Mark R. Etzel

Alternatives to Chromatographic Separations

Up to now, the productivity of mammalian cell culture has been perceived as limiting the productivity of the industrial manufacture of therapeutic monoclonal antibodies. Dramatic improvements in cell culture performance have changed this picture, and the throughput of antibody purification processes is gaining increasing attention. Although chromatographic separations currently are the centerpiece of antibody purification, mostly due to their high resolving power, it becomes more and more apparent that there may be limitations at the very large scale. This review will discuss a number of alternatives to chromatographic antibody purification, with a particular emphasis on the ability to increase throughput and overcome traditional drawbacks of column chromatography. Specifically, precipitation, membrane chromatography, high‐resolution ultrafiltration, crystallization, and high‐pressure refolding will be evaluated as potential large scale unit operations for industrial antibody production.

A mathematical analysis of affinity membrane bioseparations

Abstract A mathematical model including convection, diffusion and Langmuir adsorption is formulated to analyze the design and operation of affinity membrane bioseparations. A key prediction from the analysis is that the flow velocity through affinity membranes may be limited not by pressure drop, but rather by association kinetics between soluble protein and immobilized ligand. Furthermore, for thin membranes with Peclet numbers less than 40, slow association kinetics may limit the flow velocity to an extent where diffusion axial to flow undesirably becomes a dominant mechanism for transport of protein through the membrane. Lastly, even relatively small thickness variations of 3% and porosity variations of 1% may severely degrade membrane of performance. Practical consequences of these results are presented, e.g. constructing thicker membranes from stacks of thinner membranes will tend to reduce the effect of thickness and porosity variations, sharpen breakthrough curves, raise allowable flow velocities and increase loading capacities.

Mass transfer limitations in protein separations using ion-exchange membranes

Sorption of bovine serum albumin to commercial 150-micron pore size membranes was measured in batch and flow experiments. For residence times of 2-40 min, early and broad breakthrough curves and broad asymmetric elution peaks were observed that depended strongly on flow-rate. System dispersion could not explain the flow-rate dependence. Breakthrough and elution curves were analyzed using new models that included Langmuir sorption, convection and diffusion. From the analysis, film mass transfer resistance was found to be the rate-limiting factor. The maximum allowable pore size that eliminates this limitation was calculated for different molecular weight solutes.

Evaluation of an ion-exchange membrane for the purification of plasmid DNA.

Separation and purification of large quantities of plasmid DNA (pDNA) is a particularly difficult manufacturing issue because of the relatively low capacity, flow rate and purity observed using traditional bead‐based chromatography. The objective of the present study was to evaluate the performance of anion‐exchange membranes for the purification of pDNA from Escherichia coli lysate solution. The fate of host‐cell protein and endotoxin relative to pDNA was measured and used to calculate recoveries, mass balances, dynamic capacities and purification factors as a function of the flow rate and loading volume of the lysate solution. Breakthrough curves were not sigmoidal and symmetric in shape. They rose sharply at first, and then slowly towards, but never reaching, saturation. Conversely, elution curves were independent of flow rate. pDNA bound tightly to the membranes, whereas protein and endotoxin did not. Dynamic binding capacity for pDNA was 20–25 times greater, and the flow rate was 55–550 times greater, than values observed for beads. However, some pDNA bound irreversibly to the membrane surface and was not removed completely during elution. The intrinsic rate of pDNA adsorption to the membrane was found to be rate‐limiting, whereas effects of liquid‐phase mass transfer and flow non‐idealities were negligible. These results were interpreted using models of adsorption that included steric effects using the ‘car‐parking‐problem’ model, and surface residence time effects using the spreading model. This work demonstrated the advantages of ion‐exchange membranes compared with beads for the purification of large biomolecules such as pDNA.

Improved nutritional management of phenylketonuria by using a diet containing glycomacropeptide compared with amino acids.

BACKGROUND Phenylketonuria (PKU) requires a lifelong low-phenylalanine diet that provides the majority of protein from a phenylalanine-free amino acid (AA) formula. Glycomacropeptide (GMP), an intact protein formed during cheese production, contains minimal phenylalanine. OBJECTIVE The objective was to investigate the effects of substituting GMP food products for the AA formula on acceptability, safety, plasma AA concentrations, and measures of protein utilization in subjects with PKU. DESIGN Eleven subjects participated in an inpatient metabolic study with two 4-d treatments: a current AA diet (AA diet) followed by a diet that replaced the AA formula with GMP (GMP diet) supplemented with limiting AAs. Plasma concentrations of AAs, blood chemistries, and insulin were measured and compared in AA (day 4) and GMP diets (day 8). RESULTS The GMP diet was preferred to the AA diet in 10 of 11 subjects with PKU, and there were no adverse reactions to GMP. There was no significant difference in phenylalanine concentration in postprandial plasma with the GMP diet compared with the AA diet. When comparing fasting with postprandial plasma, plasma phenalyalanine concentration increased significantly with the AA but not with the GMP diet. Blood urea nitrogen was significantly lower, which suggests decreased ureagenesis, and plasma insulin was higher with the GMP diet than with the AA diet. CONCLUSIONS GMP, when supplemented with limiting AAs, is a safe and highly acceptable alternative to synthetic AAs as the primary protein source in the nutritional management of PKU. As an intact protein source, GMP improves protein retention and phenylalanine utilization compared with AAs.

