Effect of salt modulators on the elution behavior of insulin and the separation of product-related impurities in reversed-phase chromatography.

In this work, the effect of the salt modulators potassium chloride, ammonium chloride, ammonium sulfate, and potassium sulfate on the elution behavior of insulin in reversed-phase chromatography with ethanol as the organic modifier was investigated. Without the addition of salt modulators, insulin shows the formation of multiple peaks under non-linear loading conditions, presumably due to an aggregate formation equilibrium. Flow rate and temperature did not influence the appearance of multiple peaks. The addition of chloride and sulfate salt modulators changed the monomer-multimer equilibrium, and multi-peak formation no longer occurred. Chloride salts induce a Langmuirian elution behavior, whereas sulfate salts induce additional insulin-insulin interactions resulting in an anti-Langmuirian elution behavior. The elution behavior can be influenced by the combination of both chloride and sulfate salts and by varying the concentration ratio. The separation with respect to two product-related impurities also showed significant differences under Langmuirian and anti-Langmuirian elution conditions and the purification of insulin could be optimized. Induced anti-Langmuirian elution by lowering the chloride/sulfate ratio suppresses an observed tag-along effect of one variant resulting in a slightly smaller pool volume with increased insulin concentration and a significantly increased insulin recovery.

Mechanistic modeling of cation exchange chromatography scale-up considering packing inhomogeneities.

In process development and characterization, the scale-up of chromatographic steps is a crucial part and brings a number of challenges. Usually, scale-down models are used to represent the process step, and constant column properties are assumed. The scaling is then typically based on the concept of linear scale-up. In this work, a mechanistic model describing an anti-Langmuirian to Langmuirian elution behavior of a polypeptide, calibrated with a pre-packed 1 ml column, is applied to demonstrate the scalability to larger column volumes up to 28.2 ml. Using individual column parameters for each column size, scaling to similar eluting salt concentrations, peak heights, and shapes is experimentally demonstrated by considering the model's relationship between the normalized gradient slope and the eluting salt concentration. Further scale-up simulations show improved model predictions when radial inhomogeneities in packing quality are considered.

Mechanistic modeling and simulation of a complex low and high loading elution behavior of a polypeptide in cation exchange chromatography.

The mechanistic modeling of preparative liquid chromatography is still a challenging task. Nonideal thermodynamic conditions may require activity coefficients for the mechanistic description of preparative chromatography. In this work, a chromatographic cation exchange step with a polypeptide having a complex elution behavior in low and high loading situations is modeled. Model calibration in the linear range of the isotherm is done by applying counterion-induced linear gradient elution experiments between pH 3.3 and 4.3. Inverse fitting with column loads up to 25 mg/mLCV is performed for parameter estimation in the nonlinear range. The polypeptide elution peak shows an anti-Langmuirian behavior with fronting under low loading conditions and a switch to a Langmuirian behavior with increasing load. This unusual elution behavior could be described with an extended version of the sigmoidal Self-Association isotherm including two activity coefficients for the polypeptide and counterion in solution. The activity coefficient of the solute polypeptide shows a strong influence on the model parameters and is crucial in the linear and nonlinear range of the isotherm. The modeling procedure results in a unique and robust model parameter set that is sufficient to describe the complex elution behavior and allows modeling over the full isotherm range.

Expression and purification of the extracellular domains of human glycoprotein VI (GPVI) and the receptor for advanced glycation end products (RAGE) from Rattus norvegicus in Leishmania tarentolae.

Glycosylation is one of the most complex post-translational modifications and may have significant influence on the proper function of the corresponding proteins. Bacteria and yeast are, because of easy handling and cost reasons, the most frequently used systems for recombinant protein expression. Bacteria generally do not glycosylate proteins and yeast might tend to hyperglycosylate. Insect cell- and mammalian cell-based expression systems are able to produce complex N-glycosylation structures but are more complex to handle and more expensive. The nonpathogenic protozoa Leishmania tarentolae is an easy-to-handle alternative expression system for production of proteins requiring the eukaryotic protein folding machinery and post-translational modifications. We used and evaluated the system for the secretory expression of extracellular domains from human glycoprotein VI and the receptor for advanced glycation end products from rat. Both proteins were well expressed and homogeneously glycosylated. Analysis of the glycosylation pattern identified the structure as the conserved core pentasaccharide Man3GlcNac2.

Biophysical characterization of the interaction between hepatic glucokinase and its regulatory protein: impact of physiological and pharmacological effectors.

Glucokinase (GK) is a key enzyme of glucose metabolism in liver and pancreatic beta-cells, and small molecule activators of GK (GKAs) are under evaluation for the treatment of type 2 diabetes. In liver, GK activity is controlled by the GK regulatory protein (GKRP), which forms an inhibitory complex with the enzyme. Here, we performed isothermal titration calorimetry and surface plasmon resonance experiments to characterize GK-GKRP binding and to study the influence that physiological and pharmacological effectors of GK have on the protein-protein interaction. In the presence of fructose-6-phosphate, GK-GKRP complex formation displayed a strong entropic driving force opposed by a large positive enthalpy; a negative change in heat capacity was observed (Kd = 45 nm, DeltaH = 15.6 kcal/mol, TDeltaS = 25.7 kcal/mol, DeltaCp = -354 cal mol(-1) K(-1)). With k(off) = 1.3 x 10(-2) s(-1), the complex dissociated quickly. The thermodynamic profile suggested a largely hydrophobic interaction. In addition, effects of pH and buffer demonstrated the coupled uptake of one proton and indicated an ionic contribution to binding. Glucose decreased the binding affinity between GK and GKRP. This decrease was potentiated by an ATP analogue. Prototypical GKAs of the amino-heteroaryl-amide type bound to GK in a glucose-dependent manner and impaired the association of GK with GKRP. This mechanism might contribute to the antidiabetic effects of GKAs.