Semisynthetic hydrophilic polyals

Non-bioadhesive, fully biodegradable soluble polymers would be very instrumental in advanced biomedical applications, such as gene and drug delivery and tissue engineering. However, rational development of such materials is hindered by the complexity of macromolecule interactions with biological milieu. The prevalence of carbohydrates in naturally occurring interface structures suggests an alternative, biomimetic approach. Interface carbohydrates, regardless of their biological function, have common non-signaling substructures (e.g., acetal and ketal groups, secondary and primary alcohols). We hypothesized that hydrophilic polymers (polyals) consisting of acyclic units built of non-signaling carbohydrate substructures would be highly biocompatible and non-bioadhesive, while intrachain acetal or ketal groups would enable nonenzymatic biodegradation upon uptake by cells. Acyclic hydrophilic polyals can be prepared via either polymerization of suitable monomers or lateral cleavage of cyclic polyals (e.g., polysaccharides). In this study, model polyals were produced via lateral cleavage of polyaldoses and polyketoses. Best results were achieved using dextran B-512 as a precursor. The resultant poly[hydroxymethylethylene hydroxymethylformal], in agreement with the hypothesis, demonstrated excellent biological properties and technological flexibility. Materials of this type can potentially have several applications in pharmacology and bioengineering.     

Conjugation of proteins and enzymes with hydrophilic polymers and their applications.

The efficacy of a number of therapeutically active proteins and peptides is severely limited due to their instability in circulation. Of the various approaches used to stabilise these proteins, the one more successful is covalent modification of the protein or enzyme with some hydrophilic polymers such as dextran or PEG. These conjugates are more stable than the native protein both in vitro as well as in vivo. They exhibit enhanced resistant to proteolytic degradation, have a long-life in circulation and exhibit reduced immunogenicity. The therapeutic efficacy of these conjugates is also greatly enhanced compared to the native protein or enzyme.     

Pentafluorophenyl ester-functionalized phosphorylcholine polymers: preparation of linear, two-arm, and grafted polymer-protein conjugates

Novel pentafluorophenyl (PFP)-ester-functionalized phosphorylcholine (PC) polymers of different architectures were prepared and conjugated to lysozyme as a model protein. Linear and two-arm poly(2-methacryloyloxyethyl phosphorylcholine) (polyMPC) structures containing PFP functionality at the chain-end were prepared by atom transfer radical polymerization (ATRP) from novel initiators. Additional conjugates were prepared from phosphorylcholine-substituted cyclooctene (PC-COE) polymers containing PFP-ester bearing comonomers. The polymer-protein conjugates were characterized by HPLC, FPLC, and DLS and were seen to retain most (~80% or greater) of their native enzymatic activity. Pharmacokinetic profiles of the polymer-protein conjugates were studied in mice and found to increase the circulation half-life compared with lysozyme alone.     

Effects of polymer molecular weight on the size, activity, and stability of PEG-functionalized trypsin

Polymer conjugation increases an enzyme's circulation time and stability for use as a therapeutic agent, but this attachment indubitably affects its properties. Covalent attachment of multiple polyethylene glycol chains with sizes of either 2, 5, 10, or 20 kDa increases the molecular weight and hydrodynamic radius of the model enzyme trypsin. The sizes of these polymer-enzyme conjugates are increased to be within the recommended limits for PDEPT applications. The T(d) increases from 49 to 60 °C to expand the enzyme's workable range of conditions. This functionalization with PEG polymers of varying lengths maintains trypsin's enzymatic activity. Conjugate activities are 79-120% that of native trypsin at room temperature and 221-432% that of trypsin at 37 °C.     

Site-specific attachment of polyethylene glycol-like oligomers to proteins and peptides

Modification of proteins with polymers is a viable method to tune protein properties, e.g., to render them more water-soluble by using hydrophilic polymers. We have utilized precision-length, polyethylene glycol-based oligomers carrying a thioester moiety in transthioesterification and native chemical ligation reactions with internal and N-terminal cysteine residues in proteins and peptides. These reactions lead to uniquely modified proteins with an increased solubility in chaotrope- and detergent-free aqueous systems. Polymer modification of internal cysteines is fully reversible and allows generation of stable protein-polymer conjugates for enzymatic manipulations as demonstrated by proteolytic cleavage of a protein construct that was only soluble in buffers incompatible with protease activity before polymer modification. The permanent polymer modification of a Rab protein at its N-terminal cysteine produced a fully active Rab variant that was efficiently prenylated. Thus, PEGylation of prenylated proteins might be a viable route to increase water solubility of such proteins in order to carry out experiments in detergent- and lipid-free systems.     

