Ultrafiltration/diafiltration Research Articles

Multi-attribute PAT for UF/DF of Proteins-Monitoring Concentration, particle sizes, and Buffer Exchange.

Ultrafiltration/diafiltration (UF/DF) plays an important role in the manufacturing of biopharmaceuticals. Monitoring critical process parameters and quality attributes by process analytical technology (PAT) during those steps can facilitate process development and assure consistent quality in production processes. In this study, a lab-scale cross-flow filtration (CFF) device was equipped with a variable pathlength (VP) ultraviolet and visible (UV/Vis) spectrometer, a light scattering photometer, and a liquid density sensor (microLDS). Based on the measured signals, the protein concentration, buffer exchange, apparent molecular weight, and hydrodynamic radius were monitored. The setup was tested in three case studies. First, lysozyme was used in an UF/DF run to show the comparability of on-line and off-line measurements. The corresponding correlation coefficients exceeded 0.97. Next, urea-induced changes in protein size of glucose oxidase (GOx) were monitored during two DF steps. Here, correlation coefficients were ≥ 0.92 for static light scattering (SLS) and dynamic light scattering (DLS). The correlation coefficient for the protein concentration was 0.82, possibly due to time-dependent protein precipitation. Finally, a case study was conducted with a monoclonal antibody (mAb) to show the full potential of this setup. Again, off-line and on-line measurements were in good agreement with all correlation coefficients exceeding 0.92. The protein concentration could be monitored in-line in a large range from 3 to 120 g L- 1. A buffer-dependent increase in apparent molecular weight of the mAb was observed during DF, providing interesting supplemental information for process development and stability assessment. In summary, the developed setup provides a powerful testing system for evaluating different UF/DF processes and may be a good starting point to develop process control strategies. Graphical Abstract Piping and instrumentation diagram of the experimental setup and data generated by the different sensors. A VP UV/Vis spectrometer (FlowVPE, yellow) measures the protein concentration. From the data of the light scattering photometer (Zetasizer, green) in the on-line measurement loop, the apparant molecular weight and z-average are calculated. The density sensor (microLDS) measures density and viscosity of the fluid in the on-line loop.

Continuous ultrafiltration/diafiltration using a 3D-printed two membrane single pass module.

A 3D printed ultrafiltration/diafiltration (UF/DF) module is presented allowing the continuous, simultaneous concentration of retained (bio-)molecules and reduction or exchange of the salt buffer. Differing from the single-pass UF concepts known from the literature, DF operation does not require the application of several steps or units with intermediating dilution. In contrast, the developed module uses two membranes confining the section in which the molecules are concentrated while the sample is passing. Simultaneously to this concentration process, the two membranes allow a perpendicular in and outflow of DF buffer reducing the salt content in this section. The module showed the continuous concentration of a dissolved protein up to a factor of 4.6 while reducing the salt concentration down to 47% of the initial concentration along a flow path length of only 5 cm. Due to single-pass operation the module shows concentration polarization effects reducing the effective permeability of the applied membrane in case of higher concentration factors. However, because of its simple design and the capability to simultaneously run UF and DF processes in a single module, the development could be economically beneficial for small scale UF/DF applications.

Modeling and prediction of excipient and pH drifts during ultrafiltration/diafiltration of monoclonal antibody biotherapeutic for high concentration formulations

High concentration formulation is advantageous over conventionally used intravenous formulation. A model for the drift in the excipient concentration in the ultrafiltration/diafiltration (UF/DF) operation by including the flux of each of the species through the membrane module of the system was developed. Nernst-Planck equation was used to model the flux of the charged species, and these were coupled due to the requirement of zero electric current flow associated with the permeation of the ions. In addition, volume exclusion effect due to high protein concentration in the retentate has also been accounted for in the model. The variation of pH was also calculated using a detailed balance on the H+ ions incorporating the applicable ionic equilibrium with the concentration of relevant ions in the reservoir obtained using mass balance and permeate flux and concentration of these ions. The model was validated with the experimental data on constant volume DF and volume-reducing UF experiments on a monoclonal antibody (mAb). The exchange buffer contained histidine and sodium acetate. The model results agreed quantitatively with both the DF data at different mAb as well as exchange buffer concentrations and with the UF data for over the complete range of mAb concentrations (20–200 mg/ml) for different buffer concentrations. The model also predicted the change in the pH as a function of mAb concentration to within ±0.3 units. The model could be used for both the constant volume, buffer exchanging DF as well as the reducing reservoir volume, mAb concentrating UF operations.

