 MultiFlow Field Flow Fractionation System, Hitachi size-exclusion High Performance Liquid Chromatography system, Malvern Zetasizer Nano ZS Dynamic Light Scattering (DLS) Instrument, Thermo Scientific Multiskan™ Microplate Spectrophotometer, World Precision Instruments EVOM™ volt-ohm meter and STX100 electrodes, PerkinElmer 2470 Wizard2gamma counter, Franz vertical diffusion cells for studying controlled release, fluorescent and inverted phase microscopes (including Olympus IX81 inverted 3-axe automatic fluorescence microscope, Media Cybernetics Image-Pro 6.3 with customized algorithms to track nanoparticle interaction with cells), capillary-air flow assisted microcapsule formation, microneedle fabrication micro-positioning apparatus with optical control.

Fermentation and cell culture

The UMD has longstanding expertise in the production and purification of recombinant proteins, including human protein therapeutics. In collaboration with the Maryland Technology Enterprise Institute (MTech), IBBR provides the biotechnology community with comprehensive bench and pilot scale bioprocess development capability. In addition, in partnership with the biomanufacturing initiative at NIST, new process analytical measurements and industry “standard” materials are being developed that help to maintain the US biopharmaceutical industry’s world leadership in human biotherapeutics.