The conformation of the poly(ethylene glycol) chain in mono-PEGylated lysozyme and mono-PEGylated human growth hormone

Covalent conjugation of poly(ethylene glycol) or "PEGylation" has proven an effective strategy to improve pharmaceutical protein efficacy by hindering recognition by proteases, inhibitors, and antibodies and by retarding renal clearance. Because it determines the strength and range of intermolecular steric forces and the hydrodynamic properties of the conjugates, the configuration of protein-conjugated PEG chains is the key factor determining how PEGylation alters protein in vivo circulation time. Mono-PEGylated proteins are typically described as having a protective PEG shroud wrapped around the protein, but recent dynamic light scattering studies suggested that conjugates adopt a dumbbell configuration, with a relatively unperturbed PEG random coil adjacent to the globular protein. We used small-angle neutron scattering (SANS) to distinguish between the dumbbell model and the shroud model for chicken-egg lysozyme and human growth hormone covalently conjugated to a single 20 kDa PEG chain. The SANS contrast variation technique was used to isolate the PEG portion of the conjugate. Scattering intensity profiles were well described by the dumbbell model and inconsistent with the shroud model.     

A novel family of L-amino acid-based biodegradable polymer-lipid conjugates for the development of long-circulating liposomes with effective drug-targeting capacity

The objective of this study was to develop biodegradable polypeptide-lipid conjugates for the design of polymer-coated long-circulating liposomes (LCL). Lipid conjugates of poly(hydroxyalkyl L-asparagine/L-glutamine) were synthesized and incorporated into 0.15 microm dipalmitoyl phosphatidylcholine (DPPC)-cholesterol liposomes. Circulation times and biodistribution were assessed in rats using a radioactive lipid marker. Evaluation of the therapeutic activity of prednisolone phosphate loaded in 0.1 microm PHEA-DPPC-cholesterol liposomes in a rat experimental arthritis model was performed to demonstrate the drug-targeting potential of the polymer-coated liposomes. Coating of liposomes with poly(hydroxyethyl L-asparagine) (PHEA) and poly(hydroxyethyl L-glutamine) (PHEG) extended the circulation half-life to a similar extent as poly(ethylene glycol) (PEG), which is normally used for the preparation of LCL. Glutamine polymers with a hydroxypropyl or a hydroxybutyl group instead of hydroxyethyl group also yield prolonged circulation, however, not to the same extent as PHEA/G. The pharmacokinetic properties of PHEA-liposomes were independent of the lipid dose even at very low lipid doses of around 50 nmol per rat. PLP was successfully entrapped in PHEA-liposomes. These liposomes were shown to be stable in the circulation and equally effective in rat experimental arthritis as PLP encapsulated in PEG-liposomes. PHEA and PHEG are attractive alternative polymers for the design of LCL: their performance is similar to that of PEG-liposomes but they have the advantage of being biodegradable.     

Zwitterionic polymers in drug delivery: A review

Developments of novel drug delivery vehicles are sought-after to augment the therapeutic effectiveness of standard drugs. An urgency to design novel drug delivery vehicles that are sustainable, biocompatible, have minimized cytotoxicity, no immunogenicity, high stability, long circulation time, and are capable of averting recognition by the immune system is perceived. In this pursuit for an ideal candidate for drug delivery vehicles, zwitterionic materials have come up as fulfilling almost all these expectations. This comprehensive review is presenting the progress made by zwitterionic polymeric architectures as prospective sustainable drug delivery vehicles. Zwitterionic polymers with varied architecture such as appending protein conjugates, nanoparticles, surface coatings, liposomes, hydrogels, etc, used to fabricate drug delivery vehicles are reviewed here. A brief introduction of zwitterionic polymers and their application as reliable drug delivery vehicles, such as zwitterionic polymer–protein conjugates, zwitterionic polymer-based drug nanocarriers, and stimulus-responsive zwitterionic polymers are discussed in this discourse. The prospects shown by zwitterionic architecture suggest the tremendous potential for them in this domain. This critical review will encourage the researchers working in this area and boost the development and commercialization of such devices to benefit the healthcare fraternity.     