Sorption kinetics and breakthrough curves for pepsin and chymosin using pepstatin A affinity membranes

Isotherms and kinetic parameters for pepsin and chymosin sorption to immobilized pepstatin A were measured in batch experiments. The measured single-solute parameters were used in an affinity-membrane model which included competitive sorption kinetics, axial diffusion and dead volume mixing. The predictions made using the affinity-membrane model matched the experimental breakthrough curves, whereas predictions made using local-equilibrium theory were a distinct mismatch. The performance of affinity-membrane separations was dominated by slow sorption kinetics.

Competitive adsorption of α-lactalbumin and bovine serum albumin to a sulfopropyl ion-exchange membrane

Abstract Breakthrough curves were measured for pure solutions of α-lactalbumin (ALA) and bovine serum albumin (BSA) individually, and for a binary mixture of the proteins, using a sulfopropyl ion-exchange membrane. The breakthrough curves were qualitatively consistent with local-equilibrium theory predictions. Competitive adsorption caused displacement of bound BSA monomer by the more strongly binding BSA dimer, illustrating that even apparently single-protein systems may display multicomponent competitive behavior. In the two-protein experiment, ALA was competitively displaced by the more strongly binding BSA monomer and dimer, indicating that the binding strength was in the order: BSA dimer > BSA monomer > ALA.

Design of salt‐tolerant membrane adsorbers for viral clearance

Strong anion exchange chromatography has frequently been employed as a viral clearance step during downstream processing of biological therapeutics. When challenged with viruses having only slightly acidic isoelectric points, the performance of the anion exchange operation becomes highly dependent on the buffer salt concentration, with the virus log reduction value (LRV) dropping dramatically in buffers with 50–150 mM salt. In this work, a series of anion exchange membrane adsorbers utilizing alternative ligand chemistries instead of the traditional quaternary amine (Q) ligand have been developed that overcome this limitation. Four different ligands (agmatine, tris‐2‐aminoethyl amine, polyhexamethylene biguanide, and polyethyleneimine) achieved >5 LRV of bacteriophage ΦX174 (pI ∼ 6.7) at pH 7.5 and up to 150 mM salt, compared to 0 LRV for the Q ligand. By evaluating structural derivatives of the successful ligands, three factors were identified that contributed to ligand salt tolerance: ligand net charge, ligand immobilization density on the membrane, and molecular structure of the ligand‐binding group. Based on the results of this study, membrane adsorbers that incorporate alternative ligands provide a more robust and salt tolerant viral clearance‐processing step compared to traditional strong anion exchange membrane adsorbers. Biotechnol. Bioeng. 2009;103: 920–929.

Purification and use of glycomacropeptide for nutritional management of phenylketonuria.

Individuals with phenylketonuria (PKU) cannot metabolize phenylalanine (Phe) and must adhere to a low-Phe diet in which most dietary protein is provided by a Phe-free amino acid formula. Glycomacropeptide (GMP) is the only naturally occurring protein that does not contain Phe, and is of interest as a source of protein for dietary management of PKU. However, commercially available GMP contains too much Phe from residual whey proteins and does not contain adequate levels of all the indispensable amino acids to provide a nutritionally complete protein. The aim of this study was to increase purity of GMP and develop a mass balance calculation for indispensable amino acid supplementation of GMP foods. Cation exchange chromatography, ultrafiltration/diafiltration, and lyophilization were used at the pilot plant scale to decrease Phe. Enough purified GMP (5 kg) was manufactured to provide 15 PKU subjects with a 4-d diet in which the majority of protein was from GMP foods. A mass balance was used to supplement GMP foods so that all indispensable amino acids met or exceeded the daily recommended intake. GMP foods were tested in a human clinical trial as a replacement for the traditional amino acid formula. Nutritionally complete GMP foods created with high purity GMP provide individuals with PKU with more options to manage PKU, which may lead to improved compliance and quality of life.

Equilibrium adsorption of LDL and gold immunoconjugates to affinity membranes containing PEG spacers.

The objective of this study was to provide insight into the effects of spacer chemistry on immunoaffinity separations for the capture of large macromolecules and biological complexes. Immunoaffinity membranes were prepared by immobilization of immunoglobulin G (IgG) to flat sheet microporous membranes. Two different systems were examined: immobilized IgG for the immunoadsorption of human low-density lipoprotein (LDL) and immobilized IgG for the immunoadsorption of gold particle immunoconjugate. The IgG was immobilized either directly to the membrane or via a polyethylene glycol (PEG) spacer. Adsorption of LDL was significantly greater for anti-LDL IgG immobilized via PEG than for IgG immobilized directly to the membrane. With the PEG spacer, the adsorption capacity for LDL matched the theoretical density of a monolayer of LDL particles on the membrane surface. The gold particle immunoconjugate, similar in size to LDL, was examined as a generalized model of restrictions to immunoaffinity adsorption of large (>20 nm) biological complexes. Adsorption of gold particles was greater for IgG immobilized via PEG than for IgG immobilized directly to the membrane. It is postulated that the PEG spacer allows lateral movement of the immobilized IgG and dense monolayer packing of adsorbed particles on the membrane surface. These results are pertinent to the removal of LDL from human plasma and the purification of gene therapy delivery vectors, viral vaccines, and other large biological complexes.