The prediction of the operating conditions on the permeate flux and on protein aggregation during membrane processing of monoclonal antibodies

The lack of available material during early stage bioprocess development poses numerous processing challenges such as limiting the number of full-scale experiments. Extended fundamental process understanding could be gained with the use of an ultra scale-down (USD) device using as little as 1.7 mL per experimental run. The USD system is used to predict diafiltration and ultrafiltration/diafiltration (UF/DF) performance of a pilot-scale tangential flow filtration (TFF) system, fitted with a flat-sheet cassette, operating at 500-fold larger scale. Both systems were designed by maintaining a volumetric loading of 8.1 L of feed per m2. Permeate flux was predicted for monoclonal antibody solutions with the USD system across a range of transmembrane pressure drops, feed concentrations and flow conditions during diafiltration, and desired retentate concentrations during UF/DF operations. The resulting USD data were in good agreement with the pilot-scale TFF when scaled based on similar shear rates over the membrane surface. Little change in soluble aggregates was observed in both systems but there were significantly higher increases in product turbidity in the USD system. A correlation was established to relate turbidity increase based on the volume fraction of high shear stress zone for USD systems and various pilot-scale TFF systems. The correlation was extended to encompass the processing time and concentration for a wide range of membrane processing challenges in both scales.

Integration of a complete downstream process for the automated lab-scale production of a recombinant protein

In this work, an automated downstream process for the purification and formulation of a recombinant protein was integrated at lab scale in a single chromatography unit. The purification chain consists of three bind-and-elute chromatography columns, a flow-through membrane chromatography step, and a final ultrafiltration-diafiltration (UFDF) step to concentrate and formulate the sample. An integrated downstream process increases productivity and decreases process time and hold-up volume. In addition, the automation of the process allows reducing the manual work and increases reproducibility. To integrate the downstream steps, all the intermediate tanks are removed, and the eluate of a column is loaded directly onto the next one. This makes it necessary to design the process in order to minimize the column volumes and the process time. A research software called Orbit was used to automate the purification process and implement a UFDF step in the chromatography unit. The whole downstream sequence was successfully implemented at lab scale, getting a pure concentrated and formulated product with a productivity of 1.09 mg mL−1 h−1, achieving a time reduction from almost two to one working day, while getting a similar yield and purity. Regarding the UFDF operation, the sample was concentrated 10 times, and 97% of the old buffer was exchanged by the formulation buffer with a sequential diafiltration.

Quantification of residual AEBSF-related impurities by reversed-phase liquid chromatography

During research of a broadly neutralizing antibody (bNAb) for HIV-1 infection, site-specific clipping was observed during cell culture incubation. Protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), was supplemented to the cell culture feeding to mitigate clipping as one of the control strategies. It led to the need and development of a new assay to monitor the free AEBSF-related impurities during the purification process. In this work, a reversed-phase liquid chromatography (RPLC-UV) method was developed to measure the total concentration of AEBSF and its major degradant product, 4-(aminoethyl) benzenesulfonic acid (AEBS-OH). This quantitative approach involved hydrolysis pre-treatment to drive all AEBSF to AEBS-OH, a filtration step to remove large molecules, followed by RPLC-UV analysis. The method was qualified and shown to be capable of measuring AEBS-OH down to 0.5 μM with good accuracy and precision, which was then applied for process clearance studies. The results demonstrated that a Protein A purification step in conjunction with a mock ultrafiltration/diafiltration (UF/DF) step could remove AEBSF-related impurities below the detection level. Overall, this study is the first to provide a unique approach for monitoring the clearance of free AEBSF and its related degradant, AEBS-OH, in support of the bNAb research.

A Combined Ultrafiltration/Diafiltration Step Facilitates the Purification of Cyanovirin-N From Transgenic Tobacco Extracts.