End-functionalized phosphorylcholine methacrylates and their use in protein conjugation

Polymer-protein conjugation was performed using N-hydroxysuccinimide and aldehyde-terminated zwitterionic polymers, and the resulting polymer-protein conjugates were characterized by gel electrophoresis and fast protein liquid chromatography. Methacryloyloxyethyl phosphorylcholine (MPC) polymers were prepared by atom transfer radical polymerization in which the requisite functional end-groups for protein conjugation were embedded within the polymerization initiators. These phosphorylcholine polymers were conjugated to lysozyme as a model protein, as well as two therapeutic proteins, granulocyte colony stimulating factor (G-CSF) and erythropoietin (EPO). These MPC polymer-protein conjugates represent alternatives to PEGylated proteins, with the potential to provide improved efficacy in a therapeutic treatment relative to the protein itself.     

Mean-field model of immobilized enzymes embedded in a grafted polymer layer

Two-dimensional mean-field lattice theory is used to model immobilization and stabilization of an enzyme on a hydrophobic surface using grafted polymers. Although the enzyme affords biofunctionality, the grafted polymers stabilize the enzyme and impart biocompatibility. The protein is modeled as a compact hydrophobic-polar polymer, designed to have a specific bulk conformation reproducing the catalytic cleft of natural enzymes. Three scenarios are modeled that have medical or industrial importance: 1), It is shown that short hydrophilic grafted polymers, such as polyethylene glycol, which are often used to provide biocompatibility, can also serve to protect a surface-immobilized enzyme from adsorption and denaturation on a hydrophobic surface. 2), Screening of the enzyme from the surface and nonspecific interactions with biomaterial in bulk solution requires a grafted layer composed of short hydrophilic polymers and long triblock copolymers. 3), Hydrophilic polymers grafted on a hydrophobic surface in contact with an organic solvent form a dense hydrophilic nanoenvironment near the surface that effectively shields and stabilizes the enzyme against both surface and solvent.     

Covalent fixation of hemoglobin to dextran phosphates decreases its oxygen affinity.

The interactions between various dextran phosphates and Hb (hemoglobin) were studied by measuring the oxygen-binding parameters of the mixtures. The effector properties of polymers were found to depend on the concentration of monoalkylmonophosphate groups on the polymers and also on their molecular weights. The covalent fixation of dextran phosphates bearing aldehydic groups to oxyHb and deoxyHb was carried out. The oxygen-binding properties of the conjugates thus obtained depended upon the initial form of the protein. Thus, only the conjugates synthesized from deoxyHb exhibited a low oxygen affinity, which means that, in this case, the linkages between the dextran phosphate and the protein allow a permanent interaction of the phosphate groups with amines of the 2,3-diphosphoglycerate binding site. The Hill coefficient values of these conjugates were smaller than that of free Hb, corresponding to a loss of the cooperativity of the protein upon fixation of polymers. However, as these new conjugates are capable of unloading more O2 than blood when subjected to oxygen pressures corresponding to physiological conditions, they can be regarded as potential erythrocyte substitutes.     

The toxicity of menadione (2-methyl-1,4-naphthoquinone) and two thioether conjugates studied with isolated renal epithelial cells

Menadione (2-methyl-1,4-naphthoquinone) was used as a model compound to test the hypothesis that thioether conjugates of quinones can be toxic to tissues associated with their elimination through a mechanism involving oxidative stress. Unlike menadione, the glutathione (2-methyl-3-(glutathion-S-yl)-1,4-naphthoquinone; MGNQ) and N-acetyl-L-cysteine (2-methyl-3-(N-acetylcysteine-S-yl)-1,4-naphthoquinone; M(NAC)NQ) thioether conjugates were not able to arylate protein thiols but were still able to redox cycle with cytochrome c reductase/NADH and rat kidney microsomes and mitochondria. Interestingly, menadione and M(NAC)NQ were equally toxic to isolated rat renal epithelial cells (IREC) while MGNQ was nontoxic. The toxicity of both menadione and M(NAC)NQ was preceded by a rapid depletion of soluble thiols and was associated with a depletion of soluble thiols and was associated with a depletion of protein thiols. Treatment of IREC with the glutathione reductase inhibitor, 1,3-bis(2-chloroethyl)-1-nitrosourea, potentiated the thiol depletion and toxicity observed with menadione and M(NAC)NQ indicating the involvement of oxidative stress in this model of renal cell toxicity. The lack of MGNQ toxicity can be attributed to an intramolecular cyclization reaction which destroys the quinone nucleus and therefore eliminates its ability to redox cycle. These findings have important implications with regard to our understanding of the toxic potential of quinone thioether conjugates and of quinone toxicity in general.     

