Biophysical Methods for Characterization of Antibody-Drug Conjugates.

Antibody-drug conjugates (ADC) are made up of three components: (1) a mAb specific to cells of choice, (2) a small molecule with desired end goal, and (3) a linker to covalently link drug molecule to the antibody. Bringing together the mAb, drug molecule, and the linker results in the formation of an immunoconjugate designed to selectively deliver the drug molecule to a cell of interest. Synergic effects of the mAb and drug molecule lead to destroying the target tumor cells while leaving the normal cells unharmed. However, the development of ADCs is associated with challenges due to the heterogeneity of the ADC molecules created from the conjugation process. Addition of the linker and drug moieties during processing as well as the hydrophobicity of the drug itself can lead to structural changes that may affect the stability and functional profile of the conjugated molecule. Furthermore, linkers site of attachment plays a major role in determining the conformational and colloidal properties of the ADCs. In this chapter, several characterization methods are introduced to determine the biophysical characteristics of the ADC. Protocols, data analysis as well as notes for circular dichroism, intrinsic fluorescence, ANS fluorescence, differential scanning calorimetry, and dynamic scanning fluorimetry are outlined in detail.

Impact of Natural Variations in Freeze-Drying Parameters on Product Temperature History: Application of Quasi Steady-State Heat and Mass Transfer and Simple Statistics.

Inter- and intra-batch variability in heat and mass transfer during the drying phase of lyophilization is well recognized. Heat transfer variability between individual vials in the same batch arise from both different positions in the vial array and from variations in the bottom contour of the vials, both effects contributing roughly equally to variations in the effective heat transfer coefficient of the vials, Kv. Both effects can be measured in the laboratory, and variations in average Kv values as a function of vial position in the array for lab and production can be calculated by use of the simple steady-state heat and mass transfer theory. Typically, in the laboratory dryer, vials on the edge of the array, "edge vials," run 2-4°C warmer than "center vials," but differences between laboratory and manufacturing temperatures are modest. The variability in mass transfer can be assigned to major variations in ice nucleation temperature (both intra-batch and inter-batch), including major differences between laboratory and manufacturing. The net effect of all random variations, for each class of vial, can be evaluated by a simple statistical model-propagation of error, which then allows prediction of the distribution in product temperatures and drying times, and therefore prediction of percent of vials dry and percent of vials collapsed and proximity to the edge of failure for a given process. Good agreement between theoretical and experimentally determined maximum temperatures in primary drying and percent collapsed product demonstrates the calculations have useful accuracy.

Effect of Controlled Ice Nucleation on Stability of Lactate Dehydrogenase During Freeze-Drying.

Several controlled ice nucleation techniques have been developed to increase the efficiency of the freeze-drying process as well as to improve the quality of pharmaceutical products. Owing to the reduction in ice surface area, these techniques have the potential to reduce the degradation of proteins labile during freezing. The objective of this study was to evaluate the effect of ice nucleation temperature on the in-process stability of lactate dehydrogenase (LDH). LDH in potassium phosphate buffer was nucleated at -4°C, -8°C, and -12°C using ControLyo™ or allowed to nucleate spontaneously. Both the enzymatic activity and tetramer recovery after freeze-thawing linearly correlated with product ice nucleation temperature (n = 24). Controlled nucleation also significantly improved batch homogeneity as reflected by reduced inter-vial variation in activity and tetramer recovery. With the correlation established in the laboratory, the degradation of protein in manufacturing arising from ice nucleation temperature differences can be quantitatively predicted. The results show that controlled nucleation reduced the degradation of LDH during the freezing process, but this does not necessarily translate to vastly superior stability during the entire freeze-drying process. The capability of improving batch homogeneity provides potential advantages in scaling-up from lab to manufacturing scale.

Freeze-Drying Process Development and Scale-Up: Scale-Up of Edge Vial Versus Center Vial Heat Transfer Coefficients, Kv.

