Adapting pharmacokinetic properties of a humanized anti-interleukin-8 antibody for therapeutic applications using site-specific pegylation.

A neutralizing anti-interleukin-(IL-)8 monoclonal antibody was humanized by grafting the complementary determining regions onto the human IgG framework. Subsequent alanine scanning mutagenesis and phage display enabled the production of an affinity matured antibody with a >100-fold improvement in IL-8 binding. Antibody fragments can be efficiently produced in Escherichia coli but have the limitation of rapid clearance rates in vivo. The Fab' fragment of the antibody was therefore modified with polyethylene glycol (PEG) in order to obtain a more desirable pharmacokinetic profile. PEG (5-40 kDa) was site-specifically conjugated to the Fab' via the single free cysteine residue in the hinge region. In vitro binding and bioassays showed little or no loss of activity. The pharmacokinetic profiles of the 20 kDa, 30 kDa, 40 kDa, and 40 kDa branched PEG-Fab' molecules were evaluated in rabbits. Relative to the native Fab', the clearance rates of the PEGylated molecules were decreased by 44-175-fold. In a rabbit ear model of ischemia/reperfusion injury, all PEGylated Fab' molecules were as efficacious in reducing oedema as the original monoclonal antibody. These studies demonstrate that it is possible to customize the pharmacokinetic properties of a Fab' while retaining its antigen binding activity.

Role of high-performance liquid chromatographic protein analysis in developing fermentation processes for recombinant human growth hormone, relaxin, antibody fragments and lymphotoxin.

Development of efficient and reliable fermentation processes for protein pharmaceuticals is aided by the availability of accurate quantitative and qualitative product analyses. We have developed a variety of single and dual column chromatographic separations that meet the needs of process development and examples will be provided of how the resulting data has been used to optimize the culture process. For single column methods, reversed-phase chromatography has been the most versatile, permitting the reliable quantitation of many yeast, Chinese hamster ovary (CHO) cell and Escherichia coli-expressed products in the matrix of culture broth or cell extract. Analysis of secreted human growth hormone synthesized in E. coli, along with clipped and unprocessed forms, will be discussed. Another reversed-phase assay for direct analysis of a peptide product (B-chain relaxin) and its degradation products secreted into E. coli fermentation medium has allowed the purification of the responsible protease. Cation-exchange has proven extremely useful for the direct analysis of antibody fragment synthesized in E. coli, allowing the separation and quantitation of the desired Fab' and Fab'2, as well as the unwanted products of glutathione addition and translational read-through. Assay development is often complicated by the presence of host proteins with chromatographic behavior that is similar to that of the product. Commercial instrumentation now permits the facile development of multidimensional chromatographic assays. We show examples of coupled receptor affinity-reversed-phase assays for a mistranslation product and for covalent multimers of E. coli-synthesized lymphotoxin.

Validation of an HPLC method for the analysis of the charge heterogeneity of the recombinant monoclonal antibody IDEC-C2B8 after papain digestion.

An HPLC procedure was validated for determining the purity with respect to the charge variant distribution of the recombinant monoclonal antibody (MAb) IDEC-C2B8 by high-performance ion-exchange chromatography. Papain was used to fragment the molecule into Fab and Fc fragments prior to chromatographic analysis. Fragmentation allowed the resolution of the variants arising from the cyclization of glutamine to pyroglutamate at the amino-terminus of the light and heavy chains (Fab-pE/Q variants) from the variants resulting from the processing of the carboxy-terminal lysine residues of the heavy chains (Fc-Lys variants). The assay demonstrated good linearity, yielding correlation coefficients of > 0.99 for total protein, Fc-Lys variants and Fab-pE/Q variants. Recovery of total protein from the column was 95.7%. Sample recovery studies demonstrated a mean accuracy of 102% for a Fab fragment over the range 2-10% of the total protein. The limit of detection was 0.2 microg and 0.1 microg for Fc and Fab variants, respectively. The repeatability of the assay and intermediate precision had relative standard deviation (RSD) values of < 1%. Parameters of the papain digest (time, digest stability, reagent stability, pH and papain vendor) and of the chromatography (mobile phase pH, stability, buffer concentration, and column lot and aging) were evaluated for robustness and determined to be acceptable. Data are presented demonstrating the suitability of the assay for determining the product purity of a recombinant MAb.

Application of pyrolysis mass spectroscopy and SDS-PAGE in the study of the epidemiology of Pseudomonas cepacia in cystic fibrosis.

Representative isolates of Pseudomonas cepacia from 15 cystic fibrosis (CF) patients attending the Respiratory Unit of Alder Hey Childrens' Hospital were investigated by SDS-PAGE of whole-cell polypeptides and by pyrolysis mass spectroscopy (PMS). SDS-PAGE was less discriminatory than PMS. Eleven isolates were indistinguishable by PMS and considered to represent re-isolates of an endemic strain; four isolates were distinct from this group, and from one another. P. cepacia was first isolated on the unit in July 1989 from a patient who had attended a UK selection meeting for a Canadian CF camp. A ward and outpatient segregation policy was introduced, but colonisation of further patients occurred. In August 1991, the Adult CF Association recommended that all social activities involving colonised patients should cease. This, and an increased awareness amongst older CF patients of the risks of person-to-person transmission, was associated with a marked decline in new cases. Social activity and hospital admissions were compared for colonised patients during the year before colonisation with P. cepacia, and matched patients who did not acquire the endemic strain. This showed a significantly higher attendance at CF social events for colonised patients, but no significant association between colonisation and hospital admission. These results are strong indirect evidence that transmission of P. cepacia occurs through social contact outside the hospital environment.

Effect of pH and CO2 on in vitro susceptibility of Pseudomonas cepacia to beta-lactams.

Inhibition of Pseudomonas cepacia (but not Pseudomonas aeruginosa) by beta-lactams was decreased in 5% CO2 in air compared with air alone. The effect of CO2 and pH (range, 6.0 to 8.0) on beta-lactam susceptibility, beta-lactamase expression, and outer membrane proteins was studied in isolates recovered from the sputum of children with cystic fibrosis. Incubation in 5% CO2 decreased the activity of piperacillin, piperacillin/tazobactam, and ceftazidime, although isolates were still clinically sensitive (minimum inhibitory concentrations < 16 mg/L). Cefpirome activity was markedly decreased from a minimum inhibitory concentration of 2.0 to greater than 64 mg/L. On highly buffered 3-(N-morpholino)-propane sulfonic acid media, beta-lactam susceptibility was eliminated at pH greater 7.5. A 2- to 13-fold increase in beta-lactamase activity was demonstrated after growth in 5% CO2 compared with basal aerobic levels for 13 of 15 clinical isolates. beta-Lactamase activity did not vary significantly with pH. Addition of imipenem to media (2.0 mg/L) resulted in hyperproduction of beta-lactamase (180-fold). Isoelectric points varied with cultural conditions, and all beta-lactamases detected were inhibited by clavulanate and tazobactam. Significant hydrolysis of piperacillin and ceftazidime could not be demonstrated. A 36-kD porin was present at all pH tested. Thus, our strains of Pseudomonas cepacia were markedly affected by cultural conditions not normally used in standardized susceptibility tests. However, such conditions may be encountered in the pathologically altered infected lung in cystic fibrosis.