The production of biopharmaceutical proteins in plants offers many advantages over traditional expression platforms, including improved safety, greater scalability and lower upstream production costs. However, most products are retained within plant cells or the apoplastic space instead of being secreted into a liquid medium, so downstream processing necessarily involves tissue and cell disruption followed by the removal of abundant particles and host cell proteins (HCPs). We investigated whether ultrafiltration/diafiltration (UF/DF) can simplify the purification of the model recombinant protein cyanovirin-N (CVN), an ~ 11 kDa HIV-neutralizing lectin, from tobacco extracts prior to chromatography. We compared different membrane types and process conditions, and found that at pH 8.0 and 50 mS cm−1 an UF step using a 100 kDa regenerated cellulose membrane removed more than 80% of the ~ 0.75 mg mL−1 total soluble protein present in the clarified plant extract. We recovered ~ 70% of the CVN and the product purity increased ~ 3-fold in the permeate. The underlying effects of tobacco HCP retention during the UF/DF step were investigated by measuring the zeta potential and particle size distribution in the 2–10,000 nm range. Combined with a subsequent 10 kDa DF step, this approach simultaneously reduced the process volume, conditioned the process intermediate, and facilitated early, chromatography-free purification. Due to the generic, size-based nature of the method, it is likely to be compatible with most products smaller than ~50 kDa.

Development of a Hydrodynamic Cleaning Cycle for Ultrafiltration/Diafiltration Processes Used for Monoclonal Antibody Formulation

Ultrafiltration/diafiltration (UFDF) processes are used for final formulation of high value biotherapeutics like monoclonal antibodies. Membrane fouling and cleaning are both critical factors governing the performance of these processes, but there is very limited data on either of these phenomena, particularly at the high monoclonal antibody concentrations used in current UFDF systems. The objective of this study was to compare the effectiveness of different cleaning regimens for composite regenerated cellulose membranes in Pellicon 3 tangential flow filtration cassettes after UFDF of a monoclonal antibody product. The water permeability after UFDF was 20 ± 5% smaller than the clean membrane permeability due to fouling. Membranes could be successfully cleaned with 0.5 N NaOH, but this caused an increase in permeability and a loss in solute rejection due to an opening of the membrane pores. Cleaning with DI water was not very effective under normal flow conditions but provided very good recovery of water p...

Multi-criteria manufacturability indices for ranking high-concentration monoclonal antibody formulations.

ABSTRACTThe need for high‐concentration formulations for subcutaneous delivery of therapeutic monoclonal antibodies (mAbs) can present manufacturability challenges for the final ultrafiltration/diafiltration (UF/DF) step. Viscosity levels and the propensity to aggregate are key considerations for high‐concentration formulations. This work presents novel frameworks for deriving a set of manufacturability indices related to viscosity and thermostability to rank high‐concentration mAb formulation conditions in terms of their ease of manufacture. This is illustrated by analyzing published high‐throughput biophysical screening data that explores the influence of different formulation conditions (pH, ions, and excipients) on the solution viscosity and product thermostability. A decision tree classification method, CART (Classification and Regression Tree) is used to identify the critical formulation conditions that influence the viscosity and thermostability. In this work, three different multi‐criteria data analysis frameworks were investigated to derive manufacturability indices from analysis of the stress maps and the process conditions experienced in the final UF/DF step. Polynomial regression techniques were used to transform the experimental data into a set of stress maps that show viscosity and thermostability as functions of the formulation conditions. A mathematical filtrate flux model was used to capture the time profiles of protein concentration and flux decay behavior during UF/DF. Multi‐criteria decision‐making analysis was used to identify the optimal formulation conditions that minimize the potential for both viscosity and aggregation issues during UF/DF. Biotechnol. Bioeng. 2017;114: 2043–2056. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Perodicals, Inc.

Biorefinery of microalgal soluble proteins by sequential processing and membrane filtration

A mild biorefinery process was investigated on the microalga Nannochloropsis gaditana, to obtain an enriched fraction of water soluble proteins free from chlorophyll. After harvesting, a 100g.L−1 solution of cells was first subjected to cell disruption by either high-pressure homogenization (HPH) or enzymatic treatment (ENZ). HPH resulted in a larger release of proteins (49%) in the aqueous phase compared to the Alcalase incubation (35%). In both cases, an ultrafiltration/diafiltration (UF/DF) was then performed on the supernatant obtained from cell disruption by testing different membrane cut-off (1000kDa, 500kDa and 300kDa). After optimising the process conditions, the combination of ENZ→UF/DF ended in a larger overall yield of water soluble proteins (24.8%) in the permeate compared to the combination of HPH→UF/DF (17.4%). A gel polarization model was implemented to assess the maximum achievable concentration factor during ultrafiltration and the mass transfer coefficient related to the theoretical permeation flux rate.