This report presents calculations of the difference between the vial heat transfer coefficient of the "edge vial" and the "center vial" at all scales. The only scale-up adjustment for center vials is for the contribution of radiation from the shelf upon which the vial sits by replacing the emissivity of the laboratory dryer shelf with the emissivity of the production dryer shelf. With edge vials, scales-up adjustments are more complex. While convection is not important, heat transfer from the wall to the bands (surrounding the vial array) by radiation and directly from the band to the vials by both radiation and conduction is important; this radiation heat transfer depends on the emissivity of the vial and the bands and is nearly independent of the emissivity of the dryer walls. Differences in wall temperatures do impact the edge vial effect and scale-up, and estimates for wall temperatures are needed for both laboratory and manufacturing dryers. Auto-loading systems (no bands) may give different edge vial heat transfer coefficients than when operating with bands. Satisfactory agreement between theoretical predictions and experimental values of the edge vial effect indicate that results calculated from the theory are of useful accuracy.

Rational design of anthracene-based DNA binders.

Achieving the goal of rational design of DNA-binding ligands is important, and many inroads have been made in this direction. Toward that goal, we report a simple, systematic, and quantitative approach to design DNA-binding anthracene derivatives. Current data show that the binding free energies (DeltaG degrees) as well as enthalpies (DeltaH degrees) are related to specific structural features of the binders. Systematic design of anthracene probes, for example, indicated that the affinity can be enhanced via the introduction of methylene groups. Each methylene group contributed, on an average, -0.08+/-0.002 kcal/mol (at 1 M ionic strength, 293 K) toward the total binding free energy. Binding of the probes to DNA depended on ionic strength, and ionic strength studies were used to factor out to parse free-energy contributions due to specific interactions. The intrinsic free-energy contributions (DeltaGMol) of the probes are obtained by factoring out contributions from ionic interactions, hydration, conformational changes, polyelectrolyte effect, and the loss of rotational/translational motion. A strong, linear correlation was noted between DeltaGMol and the number of methylene groups present in the probe, and the correlation indicated free-energy contributions of -1.49 kcal/mol per methylene (at 50 mM NaCl, 293 K). This important observation provides a convenient handle to systematically fine-tune the intrinsic affinities of DNA binders. DeltaH values also showed clear trends, and each methylene contributed +0.28 kcal/mol toward the overall binding enthalpy (at 50 mM NaCl, 293 K), and this aspect is useful to fine-tune DeltaH contributions to binding. These important physical insights, derived from systematic modifications of the side chains of the DNA binders, are useful in the rational design of novel DNA binders.

Novel enzyme/DNA/inorganic nanomaterials: a new generation of biocatalysts.

The design, synthesis and properties of a new class of enzyme/DNA/inorganic nanobiomaterials are described here. DNA has been used to stabilize the enzymes intercalated in the galleries of the inorganic solid, alpha-Zr(iv) phosphate (alpha-Zr(HPO(4))(2).H(2)O, abbreviated as alpha-ZrP). Interestingly, the presence of DNA improved the activity and stability of the bound enzymes. Key studies leading to the current strategy are presented initially, and these are followed by more recent developments. Several enzymes and proteins, including horseradish peroxidase, lysozyme, glucose oxidase, chymotrypsin, bovine serum albumin, cytochrome c, met-hemoglobin and met-myoglobin are successfully intercalated in the galleries of alpha-ZrP, under benign ambient conditions (aqueous buffered solutions, at room temperature and neutral pH). These novel materials are characterized by XRD, SEM and TEM as well as by biochemical, calorimetric and spectroscopic methods. Spectroscopic studies (circular dichroism, CD), for example, indicated that co-intercalation of DNA improved the retention of bound enzyme structure. The activity was enhanced markedly (five-fold) when DNA is co-intercalated, when compared to the activity in the absence of DNA. Addition of DNA to the sample, after enzyme intercalation, did not make any improvements. Our hypothesis is that enzyme-DNA supramolecular complex binds to the solid and the unfavorable interactions between the enzyme and the solid are minimized. These novel nanobiocomposite materials provide a simple method for packaging DNA and aid in engineering more effective synthetic materials for gene/RNA-delivery and drug delivery applications.