Biomedical subjects

Raja Ghosh

Publications and source records attributed to Raja Ghosh.

13 recordsLinked to original sources

PubMed2006

Non-size-based membrane chromatographic separation and analysis of monoclonal antibody aggregates.

Humanized monoclonal antibody produced by mammalian cell culture may contain significant amounts of antibody dimers and smaller amounts of higher order aggregates. These are undesirable in therapeutic formulations, and their content should be lower than specific allowable limits. Quantitative analysis of aggregate content is usually carried out by size exclusion chromatography (SEC), which is slow and often gives poorly resolved peaks. We describe a novel hydrophobic interaction membrane chromatography-based technique for rapid, non-size-based separation and analysis of the aggregate content in monoclonal antibody samples. The typical sample analysis time using this technique is less than 3 min, this being significantly faster than SEC. The technique gives excellent resolution of the antibody, its dimer, and higher order aggregates and could potentially be scaled up for large-scale manufacture of aggregate-free monoclonal antibody. This work also clearly shows that monoclonal antibody aggregates are more hydrophobic than the monomer form, a fact that could have significant theoretical and practical implications.

Antibodies, Monoclonal↗

PubMed2006

Rapid antibody screening by membrane chromatographic immunoassay technique.

This paper describes a novel membrane chromatographic immunoassay technique suitable for rapid screening of antibodies in serum samples. This technique could potentially be utilized for antibody screening in situations where screening for exposure to one of several possible antigens is required. A synthetic microporous membrane is first selectively loaded with antibodies from the test serum sample by hydrophobic interaction. The in situ affinity membrane thus formed is sequentially pulsed with the antigens corresponding to the antibodies being screened. From the antigen chromatogram peak profile thus obtained, inferences about the antibodies present in the serum sample can then easily be made. This technique in addition to being rapid and direct is conceptually simple, and does not use any expensive media or reagents. It would potentially be useful for rapid diagnosis of infections as well as for rapid assessment of conditions such as envenomation or exposure to toxic substances.

Antibodies↗

PubMed2006

Purification of humanized monoclonal antibodies by membrane-based hybrid bioseparation technique.

We describe a rapid, inexpensive and scalable hybrid bioseparation technique for one-step purification of humanized monoclonal antibodies from mammalian cell culture supernatant. It involves the selective and reversible retention of the monoclonal antibody within a membrane module utilizing a combination of microfiltration of precipitated antibody and simultaneous hydrophobic interaction-based membrane chromatography of soluble antibody on the same microfiltration membrane. The retained monoclonal antibody is subsequently recovered from the membrane module in a highly pure form by changing the solution condition to that which favours simultaneous antibody dissolution and elution from the membrane. This combination of separation principles results in high-capacity, high-resolution separation. The monoclonal antibody purities and recoveries obtained in the experiments described in this paper were very high.

Animals↗

PubMed2006

Membrane chromatographic immunoassay method for rapid quantitative analysis of specific serum antibodies.

This paper discusses a membrane chromatographic immunoassay method for rapid detection and quantitative analysis of specific serum antibodies. A type of polyvinylidine fluoride (PVDF) microfiltration membrane was used in the method for its ability to reversibly and specifically bind IgG antibodies from antiserum samples by hydrophobic interaction. Using this form of selective antibody binding and enrichment an affinity membrane with antigen binding ability was obtained in-situ. This was done by passing a pulse of diluted antiserum sample through a stack of microporous PVDF membranes. The affinity membrane thus formed was challenged with a pulse of antigen solution and the amount of antigen bound was accurately determined using chromatographic methods. The antigen binding correlated well with the antibody loading on the membrane. This method is direct, rapid and accurate, does not involve any chemical reaction, and uses very few reagents. Moreover, the same membrane could be repeatedly used for sequential immunoassays on account of the reversible nature of the antibody binding. Proof of concept of this method is provided using human hemoglobin as model antigen and rabbit antiserum against human hemoglobin as the antibody source.

Animals↗

PubMed2006

Purification of humanized monoclonal antibody by hydrophobic interaction membrane chromatography.

