Low pKa of Lys promotes glycation at one complementarity-determining region of a bispecific antibody.

Protein glycation is a common, normally innocuous, post-translational modification in therapeutic monoclonal antibodies. However, when glycation occurs on complementarity-determining regions (CDRs) of a therapeutic monoclonal antibody, its biological activities (e.g., potency) may be impacted. Here, we present a comprehensive approach to understanding the mechanism of protein glycation using a bispecific antibody. Cation exchange chromatography and liquid chromatography-mass spectrometry were used to characterize glycation at a lysine residue within a heavy chain (HC) CDR (HC-CDR3-Lys98) of a bispecific antibody. Thermodynamic analysis revealed that this reaction is reversible and can occur under physiological conditions with an apparent affinity of 8-10 mM for a glucose binding to HC-CDR3-Lys98. Results from kinetic analysis demonstrated that this reaction follows Arrhenius behavior in the temperature range of 5°C-45°C and can be well predicted in vitro and in a non-human primate. In addition, this glycation reaction was found to be driven by an unusually low pKa on the ε-amino group of HC-CDR3-Lys98. Van't Hoff analysis and homology modeling suggested that this reaction is enthalpically driven, with this lysine residue surrounded by a microenvironment with low polarity. This study provides, to our knowledge, new insights toward a mechanistic understanding of protein glycation and strategies to mitigate the impact of protein glycation during pharmaceutical development.

Container Closure and Delivery Considerations for Intravitreal Drug Administration.

Intravitreal (IVT) administration of therapeutics is the standard of care for treatment of back-of-eye disorders. Although a common procedure performed by retinal specialists, IVT administration is associated with unique challenges related to drug product, device and the procedure, which may result in adverse events. Container closure configuration plays a crucial role in maintaining product stability, safety, and efficacy for the intended shelf-life. Careful design of primary container configuration is also important to accurately deliver small volumes (10-100 µL). Over- or under-dosing may lead to undesired adverse events or lack of efficacy resulting in unpredictable and variable clinical responses. IVT drug products have been traditionally presented in glass vials. However, pre-filled syringes offer a more convenient administration option by reducing the number of steps required for dose preparation there by potentially reducing the time demand on the healthcare providers. In addition to primary container selection, product development studies should focus on, among other things, primary container component characterization, material compatibility with the formulation, formulation stability, fill volume determination, extractables/leachables, and terminal sterilization. Ancillary components such as disposable syringes and needles must be carefully selected, and a detailed administration procedure that includes dosing instructions is required to ensure successful administration of the product. Despite significant efforts in improving the drug product and administration procedures, ocular safety concerns such as endophthalmitis, increased intraocular pressure, and presence of silicone floaters have been reported. A systematic review of available literature on container closure and devices for IVT administration can help guide successful product development.

Identification of Low Abundant Isomeric N-Glycan Structures in Biological Therapeutics by LC/MS.

An effective LC-MS based method for online characterization of low abundant structural isomers of N-linked glycans in biological therapeutics was developed. N-linked glycans of a recombinant monoclonal antibody were released by PNGase F and labeled with 2-aminobenzamide (2-AB) fluorescent tag. The labeled glycans were analyzed by online ultraperformance liquid chromatography-hydrophilic interaction liquid chromatography (UPLC-HILIC) coupled with mass spectrometry (MS). The glycan structure was characterized by MS(n) fragmentation in negative ion mode followed by identification of the signature D ions. The assignment included monosaccharide sequence and linkage information. The developed method successfully characterized structural isomers of A1G1F (assigned as terminal sialic acid attached in the 1,6 branch at 2,3 position), and A1G1F' (assigned as terminal sialic acid attached in the 1,3 branch at 2,3 position). Moreover, using the same approach, previously unknown low abundant species were identified unambiguously. One such structural isomer at low level, terminal GlcNAc of G1F+GlcNAc, was identified to be linked at the 1,6 branch. Additionally, another low level structural isomer, previously assigned as Man8 glycan, was found to be Man7+Glc glycan as its 1,3 branch containing three mannoses and one terminal glucose. The identification was further confirmed by a purified α-1,2-endomannosidase enzyme to generate the cleavage of α-1,3 linked terminal disaccharides (Man+glucose). Using this approach, different lots or different CHO produced mAbs was thoroughly examined and found that the newly identified "Man8" (Man7+Glc) was also present in different batches and in some commercially available therapeutic mAbs.

Development of a sample preparation method for monitoring correct disulfide linkages of monoclonal antibodies by liquid chromatography-mass spectrometry.