Site-specific conjugation of chain-terminal chelating polymers to Fab' fragments of anti-CEA mAb: effect of linkage type and polymer size on conjugate biodistribution in nude mice bearing human colorectal carcinoma.

Polylysine-based chelating polymers were used for site-specific modification of anti-CEA mAb Fab' fragments via their SH group distal to the antigen-binding site of the antibody molecule. Conjugation was performed using chain-terminal (pyridyldithio)propionate or 4-(p-maleimidophenyl)butyrate moieties to form reducible (S-S) or stable (S-C) bonds between a polymer and Fab' molecule, respectively. One S-S conjugate (S-S9) and two different S-C conjugates (S-C3 and S-C9) were prepared using 3- and 9-kDa molecular weight polymers. No significant loss of immunoreactivity was observed in solid-phase immunoassay, 90-95% of 111In-labeled conjugates being bound to CEA-coated Sepharose beads. After labeling with 111In, the conjugates had a specific radioactivity of 90-120 microCi/micrograms. Injected in nude mice bearing LS 174T carcinoma, the conjugates produced different biodistribution patterns. S-S9 was practically unable to accumulate in tumor and produced very rapid blood clearance of radioactivity and high uptake of radioactivity in liver, spleen, and especially kidneys (225% ID/g 24 h postinjection). S-C3 and S-C9 produced practically the same blood clearances (much slower than that of S-S9) and significant tumor uptake (9-10% ID/g at 24 h). S-C3 gave significantly lower radioactivity in spleen, skin, and bones, and cleared more rapidly from liver and kidneys. Renal uptake for S-C3 and S-C9 was rather high (45% ID/g at 24 h), but much lower than for S-S9.     

Squaric acid mediated synthesis and biological activity of a library of linear and hyperbranched poly(glycerol)-protein conjugates

Polymer-protein conjugates generated from side chain functional synthetic polymers are attractive because they can be easily further modified with, for example, labeling groups or targeting ligands. The residue specific modification of proteins with side chain functional synthetic polymers using the traditional coupling strategies may be compromised due to the nonorthogonality of the side-chain and chain-end functional groups of the synthetic polymer, which may lead to side reactions. This study explores the feasibility of the squaric acid diethyl ester mediated coupling as an amine selective, hydroxyl tolerant, and hydrolysis insensitive route for the preparation of side-chain functional, hydroxyl-containing, polymer-protein conjugates. The hydroxyl side chain functional polymers selected for this study are a library of amine end-functional, linear, midfunctional, hyperbranched, and linear-block-hyperbranched polyglycerol (PG) copolymers. These synthetic polymers have been used to prepare a diverse library of BSA and lysozyme polymer conjugates. In addition to exploring the scope and limitations of the squaric acid diethylester-mediated coupling strategy, the use of the library of polyglycerol copolymers also allows to systematically study the influence of molecular weight and architecture of the synthetic polymer on the biological activity of the protein. Comparison of the activity of PG-lysozyme conjugates generated from relatively low molecular weight PG copolymers did not reveal any obvious structure-activity relationships. Evaluation of the activity of conjugates composed of PG copolymers with molecular weights of 10000 or 20000 g/mol, however, indicated significantly higher activities of conjugates prepared from midfunctional synthetic polymers as compared to linear polymers of similar molecular weight.     

Protein-polymer conjugates: synthetic approaches by controlled radical polymerizations and interesting applications

Protein-polymer conjugates are of interest to researchers in diverse fields. Attachment of polymers to proteins results in improved pharmacokinetics, which is important in medicine. From an engineering standpoint, conjugates are exciting because they exhibit properties of both the biomolecules and synthetic polymers. This allows the activity of the protein to be altered or tuned, anchoring to surfaces, and supramolecular self-assembly. Thus, there is broad interest in straightforward synthetic methods to prepare protein-polymer conjugates. Controlled radical polymerization (CRP) techniques have emerged as excellent strategies to make conjugates because the resulting polymers have narrow molecular weight distributions, targeted molecular weights, and attach to specific sites on proteins. Herein, recent advances in the synthesis and application of protein-polymer conjugates by CRP are highlighted.     