Humanized monoclonal antibodies (mAbs) hold significant promise as biopharmaceuticals. One of the main challenges faced in the purification of mAbs is their separation from bovine serum albumin, which is the main protein present in most mammalian cell culture media. This paper discusses the purification of humanized mAb hIgG1-CD4 from CHO cell culture media by hydrophobic interaction membrane chromatography using a stack of microporous synthetic membranes. The effects of solution conditions on mAb solubility and binding on the membrane were first studied. The separation of a simulated mixture of bovine albumin and the mAb was then carried out to examine the feasibility of mAb purification. Separation experiments carried out under optimized conditions demonstrated that this membrane-based technique could be used for mAb purification from cell culture media. High purity (97%) and recovery (in excess of 97%) were obtained.

Animals↗

PubMed2005

Effects of protein-protein interaction in ultrafiltration based fractionation processes.

This paper discusses the use of pulsed sample injection ultrafiltration (UF) for investigating protein-protein interaction, particularly its effect on protein transmission through UF membranes. Several binary protein mixtures were investigated; the proteins in each mixture being selected such that one of the proteins in the pair would be preferentially transmitted while the other would be either totally or substantially retained. The "retained" protein either decreased or increased or did not affect the sieving coefficient of the "transmitted" protein, this depending the type of protein-protein interaction, that is, associative, repulsive, or neutral. The type of protein-protein interaction depended on the particular protein pair under investigation as well as on the operating conditions used (pH and salt concentration). The magnitude of either decrease or increase in transmission of a preferentially transmitted protein due to the presence of a retained protein was found to be independent of the manner in which the proteins were injected into the system, that is, simultaneous or sequential. These magnitudes however correlated well with the ratio of the two proteins present in the feed.

Chemical Fractionation↗

PubMed2005

Separation of monoclonal antibody alemtuzumab monomer and dimers using ultrafiltration.

This article examines the feasibility of using ultrafiltration to separate the monomer of the monoclonal antibody alemtuzumab (Campath or Campath-1H) from a mixture of dimer and higher-order oligomers (collectively called "dimers" here). Using parameter scanning ultrafiltration, we initially assessed the suitability of the following membranes: 100 kDa and 300 kDa polyethersulfone (PES) membranes, and a 100 kDa polyvinylidene fluoride (PVDF) membrane. A detailed study was then carried out to examine the effects of operating conditions (such as solution pH, ionic strength, stirring speed, and permeate flux) on the separation of the monomer from the dimers using 300 kDa PES and 100 kDa PVDF membranes. Results of the experiments carried out in the carrier phase ultrafiltration (CPUF) mode indicate that the size-based protein-protein separation critically depends on the membrane used as well as the system hydrodynamics. The separation of the monoclonal antibody monomer and dimers using 100 kDa PVDF membranes in the diafiltration mode was also examined. Experimental results demonstrate that under suitable conditions, it is feasible to obtain the alemtuzumab monomer with a purity of more than 93% and a yield of more than 85% (from a mixture of 75% monomer and 25% dimers, which is the typical composition obtained after affinity chromatography). Simulation study indicates that this could be further improved to a purity of more than 96% and a monomer yield of more than 96% by increasing the selectivity of separation or by employing a two-stage diafiltration process.

Alemtuzumab↗

PubMed2005

Fractionation of bovine serum albumin and monoclonal antibody alemtuzumab using carrier phase ultrafiltration.

Protein transmission and hence selectivity of separation can be significantly affected by solution pH and ionic strength in protein fractionation using ultrafiltration. Using parameter scanning ultrafiltration, the transmission of bovine serum albumin (BSA) and monoclonal antibody alemtuzumab (Campath-1H) through 300 kDa polyethersulfone (PES) ultrafiltration membranes were studied over a range of pH and salt concentrations, with focus on the likely conditions for achieving "reverse selectivity," i.e., obtaining purified alemtuzumab (approximately 155 kDa) in the permeate. Experimental results demonstrate that the reverse selectivity could be obtained by manipulating the operating conditions such as the solution pH, ionic strength, permeate flux, and system hydrodynamics. With a two-stage batch ultrafiltration process under suitable conditions, the monoclonal antibody alemtuzumab with a purity of > 98% was obtained in the permeate from a feed solution initially containing 0.50 g/l each of BSA and alemtuzumab. Further purity can be expected by selecting more suitable membranes and optimizing operating conditions.