The number and positions of disulfide linkages in a therapeutic monoclonal antibody (mAb) play a crucial role in forming and stabilizing a correct mAb structure that is critical to its function. Peptide mapping by liquid chromatography-mass spectrometry (LC-MS) analysis of enzymatically digested mAb under nonreducing condition is a powerful method for disulfide linkage characterization to ensure mAb drug function and quality. However, the development of a robust sample preparation method with improved digestion efficiency and minimized disulfide scrambling for disulfide linkage analysis is essential but challenging. In this study, a sample preparation method for analysis of correct disulfide linkages in therapeutic mAbs was developed. Instead of common trypsin digestion, Lys-C plus trypsin was used in this approach to improve digestion efficiency. In addition, lower digestion temperature (25 °C) and lower digestion pH (pH 6.8) were also examined to minimize disulfide scrambling. Our results showed that Lys-C plus trypsin digestion at pH 6.8 and 25 °C is a better sample preparation condition for all therapeutic mAbs tested in this study because of a better digestion efficiency (all expected disulfide linkages can be confidently observed) and minimal disulfide scrambling.

Characterization of drug-product-related impurities and variants of a therapeutic monoclonal antibody by higher energy C-trap dissociation mass spectrometry.

Mass spectrometry (MS) characterization of recombinant monoclonal antibody (mAb) drugs and their degraded and/or post-translationally modified counterparts, drug-product-related impurities and variants, is critical for successful development of biotherapeutics. Specifically in this study, drug-product-related impurities of an anti-Clostridium difficile IgG1 mAb drug substance were profiled by cation-exchange liquid chromatography (CEX) followed by the CEX peaks being fraction-collected for MS characterization. A reversed-phase liquid chromatography/mass spectrometry (LC/MS) methodology was developed on a Thermo Q-Exactive orbitrap mass spectrometer for (1) accurate mass measurements of the mAb, its CEX fractionated impurities, and their respective heavy chains and light chains and (2) middle-down LC/MS/MS of the light chains and the heavy chains using higher energy C-trap dissociation (HCD). The accurate mass measurements and the HCD middle-down MS/MS experiments identify that major impurities and variants of the anti-C. difficile mAb are degradation species of the heavy chains at residue Asn101 as well as at the hinge region amino acids, including Cys222, Lys224, His226, and Thr227, with levels ranging from 0.3% to 6.2% of the total drug substance. Additional impurities were identified as light chain C-terminal truncation at Gly93 and oxidized heavy chains at Met40, Met93, and Met430. Our impurity characterization results demonstrate that the middle-down MS method allows direct and accurate identification of drug-product-related impurities of therapeutic mAbs. It is particularly useful for those low-level impurities and variants that are not suitable for further fractionation and characterization by bottom-up MS.

Analysis and purification of IgG4 bispecific antibodies by a mixed-mode chromatography.

Therapeutic non-hinge-modified IgG4 molecules form bispecific hybrid antibodies with endogenous human IgG4 molecules via a process known as Fab-arm exchange (or called half molecule exchange). Analysis of the bispecific hybrids is critical for studies of half molecule exchange. A number of analytical methods are available to detect IgG4 hybrids. These methods mostly necessitate labeling or alteration of the model IgG4 molecules, or rely on time-consuming immunoassays and mass spectrometry. In addition, these methods do not allow isolation of hybrid antibodies. We report here the only analytical method to date that relies on chromatographic separation for detection of hybrids formed from intact antibodies in their native forms using pembrolizumab as an example. This method employs a mixed-mode chromatography using a Sepax Zenix SEC-300 column to separate a bispecific hybrid from the parental antibodies. The simultaneous quantitative monitoring of the newly formed hybrid and parental antibodies was achieved by UV absorption and/or protein fluorescence. The bispecific hybrid antibodies were purified with the same method for further biochemical characterization. The method has allowed monitoring of half molecule exchange between a human serum IgG4 and a tested IgG4 molecule, and has been implemented for the analysis of in vitro as well as in vivo samples.

Effect of pegylation on self-association of IFN-α2b.

