Derivatives of human growth hormone (hGH) of increasing size were produced by reaction with the Nhydroxysuccinimide ester of polyethylene glycol-5000 (PEG 5000 ), a 5-kDa reagent that selectively conjugates to primary amines. By adjusting the reaction conditions and purification procedure, it was possible to isolate hGH derivatives containing up to seven PEG moieties that altered the Stokes radius and thereby the effective molecular masses of the unmodified hormone from 22 to 300 kDa. Fortunately, the most reactive amines were ones that did not lie in either of the two sites important for receptor binding. Nonetheless, increasing the level of PEG modification linearly reduced the affinity of hGH for its receptor and increased the EC 50 in a cellbased assay up to 1500-fold. Most of the reduction in affinity was the result of slowing the association rate for the receptor. The clearance rate of hGH in rats was inversely proportional to effective molecular weight and closely fit a filtration model. We have tested the potency of these analogs by injecting them daily or every 6 days into hypophysectomized rats and determining the effects on body and organ growth. The efficacy of these analogs was optimal for hGH conjugated with 5 eq of PEG 5000 , and the potency was increased by about 10-fold compared with unmodified hGH. Such PEG-hGH derivatives show promise as long-acting alternatives to daily injections of hGH. More generally these studies show that improving hormone clearance properties, even at the expense of reducing receptor binding affinity, can lead to dramatic increases in hormone efficacy.The ability of a hormone to elicit a biological effect in vivo depends on many factors including the affinity for its receptor and the rate at which it is cleared from the circulation. Some hormones, like atrial natriuretic peptide, have a very high affinity for their receptor (10 pM) and are cleared very rapidly (t1 ⁄2 ϳ0.5 min) by receptor and protease-mediated events (1). Other hormones, like human growth hormone (hGH), 1 have lower affinity for their receptor (300 pM) but are cleared more slowly (t1 ⁄2 ϳ30 min in rats), primarily via the kidney (2, 3).Understanding the relationships between hormone affinity, clearance, and efficacy is important in optimizing hormone therapy. To study this systematically one would like to vary these parameters and evaluate their relative importance in regulating biopotency. hGH is a good model system in this regard as much is known about its structure and function (for review see Ref. 4). Simple receptor binding (5, 6), cell-based assays (7,8), and growth parameters in rodents (9) can be used to determine biopotency in vitro and in vivo. The properties of proteins such as hGH that are cleared by kidney filtration can be modulated by attachment of polyethylene glycol (PEG) polymers, which increases the hydrodynamic volume of the hormone and thereby slows its clearance (10, for recent review see Ref. 11).Here, we describe a set of hGH derivatives conjugated with increasing numbers of PEG ...

This paper examines the retention behavior of recombinant DNA-derived human growth hormone (rhGH) in reversed-phase chromatography and its separation from the closely related N-methionyl variant (Met-hGH). It is first shown that retention for rhGH decreases with increasing column temperature when 1-propanol (1-PrOH) is used as organic modifier. On the other hand, retention increases with temperature when acetonitrile (CH3CN) is employed. The differences in behavior for the two organic modifiers could be related to conformational changes in the protein as determined by solution and adsorption intrinsic fluorescence spectroscopy. Specifically, desorption and elution of rhGH using 1-PrOH could be correlated with a solvent-induced conformational change, with retention decreasing with increasing temperature due to the increasing ease of structural alteration. On the other hand for CH3CN the increase in retention correlated with temperature rise was related to a partial structural change yielding a more hydrophobic species. In this case, a surface-driven process is suggested. The work then turned to the separation of rhGH and Met-hGH where it was found for both organic modifiers optimum separation occurred at 45 degrees C and pH 6.5. Separate studies revealed that during the conformational change Met-hGH appeared more hydrophobic than rhGH since protein-protein aggregation was observed at a lower 1-PrOH concentration. It is suggested that this hydrophobic difference, which was optimized under the conditions cited above, resulted in the separation. The study demonstrates the importance of conformational changes in retention behavior and separation of protein samples.

Monoclonal antibodies are subject to a variety of degradation mechanisms, therefore orthogonal techniques are required to demonstrate product quality. In this study, the three individual antibodies comprising a multi-antibody drug product, XOMA 3AB were evaluated by both cationexchange (CEX) and anion-exchange chromatography (AEX).In contrast to CEX analysis which showed only a single, broad peak for the force-oxidized antibodies, AEX analysis of Ab-A (pI=7.6) revealed two more basic peaks. Ab-B (pI=6.7) bound but exhibited only a single major peak while Ab-C (pI=8.6) flowed through. Peptide mapping LC/ MS analysis of the isolated Ab-A fractions demonstrated that the basic peaks resulted from oxidation in a complementary determining region (CDR). Differential scanning calorimetry (DSC) analysis of the oxidized Ab-A species showed a decrease in the Fab melting point for the oxidized species consistent with unfolding of the molecule. Greater/lesser surface exposure of ionic residues resulting from a conformational change provides a likely explanation for the dramatic shift in retention behavior for the Ab-A oxidized variants. Peptide mapping analysis of the Ab-B antibody showed, in contrast to Ab-A, no detectable CDR oxidation. Hence, the lack of separation of oxidized variants in Ab-B can be explained by the absence of CDR oxidation and the associated changes in secondary/tertiary structure which were observed for oxidized AbA. In summary, anion-exchange HPLC shows potential as an orthogonal analytical technique for assessing product quality of monoclonal antibody therapeutics. In the case of the XOMA 3AB drug product, two of the antibodies bound and one, Ab-A, exhibited separation of CDR oxidized variants.

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High-performance cation-exchange chromatography of recombinant soluble CD4 (rCD4) allowed the resolution of four charge variants. This charge heterogeneity could be eliminated by neuraminidase treatment of rCD4 and therefore can be attributed to different degrees of sialylation of the carbohydrate portion of this glycoprotein. A single acidic variant was observed upon cation-exchange chromatography of neuraminidase-treated rCD4 that had been stored in liquid solution, pH 7.2, at 25 degrees C for 6 months. This acidic variant was isolated by semipreparative cation-exchange chromatography and subjected to tryptic mapping analysis. Tryptic peptides were characterized by fast atom bombardment mass spectrometry (FABMS). The results of this analysis demonstrated that the acidic variant of neuraminidase-treated rCD4 is generated from deamidation at Asn-52. Digestion of the deamidated rCD4 with endoproteinase Asp-N confirmed Asn-52 as the primary site of deamidation. The ability of the deamidated rCD4 variant to bind gp120 was assessed by use of an ELISA-based binding assay. The binding capacity of the deamidated variant was 24% of the binding capacity of unmodified rCD4. The overall structure of the V1 domain in the deamidated variant was not markedly different from that of the native protein as probed with eight conformationally dependent anti-V1 monoclonal antibodies. Therefore, it appears that Asn-52 is directly involved in binding to gp120.