Effect of channel-induced shear on biologics during ultrafiltration/diafiltration (UF/DF)

Ultrafiltration/diafiltration (UF/DF) is the standard unit operation in place in biotechnology industry for the final protein concentration and buffer exchange to produce drug substance. Several researchers have observed and published on the formation of higher order aggregates and particulates during UF/DF as a result of recirculation shear and potential exposure to air-liquid interface. This work examines the effect of screen channel-induced shear on particulate formation during UF/DF. To this end, different types of screened channels were examined for processing of monoclonal antibodies and a fusion protein during UF/DF at a fixed crossflow flux. It was found that channel-induced shear significantly affected the product quality for certain biologics, measured using various techniques, such as size-exclusion chromatography (SEC), Micro-Flow Imaging (MFI), dynamic light scattering (DLS), and filterability through a 0.2µm filter. It was demonstrated that cassettes with a more open screened-channel or “lightly suspended” feed screens resulted in consistently better product quality than the corresponding tighter or medium screen cassettes. The mass transfer coefficient for the lightly suspended screened channel was 260% higher than an open non-screened channel and only 22% lower than commercially available medium screen cassettes. The lightly suspended screen channel cassette thus offered the hydraulic advantage of lower differential pressure that an open channel offered, and the process advantage (processing time) that a medium screen channel offered along with consistently better product quality for the different biologics evaluated. The results of this study are useful in UF/DF process development of biologics to produce drug substance (DS) that has a better product quality and potentially lesser particulates.

Clearance of extractables and leachables from single-use technologies via ultrafiltration/diafiltration operations.

Quantifying the clearance of extractables and leachables (E/L) throughout ultrafiltration/diafiltration (UFDF) operations allows for greater flexibility in the implementation of single-use technologies in steps upstream of the UFDF process. A proof-of-concept study was completed in which the clearance of 7 E/L from single-use technologies (trimethylsilanol, hexanoic acid, butyrolactone, t-butyl alcohol, caprolactam, acetonitrile, and benzyl alcohol) in four representative proteins were measured and monitored during the UFDF process using quantitative NMR. This study demonstrated that the defined E/L spiked into a variety of protein solutions can be cleared to <1 ppm by 9 diavolumes from a maximum initial load concentration of 1,000 ppm. However, in some cases a rebound effect was observed in the recovered pool to >1 ppm, which is explained in detail. The overall clearance trend observed for both buffer control and protein-containing solutions resembled the ideal clearance trend where no apparent interactions were observed between E/L with the protein, UFDF system, or with other defined E/L which may be present in the system. Additionally, the UFDF system is capable of clearing these potential E/L from single-use technologies below 1 ppm irrespective of initial concentrations in the load (1,000 or 100 ppm), independently from the type of protein. In general, mass recoveries were within ±15% of each spiked compound in protein solutions and their respective buffer controls, suggesting spiked E/L do not interact strongly with protein. By demonstrating the product independent clearance trends of the spiked E/L across UFDF, these results will contribute to the simplification of the E/L toxicology assessment and allow modular manufacturing approach for single-use technologies in biopharmaceutical manufacturing. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:718-724, 2016.

Utilisation of chitosan flocculation of residual lipids and microfiltration for the production of low fat, clear WPC80

Annato coloured cheese whey was adjusted to pH 4.5 and treated with 0.01% (w/w) chitosan to selectively precipitate residual lipids, which were removed by gravity settling and microfiltration (MF). MF permeate was concentrated by ultrafiltration/diafiltration (UF/DF) to produce whey protein concentrate with 80% protein (WPC80-Chitosan). WPC80 samples were also produced by UF/DF only (Control), and by MF without chitosan treatment (MF). Both WPC80-Chitosan and WPC80-MF samples had lower fat, lower turbidity, higher foam overrun/stability and lower quantities of volatile compounds than WPC80-Control before and after storage. WPC80-Chitosan samples have an additional advantage of annatto removal (excellent clarity).