Alemtuzumab↗

PubMed2004

Polarization-based compensation of astigmatism.

One approach to aberration compensation of an imaging system is to introduce a suitable phase mask at the aperture plane of an imaging system. We utilize this principle for the compensation of astigmatism. A suitable polarization mask used on the aperture plane together with a polarizer-retarder combination at the input of the imaging system provides the compensating polarization-induced phase steps at different quadrants of the apertures masked by different polarizers. The aberrant phase can be considerably compensated by the proper choice of a polarization mask and suitable selection of the polarization parameters involved. The results presented here bear out our theoretical expectation.

Astigmatism↗

PubMed2003

Possibility of an optical focal shift with polarization masks.

The polarization phase shift (PPS) has emerged as an important analytical tool in optical metrology. The present study utilizes the concept of controlling the polarization phase in applications such as focal shift and automatic focusing. When elliptically polarized light, in general, is incident upon a circularly symmetric polarization mask consisting of circular and annular zones with each zone having a unique linear polarizability, the polarization-phase difference introduced between the polarization-masked zones is also circularly symmetric. With the mask at the lens aperture, the polarization phase introduced is multiplicative with the lens function and is shown to result in a shift of the Gaussian focus plane. Because the polarization phase can be controlled by variation of the polarization parameters, the effective focal length of the imaging system can be varied within a small range. A study of the point-spread functions at the shifted focal planes has shown that the quality of the focal patch in these planes is comparable with that produced by a diffraction-limited imaging system at Gaussian focus. The shift of focus can be achieved by control of the polarization of the input beam. It is anticipated that this technique may find application in areas for which dynamic focusing within a small range is required.

Journal Article↗

PubMed2003

Parameter scanning ultrafiltration: rapid optimisation of protein separation.

High-resolution fractionation of proteins using ultrafiltration is feasible only at highly optimised conditions. Conventional process optimisation methodology demands both time and material. Pulsed sample injection ultrafiltration has been suggested as a rapid process optimisation technique. In the present work the scope of this technique is further extended by "parameter scanning ultrafiltration," which involves continuous change of a process parameter (e.g., pH, salt concentration). The time and material consumption are thus further reduced. The technique was validated using different proteins and membranes. Sieving coefficients at different pH and salt concentration were compared to those obtained in fixed parameter ultrafiltration experiments. As fractionation case studies the separation of monoclonal antibody from bovine serum albumin and separation of human IgG from human serum albumin were examined.

Alemtuzumab↗

PubMed2002

Protein separation using membrane chromatography: opportunities and challenges.

Some of the problems associated with packed bed chromatography can be overcome by using synthetic macroporous and microporous membranes as chromatographic media. This paper reviews the current state of development in the area of membrane chromatographic separation of proteins. The transport phenomenon of membrane chromatography is briefly discussed and work done in this area is reviewed. The various separation chemistries which have been utilised for protein separation, along with different applications, are also reviewed. The technical challenges facing membrane chromatography are highlighted and the scope for future work is discussed.

Chromatography, Liquid↗

PubMedDate not supplied

Separation of human serum albumin and human immunoglobulins using carrier phase ultrafiltration.

The fractionation of the plasma proteins human serum albumin (HSA) and human immunoglobulins (HIgG) using the combination of two newly developed techniques, pulsed sample injection technique and carrier phase ultrafiltration (CPUF), is discussed in this paper. The effects of pH and ionic strength on the transmission of a single protein (i.e., either HSA or HIgG) through 100 and 300 kDa MWCO polyethersulfone (PES) membranes were quantified using the pulsed sample injection technique. The experimental results thus obtained suggested that it would be possible to fractionate these proteins by optimizing the solution pH and ionic strength. With 100 and 300 kDa PES membranes, effective separation of HSA and HIgG was achieved by CPUF using suitable conditions, i.e., pH 4.7 and low salt concentration. The fractionation of HSA and HIgG by "reverse selectivity" using 300 kDa membranes was also examined.

Electrophoresis, Polyacrylamide Gel↗