Pegylation of therapeutic proteins is an established technology used to enhance the bioavailability of an active pharmaceutical ingredient in the body of patients. While the physiochemical properties of pegylated monomeric proteins have been extensively described, there is still limited information on the characterization of pegylated oligomeric proteins. In this study, we report the characterization of a pegylated interferon alpha2b (PEGIFN-α2b) concentration-dependent oligomerization by a series of orthogonal biochemical and biophysical methods. These methods include sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation, matrix-assisted laser desorption ionization, and size exclusion chromatography of bissulfosuccinimidyl suberate cross-linked PEGIFN. We report here that PEGIFN-α2b self-associates in a concentration-dependent manner into mainly monomers, dimers, and trimers. In the presence of the chemical cross-linker, PEGIFN-α2b is primarily monomeric (57%) at concentration lower than 0.3 mg/mL and contains about equal amount of monomers and dimers (47.0% and 37.7%, respectively), about 15% of trimers, and up to 4% of higher molecular weight species at 0.7 mg/mL and above.

Antibody association in solution: cluster distributions and mechanisms.

Understanding factors that affect the clustering and association of antibodies molecules in solution is critical to their development as therapeutics. For 19 different monoclonal antibody (mAb) solutions, we measured the viscosities, the second virial coefficients, the Kirkwood-Buff integrals, and the cluster distributions of the antibody molecules as functions of protein concentration. Solutions were modeled using the statistical-physics Wertheim liquid-solution theory, representing antibodies as Y-shaped molecular structures of seven beads each. We found that high-viscosity solutions result from more antibody molecules per cluster. Multi-body properties such as viscosity are well predicted experimentally by the 2-body Kirkwood-Buff quantity, G22, but not by the second virial coefficient, B22, and well-predicted theoretically from the Wertheim protein-protein sticking energy. Weakly interacting antibodies are rate-limited by nucleation; strongly interacting ones by propagation. This approach gives a way to relate micro to macro properties of solutions of associating proteins.

Water loss from silicone tubing and effect on protein concentration during drug product manufacturing.

Silicone tubing is used in various unit operations during drug product (DP) manufacturing. Hold of protein formulations in silicone tubing over time may have an impact on product quality, particularly protein concentration. This study evaluated the change in protein concentration of a test monoclonal antibody (mAb) formulation over various hold times in silicone tubing as a function of tubing internal diameter (ID) and wall thickness. It was hypothesized that the rate of water diffusion through the semi-permeable membrane is a function of the tubing ID and wall thickness. The weight and protein concentration of various formulation-filled tubings over time was measured. The weight of water lost varied linearly with the change in protein concentration. It was observed to be independent of mAb type, formulation composition, and initial protein concentration for a given tubing ID and wall thickness. The effect of formulation water activity on the water loss rate was investigated. A mechanistic diffusion-based model was developed that predicts the change in tubing weight and therefore protein concentration over various hold times for a given formulation and tubing. Overall, this study suggests that water loss from silicone tubing affects protein concentration and should be monitored during DP process development and manufacturing.

Evaluation of the In-Use Stability of Monoclonal Antibody IV Admixtures Prepared from Drug Products Containing Polysorbate 20 Degraded by Host-Cell Lipases.

Host-cell lipases can be present in monoclonal antibody drug products and can degrade polysorbates present in the formulations as stabilizers. We hypothesized that the in-use stability of the IV admixture prepared from such a drug product might be impacted by decreasing levels of polysorbate 20. Host-cell lipase activity has, in fact, been observed during development of one of our therapeutic monoclonal antibody drug products. Throughout the course of the product shelf life, polysorbate 20 levels decreased but no other quality attributes of the drug product were impacted. An experimental approach was developed to simulate how the prepared IV admixture in-use stability is affected as polysorbate 20 concentration in the drug product decreased over the shelf life, and from that a minimum level of polysorbate 20 required in the drug product was determined to estimate the in-use stability of the IV admixture as the polysorbate 20 in the drug product degrades. The results indicate that although the observed degradation of polysorbate 20 does not affect quality attributes of this drug product, in-use stability of the IV admixture as a function of polysorbate degradation can be impacted and should be assessed to ensure sufficient quality.

Vaporized Hydrogen Peroxide Sterilization in the Production of Protein Therapeutics: Uptake and Effects on Product Quality.

The aseptic filling of drug products is carried out in pharmaceutical isolators that have been sterilized. A commonly used method for achieving a high level of sterility assurance is vaporized hydrogen peroxide (VHP) sterilization, which is favorable to other methods, such as ethylene oxide sterilization, due to its low cycle times and nontoxic residuals. While VHP cycles are often employed to create a sterile environment within an isolator, they can leave residual levels of hydrogen peroxide behind that can enter the product during fill-finish operations. Due to the oxidizing potential of hydrogen peroxide and the multiple possible sources of uptake along filling lines, the extent of the potential impact on product quality needs to be understood during pharmaceutical development. Herein, different factors affecting hydrogen peroxide uptake, points of entry along the filling line, and possible impacts on product quality are reviewed.