Contribution of linker stability to the activities of anticancer immunoconjugates

The linker component of antibody-drug conjugates (ADC) is a key feature in developing optimized therapeutic agents that are highly active at well tolerated doses. For maximal intratumoral drug delivery, linkers are required that are highly stable in the systemic circulation, yet allow for efficient drug release at the target site. In this respect, amide bond-based technologies constitute a technological advancement, since the linker half-lives in circulation ( t 1/2 approximately 7 days) are much longer than earlier generation linkers that break down within 1-2 days. The amide linkers, some of which contain peptides, are appended to the mAb carriers through thioether/maleimide adducts. Here, we describe that use of a bromoacetamidecaproyl (bac) in place of the maleimidocaproyl (mc) increases the plasma stability of resulting thioether ADCs. One such ADC, 1F6-C4v2-bac-MMAF, which is directed against the CD70 antigen on lymphomas and renal cell carcinoma, was prepared containing a bac thioether spacer between the drug (MMAF) and the mAb carrier (1F6-C4v2). There was no measurable systemic drug release from this ADC for 2 weeks postadministration in mice. In order to assess the impact of improving linker stability beyond mc containing ADCs, a series of mc and bac-linked 1F6-MMAF conjugates were compared for tolerability, intratumoral drug delivery, and therapeutic efficacy in nude mice with renal cell carcinoma xenografts. There were no statistically significant efficacy differences between sets of mc and bac containing ADCs, although the bac linker technology led to 25% higher intratumoral drug exposure over a 7 day period compared to the corresponding mc linker. The mechanism of drug release from maleimide-adducts likely involves a retro-Michael reaction that takes place in plasma, based on in vitro studies demonstrating that some of the released drug-maleimide derivative became covalently bound to cysteine-34 of serum albumin. In summary, the data indicate that new linkers can be obtained with improved in vivo stability by replacing the maleimide with an acetamide, but the resulting ADCs had similar tolerability and activity profiles.     

PEG-doxorubicin conjugates: influence of polymer structure on drug release, in vitro cytotoxicity, biodistribution, and antitumor activity

Polymer-drug conjugates (polymer therapeutics) are finding increasing use as novel anticancer agents. Here a series of poly(ethylene glycol) PEG-doxorubicin (Dox) conjugates were synthesized using polymers of linear or branched architecture (molecular weight 5000-20000 g/mol) and with different peptidyl linkers (GFLG, GLFG, GLG, GGRR, and RGLG). The resultant conjugates had a drug loading of 2.7-8.0 wt % Dox and contained <2.0% free drug (% total drug). All conjugates containing a GFLG linker showed approximately 30% release of Dox at 5 h irrespective of PEG molecular weight or architecture. The GLFG linker was degraded more quickly (approximately 57% Dox release at 5 h), and the other linkers more slowly (<16% release at 5 h), by lysosomal enzymes in vitro. In vitro there was no clear relationship between cytotoxicity toward B16F10 cells and the observed Dox release rate. All PEG conjugates were more than 10-fold less toxic (IC50 values > 2 microg/mL) than free Dox (IC50 value = 0.24 microg/mL). Biodistribution in mice bearing sc B16F10 tumors was assessed after administration of PEGs (5000, 10000, or 20000 g/mol) radioiodinated using the Bolton and Hunter reagent or PEG-Dox conjugates by HPLC. The 125I-labeled PEGs showed a clear relationship between Mw and blood clearance and tumor accumulation. The highest Mw PEG had the longest plasma residence time and consequently the greatest tumor targeting. The PEG-Dox conjugates showed a markedly prolonged plasma clearance and greater tumor targeting compared to free Dox, but there was no clear molecular weight-dependence on biodistribution. This was consistent with the observation that the PEG-Dox conjugates formed micelles in aqueous solution comprising 2-20 PEG-Dox molecules depending on polymer Mw and architecture. Although PEG-Dox showed greater tumor targeting than free Dox, PEG conjugation led to significantly lower anthracycline levels in heart. Preliminary experiments to assess antitumor activity against sc B16F10 in vivo showed the PEG5000linear (L)-GFLG-Dox and PEG10000branched (B)-GLFG-Dox (both 5 mg/kg Dox-equiv) to be the most active with T/C values of 146 and 143%, respectively. Free Dox did not show significant activity in this model (T/C = 121%). Dose escalation of PEG5000(L)-GFLG-Dox to 10 mg/kg Dox-equiv prolonged further animal survival (T/C = 161%). Using the Dox-sensitive model ip L1210 (where Dox displayed a T/C = 150% after single ip dose), the PEG5000(L)-GFLG-Dox displayed a maximum T/C of 141% (10 mg/kg Dox-equiv) using a once a day (x3) schedule. Further studies are warranted with PEG5000(L)-GFLG-Dox to determine its spectrum of antitumor activity and also the optimum dosing schedule before clinical testing.     