Non-chromatographic preparative purification of enhanced green fluorescent protein

Recombinant enhanced green fluorescent protein (eGFP) is used as a marker in numerous applications in biomedical research and diagnostics. For these applications, the macromolecule needs to be provided in a highly purified form. The conventional purification process of eGFP usually consists of multiple subsequent preparative chromatography steps. Since this procedure is costly and time-consuming, an alternative chromatography-free purification process was investigated. This process was a combination of three-phase partitioning (TPP) and preparative crystallization including an ultrafiltration/diafiltration (UF/DF) intermediate step. After the TPP step, eGFP with a purity level suitable for preparative crystallization of 82.5–85.0% and a yield of 84–92% was obtained depending on the scale. After cross-flow UF/DF, the crystallization was performed in parallelized mL-scale stirred tanks. A favorable robust crystal morphology was obtained combined with fast crystallization kinetics when two polyethylenglycols and ethanol were used simultaneously as crystallization additives. The crystallization process can easily be scaled-up to obtain large amounts of highly purified, concentrated eGFP with a purity >99% after a crystal wash step and resolubilization. The proposed chromatography-free purification procedure gives reason to expect significant reductions of costs and required process time compared to conventional preparative chromatography.

Properties of heat-induced gels of caprine whey protein concentrates obtained from clarified cheese whey

Rheological properties, color and microstructure of heat-induced gels of caprine whey protein concentrates manufactured by means of clarification by thermocalcic precipitation followed by ultrafiltration–diafiltration were evaluated. These properties were compared with those of unclarified ultrafiltrated/diafiltrated whey protein concentrates and a commercial bovine whey protein concentrate. At pH 7, the clarified whey protein concentrate gels were significantly firmer than unclarified WPC gels. Values of L* of gels were also influenced by the clarification procedure. At pH 7, caprine whey protein concentrates were able to gel at lower protein concentration than the bovine whey protein concentrate. Color differences were also found between caprine and bovine WPC gels. All gels were formed by particulate networks; however, differences in the microstructural observations were found among caprine WPC and bovine WPC gels. According to their gelation properties, caprine WPC could be very useful in the food industry.

Processing and bread-making potential of proteins isolated from malted and non-malted pea seeds by ultrafiltration/diafiltration

Abstract The bread-making potential of a pea protein isolate produced by an ultrafiltration/diafiltration (UF/DF) process applied to malted seed has not been studied, to date. Thus, the objective of this work was to study the impact of supplementing breads, at a 10% level, with malted pea protein isolate produced by an UF/DF processes with a 50 kDa hollow fibers membrane. Malted pea protein isolate with approximately 90% of protein (dry basis) was obtained. Flour substituted with this isolate showed good bread-making potential allowing the production of bread with protein content over 20%, which was comparable to the protein content obtained for bread produced from non-malted pea protein isolate. The bread produced from malted pea protein isolate had a specific volume of 3.9±0.1 cm 3 g −1 which was slightly lower than for the one produced from non-malted pea protein isolate (4.4±0.1 cm 3 g −1 ) but comparable to the control bread produced from 100% refined bread flour (4.2±0.1 cm 3 g −1 ). Color ( L ⁎ , a ⁎ , b ⁎ ) of the crust and of the crumb of breads produced from UF/DF isolates was barely affected by the malting process but slightly differed from the color of bread produced from 100% refined bread flour.

Improved Solubility and Emulsification of Wet‐Milled Corn Germ Protein Recovered by Ultrafiltration–Diafiltration

AbstractThis study evaluated Ultrafiltration–Diafiltration (UFDF) as a means to improve the extractability of wet‐milled corn germ protein and determined its effects on the functional properties of the recovered protein product. Wet germ and dried germ proteins were extracted by using 0.1 M NaCl at 50 °C. Major steps in the method were stirring, centrifugation, UFDF and freeze‐drying. For dried germ, the UFDF method showed marginal increase in protein extraction efficiency, while that of wet germ remained similar to what we observed for the baseline method (saline extraction with dialysis instead of UFDF). UFDF protein extracts from both germ samples were significantly more soluble than extracts recovered by the baseline method and showed atypical solubility profiles; i.e., the amount of soluble protein was essentially unchanged under acidic, neutral, and alkaline pH. UFDF‐dried germ protein was markedly more soluble than UFDF‐wet germ protein (80 vs 50 % soluble protein, respectively) between pH 2 and pH 10. Both of these UFDF‐germ proteins also had emulsifying capacities and water‐holding capacities that were superior to those of proteins extracted by the baseline method, but this favorable effect by UFDF was not observed for emulsion stability, foaming properties, and heat stability.