Synthesis of waste limestone powder-based alkali-activated binder: experimental, optimization modeling, and eco-efficiency assessment.

More than half of the CO2 emissions during the manufacturing of ordinary Portland cement (OPC) occur due to the calcination of calcium carbonate in addition to burning of fossil fuel to power the process. Consequently, there is a concerted effort to decrease the carbon footprint associated with this process, by minimizing the use of OPC. In line with this trend, an attempt was made in the reported study to synthesize a novel alkali-activated binder using CaCO3-rich waste limestone powder (WLSP) as a precursor. Utilizing the Taguchi method, four important parameters were varied at three levels to optimize the alkali-activated mixture. Analysis of variance (ANOVA) of the obtained results was performed to assess the impact of each of the factors on the properties of the developed binder. To enhance the strength further, OPC was added as a partial replacement of WLSP. The binder was characterized using scanning electron microscopy. The results have indicated that alkaline activator to binder ratio, Na2SiO3 to NaOH ratio, and sand to binder ratio of 0.575, 1.57, and 2.5, respectively, were the optimum to obtain satisfactory strength and workability with a 13.7-M NaOH activator solution. The incorporation of a small quantity of OPC in the mixture remarkably improved the density and strength of the alkali-activated-WLSP binder. Pirssonite (CaCO3.Na2CO3.2H2O) and C/N-A-S-H were the dominant mineral phases formed in the developed binder, particularly in the ones alkali-activated WLSP/OPC. In addition, the eco-efficiency assessment revealed that the WLSP is a promising low-carbon binder that can be used in developing more sustainable alkali-activated binder. The results have shown that the WLSP can be potentially utilized in developing binder that can be potentially used in the structural applications.

Prediction of long-term polysorbate degradation according to short-term degradation kinetics.

Polysorbates (PSs) are a class of surfactants commonly used in the formulation of protein therapeutic agents to provide protection against denaturation and aggregation. When the PS in these drug formulations degrades, loss of stabilization of the protein therapeutic and formulation may occur, resulting in particulate formation or other undesirable changes in product critical quality attributes. Here, we present a simplified platform to predict long-term PS20 and PS80 degradation for monoclonal antibody drugs containing the PS-degrading enzyme lysosomal acid lipase. The platform was based on a temperature-dependent equation derived from existing PS20 degradation stability data. Accurate prediction of both PS20 and PS80 hydrolysis for as long as 2 years was achieved through short-term kinetics studies performed within 2 weeks. This platform substantially shortens the time required to determine the long-term stability of PS degradation and therefore can be used to guide the purification process and optimization of antibody formulations.

Continuous monitoring of a monoclonal antibody by size exclusion chromatography reveals a correlation between system suitability parameters and column aging.

Size exclusion chromatography (SEC) is a foundational analytical method to assess product purity of biological molecules. To ensure accurate and reproducible data that meet regulatory agency standards, it is critical to monitor the chromatographic column with efficient and continuous approaches. In this study, 19 SEC columns (Waters Acquity BEH200) were evaluated using an in-house monoclonal antibody made at Regeneron. System suitability parameters (SSPs) were used to monitor the performance of the SEC assay, including USP resolution, USP plate count, USP tailing factor, asymmetry factor, elution time, peak width, and peak height. A general linear model was built and revealed that elution time, peak width, asymmetry factor, and tailing factor increased with injection number, while peak height, resolution, and plate count decreased. After 1000 injections, tailing factor and peak width increased by more than 10%, while resolution and plate count decreased by more than 10% from their respective starting values.

Characterization of Microbial Growth Potential in Antibody Drug IV Admixtures by Microbial Challenge.