Mechanisms of phase behaviour and protein partitioning in detergent/polymer aqueous two-phase systems for purification of integral membrane proteins

Detergent/polymer aqueous two-phase systems are studied as a fast, mild and efficient general separation method for isolation of labile integral membrane proteins. Mechanisms for phase behaviour and protein partitioning of both membrane-bound and hydrophilic proteins have been examined in a large number of detergent/polymer aqueous two-phase systems. Non-ionic detergents such as the Triton series (polyoxyethylene alkyl phenols), alkyl polyoxyethylene ethers (C(m)EO(n)), Tween series (polyoxyethylene sorbitol esters) and alkylglucosides form aqueous two-phase systems in mixtures with hydrophilic polymers, such as PEG or dextran, at low and moderate temperatures. Phase diagrams for these mixtures are shown and phase behaviour is discussed from a thermodynamic model. Membrane proteins, such as bacteriorhodopsin and cholesterol oxidase, were partitioned strongly to the micelle phase, while hydrophilic proteins, BSA and lysozyme, were partitioned to the polymer phase. The partitioning of membrane protein is mainly determined by non-specific hydrophobic interactions between detergent and membrane protein. An increased partitioning of membrane proteins to the micelle phase was found with an increased detergent concentration difference between the phases, lower polymer molecular weight and increased micelle size. Partitioning of hydrophilic proteins is mainly related to excluded volume effects, i.e. increased phase component size made the hydrophilic proteins partition more to the opposite phase. Addition of ionic detergent to the system changed the partitioning of membrane proteins slightly, but had a strong effect on hydrophilic proteins, and can be used for enhanced separation between hydrophilic proteins and membrane protein.     

Plasmodium falciparum expresses a multidrug resistance-associated protein

Plasmodium falciparum proteins that efflux toxic metabolic products such as oxidised glutathione (GSSG) are possible targets for anti-malarial drug development. Proteins capable of transporting GSSG and glutathione conjugates include the multidrug resistance-associated transporters (MRPs). A gene, PFA0590w, encoding a MRP homologue, has been identified in P. falciparum. Here we show the presence of full-length mRNA (5.5 kb) of this PfMRP in trophozoites by RT-PCR and Northern blotting. A polyclonal anti-PfMRP antibody generated against two unique, hydrophilic peptides in the predicted sequence produced a strong immunoreactive protein band of 210-215 kDa on Western blots of schizonts of chloroquine-sensitive and chloroquine-resistant strains, confirming expression of PfMRP protein. Using confocal microscopy the protein was seen to be localised at the edge of the schizonts with no obvious staining of the food vacuole. We suggest that PfMRP may act as the GSSG transporter in the parasite plasma membrane.     

Conjugation of horseradish peroxidase to staphylococcal protein A with benzoquinone, glutaraldehyde, or periodate as cross-linking reagents

Horseradish peroxidase was conjugated to Staphylococcal protein A by three different two-step procedures using an increasing excess of peroxidase in the second step reaction. The yield of conjugated protein A was analyzed by SDS-polyacrylamide gel electrophoresis. Conjugation of peroxidase to protein A with benzoquinone or glutaraldehyde as cross-linking reagents at a 3- to 4-fold molar excess of peroxidase resulted in a high yield of coupled protein A with conjugates of low molecular size. Conjugation of peroxidase to protein A by the periodate method resulted in a high yield of coupled protein A with polymeric conjugates of large molecular size. Based on these results, conjugates produced with glutaraldehyde as cross-linking reagents were further analyzed. The capacity of the conjugates to precipitate human immunoglobulin evaluated by radial immunodiffusion was found to be reduced to about 50% of that of native protein A. Conjugates produced with glutaraldehyde as cross-linking reagent retained 70% of the enzyme activity of native peroxidase.