Control of Protein Particle Formation During Ultrafiltration/Diafiltration Through Interfacial Protection

This study investigates the mechanism of protein particle formation during ultrafiltration/diafiltration (UF/DF), finding that agitation drives particle formation by promoting protein-interface adsorption and desorption. Low conductivity and the presence of surfactant reduced the level of particle formation in small-scale stirring studies, and the same trends were observed in pumping and UF/DF. Polysorbate 80 (PS80) and hydroxypropyl-β-cyclodextrin (HPβCD) reduced particle formation in UF/DF by factors of 15 and 4, respectively. Measurements of conformational stability, colloidal stability, and surface tension demonstrated that PS80 protects against particle formation by preventing protein-interface adsorption, low conductivity improves the colloidal stability of the protein, and the mechanism of action of HPβCD remains unclear. This work demonstrates that interfacial adsorption–desorption of the protein during UF/DF is the principal cause of particle formation, that the level of surfactant-free particle formation depends on the colloidal stability of the protein, and that the inclusion of surfactant greatly reduces in-process particle formation during UF/DF.

High-Purity Preparation of HSV-2 Vaccine Candidate ACAM529 Is Immunogenic and Efficacious In Vivo

Genital herpes is a sexually transmitted infection (STI) caused by herpes simplex virus 2 (HSV-2) and to a lesser extent herpes simplex virus 1 (HSV-1). Infection by HSV-2 is life-long and is associated with significant cost to healthcare systems and social stigma despite the highly prevalent nature of the disease. For instance, the proportion of HSV-2 seropositive to seronegative adults is approximately 1 in 5 in the US and greater than 4 in 5 in some areas of sub-Saharan Africa. The replication-defective vaccine strain virus dl5-29 was re-derived using cells appropriate for GMP manufacturing and renamed ACAM529. Immunization with dl5-29 was previously reported to be protective both in mice and in guinea pigs, however these studies were performed with vaccine that was purified using methods that cannot be scaled for manufacturing of clinical material. Here we describe methods which serve as a major step towards preparation of ACAM529 which may be suitable for testing in humans. ACAM529 can be harvested from infected cell culture of the trans-complementing cell line AV529 clone 19 (AV529-19) without mechanical cell disruption. ACAM529 may then be purified with respect to host cell DNA and proteins by a novel purification scheme, which includes a combination of endonuclease treatment, depth filtration, anion-exchange chromatography and ultrafiltration/diafiltration (UF/DF). The resultant virus retains infectivity and is ∼ 200-fold more pure with respect to host cell DNA and proteins than is ACAM529 purified by ultracentrifugation. Additionally, we describe a side-by-side comparison of chromatography-purified ACAM529 with sucrose cushion-purified ACAM529, which shows that both preparations are equally immunogenic and protective when tested in vivo.

Metal-Leachate-Induced Conjugate Protein Instability

During the scale-up of an ultrafiltration/diafiltration (UF/DF) step for a protein-based conjugate vaccine, significant precipitation was observed at room temperature. It was found that a specific type of metal hosebarb fitting used in the UF/DF system, when placed in the conjugate solution, caused the precipitation. Inductively Coupled Plasma Mass Spectrometry analysis showed significant amounts of Ni(II), Zn(II), and Cu(II) present in the conjugate solution. A kinetic study showed that the concentration of these metal ions gradually increased with increasing incubation time with a corresponding decrease in conjugate concentration. Direct spiking of trace amounts of NiCl₂, ZnCl₂, and CuCl₂ into the conjugate solution also caused precipitation, and spiking studies showed that the metal ions caused precipitation of the conjugate but not of the carrier protein, antigen, or carrier protein + linker. The precipitation was found to be significantly dependent on buffer species but not solution pH and led to an irreversible loss of tertiary structure even after dissolution in and removal of guanidine hydrochloride. The precipitation is likely the result of formation of transition-metal complexes with histidine residues on the antigen peptide, which may involve both intraconjugate and interconjugate antigens. Such complexation may lead to formation of multimers that may exceed the solubility limit.