Monoclonal antibody (mAb) drug products (DP) for IV administration are commonly diluted in a diluent such 0.9% sodium chloride (saline) or 5% dextrose (D5W) injection yielding IV admixtures before infusion or injection. During dose preparation, storage, and administration, the sterility of IV admixtures must be maintained to ensure patient safety. However, the introduction of adventitious microorganisms may occur during dose preparation, and microbial proliferation may take place during IV admixture storage. Sterility testing of IV admixtures prior to administration is not feasible in clinic due to its destructive nature. Instead, microbial growth potential assessment could be performed to ensure patient safety. To assess microbial growth potential of IV admixtures, microbial challenge studies, which evaluate the ability of IV admixtures supporting or not supporting microorganism proliferation, are often recommended. Since the initial introduction of microbial challenge studies 2009, there has been very limited data published on microbial challenge studies for IV admixtures. In this publication, data from independent microbial challenge studies for IV admixtures prepared from 10 monoclonal antibodies (mAb) were generated, pooled, and analyzed together for microbial growth trends. The results indicated that major factors impacting the microbial growth in mAb IV admixtures include temperature and time as well as protein and excipient concentration. No microbial growth was observed for IV admixtures stored at 2-8 °C for up to 14 days. At room temperature, no microbial growth was observed for 12 h in IV admixture with protein concentration ≤32 mg/mL. Growth of E. coli, P. aeruginosa, and K. pneumoniae are commonly observed in IV admixtures stored for 16-48 h at room temperature. The study results provided input for designing effective challenge studies to maximize IV admixtures in-use time as well as for potential regulatory guidance development to facilitate the drug development while ensuring patient safety.

Lyophilization as an effective tool to develop AAV8 gene therapy products for refrigerated storage.

Recombinant adeno-associated virus (rAAV) has emerged as the leading gene delivery platform for treatment of monogenic disorders. Currently, for clinical and commercial products, rAAVs are typically formulated and stored below -65 °C as frozen liquid. Their long-term storage is often far from ideal because it may result in shorter drug product (DP) shelf-life compared to recombinant protein-based biologics, and also presents challenges for supply chain and inventory management. Consequently, there is great interest in developing robust lyophilized AAV DPs that are stable at 2 to 8 °C. In this study, we evaluated formulation excipients required for stable lyophilized AAV8 products including buffers, salts, cryoprotectants/lyoprotectants, surfactants, and bulking agents, and optimized the concentrations and ratios between the excipients. This led to the identification of the lead formulation that demonstrated short-term in-solution stability at 25 °C and, upon lyophilization, sufficient long-term stability at 2 to 8 °C. Our study demonstrated that, in the presence of 110 mM salts, mannitol can serve as an effective bulking agent with the appropriate formulation and lyophilization process design, and the sucrose to mannitol ratio is critical to maintain the stability and cake appearance of the lyophilized AAV8 DP. Thorough characterization of the effect of formulation components on the properties and quality of the lyophilized DP led to an optimized AAV8 lyophilized DP. This approach could be applied to streamline the future development of lyophilized AAV gene therapy products with various target transgenes and capsid serotypes.

Solution pH that minimizes self-association of three monoclonal antibodies is strongly dependent on ionic strength.

Monoclonal antibodies display highly variable solution properties such as solubility and viscosity at elevated concentrations (>50 mg/mL), which complicates antibody formulation and delivery. To understand this complex behavior, it is critical to measure the underlying protein self-interactions that govern the solution properties of antibody suspensions. We have evaluated the pH-dependent self-association behavior of three monoclonal antibodies using self-interaction chromatography for a range of pH values commonly used in antibody formulations (pH 4.4-6). At low ionic strength (<25 mM), we find that each antibody is more associative at near-neutral pH (pH 6) than at low pH (pH 4.4). At high ionic strength (>100 mM), we observe the opposite pH-dependent pattern of antibody self-association. Importantly, this inversion in self-association behavior is not unique to multidomain antibodies, as similar pH-dependent behavior is observed for some small globular proteins (e.g., ribonuclease A and α-chymotrypsinogen). We also find that the opalescence of concentrated antibody solutions (90 mg/mL) is minimized at low ionic strength at pH 4.4 and high ionic strength at pH 6, in agreement with the self-interaction measurements conducted at low antibody concentrations (5 mg/mL). Our results highlight the complexity of antibody self-association and emphasize the need for systematic approaches to optimize the solution properties of concentrated antibody formulations.

Genome DNA leakage of Adeno-Associated virus under freeze-thaw stress.

Adeno-associated virus (AAV) has become an emerging tool for human gene therapies. Currently, AAV gene therapies are subjected to multiple freeze-thaw cycles during manufacturing, storage, transportation, and administration. While studies have shown that multiple freeze-thaw cycles led to a decrease in transduction efficiency, the AAV degradation mechanism during freeze-thaw is not well understood. Here, we have characterized the impact of freeze-thaw on AAV8 by employing a variety of assays, which revealed significant increases in the amount of free single-stranded DNA (ssDNA) in AAV8 formulations after multiple freeze-thaw cycles. Subsequent analysis using Next Generation Sequencing (NGS) revealed that the ssDNA primarily consisted of genome DNA, indicating that the increased ssDNA leaked out from AAV8. Experiments performed using different serotypes of AAV confirmed the pervasiveness of such behavior amongst AAVs. In addition, formulation screening studies were performed to understand the impact on genome DNA leakage from AAV. The formulation screening results showed that the addition of 10% sucrose and 0.1% poloxamer 188 to Dulbecco's phosphate-buffered saline (DPBS) reduced the leakage of ssDNA in AAV samples after freeze-thaw cycles compared to the base formulation of DPBS alone. These findings shed new light on the degradation mechanism of AAVs and stabilization of the AAV-based gene therapies.

Deciphering the High Viscosity of a Therapeutic Monoclonal Antibody in High Concentration Formulations by Microdialysis-Hydrogen/Deuterium Exchange Mass Spectrometry.

High concentration formulations of therapeutic monoclonal antibodies (mAbs) are highly desired for subcutaneous injection. However, high concentration formulations can exhibit unusual molecular behaviors, such as high viscosity or aggregation, that present challenges for manufacturing and administration. To understand the molecular mechanism of the high viscosity exhibited by high concentration protein formulations, we analyzed a human IgG4 (mAb1) at high protein concentrations using sedimentation velocity analytical ultracentrifugation (SV-AUC), X-ray crystallography, hydrogen/deuterium exchange mass spectrometry (HDX-MS), and protein surface patches analysis. Particularly, we developed a microdialysis HDX-MS method to determine intermolecular interactions at different protein concentrations. SV-AUC revealed that mAb1 displayed a propensity for self-association of Fab-Fab, Fab-Fc, and Fc-Fc. mAb1 crystal structure and HDX-MS results demonstrated self-association between complementarity-determining regions (CDRs) and Fc through electrostatic interactions. HDX-MS also indicated Fab-Fab interactions through hydrophobic surface patches constructed by mAb1 CDRs. Our multi-method approach, including fast screening of SV-AUC as well as interface analysis by X-ray crystallography and HDX-MS, helped to elucidate the high viscosity of mAb1 at high concentrations as induced by self-associations of Fab-Fc and Fab-Fab.

Evaluation of role of interferon gamma release assays in the diagnosis of latent tuberculosis in human immunodeficiency virus-infected patients.

INTRODUCTION: Tuberculosis (TB) is the most common opportunistic infection in human immunodeficiency virus (HIV)-infected individuals. The risk of eventually developing active TB from latent TB infection (LTBI) is about 10% per year in HIV-positive patients in contrast to 10% lifetime risk in HIV-negative patients. Until recently, the tuberculin skin test (TST) was the only tool available for diagnosing LTBI. Interferon-gamma release assays (IGRAs) were recently developed and address many of the limitations of TST test, especially in immunocompromised state. AIMS AND OBJECTIVES: (1) To determine the prevalence of latent, active pulmonary, and multidrug-resistant (MDR)-TB among HIV-positive patients in and around Aligarh region; (2) sensitivity and specificity of TST and IGRAs for diagnosis of LTBI in HIV positive patients; and (3) to assess drug resistance and mutational patterns of the clinical isolates of MDR-TB in HIV-TB co-infection. MATERIALS AND METHODS: A cross-sectional study was done on all the patients attended the ICTC centre, JNMC, AMU Aligarh, seropositive for HIV, i.e. 469 (sample size) for the study period of 2 years from October 2015 to October 2017. All 469 HIV-positive patients were screened for latent and active pulmonary TB. Diagnosis of TB (active and latent) was made using clinical, radiological, and microbiological tests. TST and IGRA testing along with CD4 cell counts were also determined. Line probe assay was also done to assess drug resistance and mutational patterns of MDR-TB in HIV patients. RESULTS: In our study, prevalence of HIV infection was 5.04%. Sixty-seven (14.28%) patients were as active TB (HIV-TB co-infection), out of which only one patient (1.49%) was confirmed as MDR-TB, 117 (24.94%) were diagnosed as LTBI. It was also evaluated that IGRA has more sensitivity (75%) and specificity (76%) than TST with sensitivity of 71.7% and specificity 66%. CONCLUSION: As there is no gold standard test for latent TB, longitudinal follow-up is needed to interpret discordant test results. There is a need to interpret negative QFT results with caution and to test for latent TB at higher CD4 counts, if possible. Interferon gamma assays can become better tool for diagnosis of especially for latent TB. However, more research study required for establish their relevance, especially in immunocompromised states.