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Article ; Online: Convolutional Neural Networks Guided Raman Spectroscopy as a Process Analytical Technology (PAT) Tool for Monitoring and Simultaneous Prediction of Monoclonal Antibody Charge Variants.

Nitika, Nitika / Keerthiveena, B / Thakur, Garima / Rathore, Anurag S

2024 Volume 41, Issue 3, Page(s) 463–479

Abstract: Background: Charge related heterogeneities of monoclonal antibody (mAb) based therapeutic products are increasingly being considered as a critical quality attribute (CQA). They are typically estimated using analytical cation exchange chromatography (CEX) ...

Abstract	Background: Charge related heterogeneities of monoclonal antibody (mAb) based therapeutic products are increasingly being considered as a critical quality attribute (CQA). They are typically estimated using analytical cation exchange chromatography (CEX), which is time consuming and not suitable for real time control. Raman spectroscopy coupled with artificial intelligence (AI) tools offers an opportunity for real time monitoring and control of charge variants. Objective: We present a process analytical technology (PAT) tool for on-line and real-time charge variant determination during process scale CEX based on Raman spectroscopy employing machine learning techniques. Method: Raman spectra are collected from a reference library of samples with distribution of acidic, main, and basic species from 0-100% in a mAb concentration range of 0-20 g/L generated from process-scale CEX. The performance of different machine learning techniques for spectral processing is compared for predicting different charge variant species. Result: A convolutional neural network (CNN) based model was successfully calibrated for quantification of acidic species, main species, basic species, and total protein concentration with R Conclusion: We demonstrate that Raman spectroscopy combined with AI-ML frameworks can deliver rapid and accurate determination of product related impurities. This approach can be used for real time CEX pooling decisions in mAb production processes, thus enabling consistent charge variant profiles to be achieved.
MeSH term(s)	Antibodies, Monoclonal/chemistry ; Spectrum Analysis, Raman/methods ; Artificial Intelligence ; Technology ; Neural Networks, Computer
Chemical Substances	Antibodies, Monoclonal
Language	English
Publishing date	2024-02-16
Publishing country	United States
Document type	Journal Article
ZDB-ID	843063-9
ISSN	1573-904X ; 0724-8741 ; 0739-0742
ISSN (online)	1573-904X
ISSN	0724-8741 ; 0739-0742
DOI	10.1007/s11095-024-03663-9
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Article: Association studies in linseed (Linum usitatissimum L.) under different production systems over locations

Thakur, Garima / Paul, Satish

Electronic journal of plant breeding. 2022 June, v. 13, no. 2

2022

Abstract: The present experiment was conducted to determine the important yield components and their association with one another and with yield. The experiment was laid out under two production systems over four different locations in the mid-hills of the North- ... ...

Abstract	The present experiment was conducted to determine the important yield components and their association with one another and with yield. The experiment was laid out under two production systems over four different locations in the mid-hills of the North-western Himalayas. Correlation studies indicated a strong positive correlation for seed yield with harvest index (EI 0.668; EII 0.472) and biological yield (EI 0.362; EII 0.532) in both the production systems. Whereas, a non-significant positive correlation was observed for the number of primary branches (EI 0.103; EII 0.122) and the number of secondary branches (EI 0.033; EII 0.160) in both production systems. Whereas, among other traits highly significant correlation was observed for days to 50% flowering with days to 75% maturity in both production systems (EI 0.837; EII 0.781) followed by plant height (EI- 0.626; EII- 0.562) and technical plant height (EI 0.617; EII 0.598). Path coefficient analysis indicated that only biological yield (EI 0.70348; EII 0.60597) and harvest index (EI 0.90210; EII 0.53283) showed a highly significant direct effect with seed yield in both production systems. This showed that selection for biological yield and harvest index would have a direct positive effect in the improvement of grain yield and are the most important determinants of yield in linseed. However, traits such as number of primary branches, number of secondary branches and 1000 seed weight should also be given significant importance since these traits also showed a positive correlation with seed yield but with indirect effects via biological yield and harvest index in EI and EII, respectively.
Keywords	Linum usitatissimum ; grain yield ; harvest index ; linseed ; plant height ; seed weight ; seed yield ; Himalayan region
Language	English
Dates of publication	2022-06
Size	p. 731-738.
Publishing place	Indian Society of Plant Breeders
Document type	Article
ZDB-ID	2534084-0
ISSN	0975-928X
ISSN	0975-928X
DOI	10.37992/2022.1302.100
Database	NAL-Catalogue (AGRICOLA)

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Article: A Rare Case of Pulmonary Cavitary Disease Caused by Mycobacterium xenopi.

Dogra, Megha / Thakur, Manish / Thakur, Garima / Kumar, Amrat

Cureus

2023 Volume 15, Issue 2, Page(s) e34561

Abstract: Mycobacterium xenopi is a slow-growing, acid-fast, non-tuberculous mycobacterium (NTM). It is often considered to be a saprophyte or an environmental contaminant. Mycobacterium xenopi has low pathogenicity and is usually seen in patients with pre- ... ...

Abstract	Mycobacterium xenopi is a slow-growing, acid-fast, non-tuberculous mycobacterium (NTM). It is often considered to be a saprophyte or an environmental contaminant. Mycobacterium xenopi has low pathogenicity and is usually seen in patients with pre-existing chronic lung diseases and immunocompromised patients. We present a case of Mycobacterium xenopi causing a cavitary lesion in a patient with chronic obstructive pulmonary disease (COPD) that was discovered incidentally during the low-dose CT scan done for lung cancer screening in a patient with COPD. The initial workup was negative for NTM. An Interventional-guided (IR) core needle biopsy was done given the high suspicion for NTM and revealed a positive culture for Mycobacterium xenopi. Our case highlights the importance of considering NTM in the differential diagnosis of at-risk patients and pursuing invasive testing if there is a high clinical suspicion.
Language	English
Publishing date	2023-02-02
Publishing country	United States
Document type	Case Reports
ZDB-ID	2747273-5
ISSN	2168-8184
ISSN	2168-8184
DOI	10.7759/cureus.34561
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Article: Tick-Borne Rhabdomyolysis: A Rare Case of Rhabdomyolysis and Acute Kidney Injury Due to Anaplasmosis.

Dogra, Megha / Thakur, Manish / Kumar, Amrat / Thakur, Garima

Cureus

2023 Volume 15, Issue 2, Page(s) e34835

Abstract: Anaplasmosis is a tick-borne illness commonly seen in the northeastern states of the United States. The most common presenting signs are fever, malaise, and body aches accompanied by leukopenia, thrombocytopenia, and transaminitis. Rhabdomyolysis and ... ...

Abstract	Anaplasmosis is a tick-borne illness commonly seen in the northeastern states of the United States. The most common presenting signs are fever, malaise, and body aches accompanied by leukopenia, thrombocytopenia, and transaminitis. Rhabdomyolysis and acute kidney injury are rare presentations that can lead to significant morbidity. We present the case of a patient who presented with non-specific symptoms of malaise, fatigue, and body aches and was found to have rhabdomyolysis and acute kidney injury on laboratory workup. A presumptive diagnosis of anaplasmosis was made, and the patient was started on treatment for the same. The patient recovered successfully. Our case highlights the rare presentation of anaplasmosis with rhabdomyolysis and acute kidney injury. Physician awareness is needed for early diagnosis and preventing morbidity.
Language	English
Publishing date	2023-02-10
Publishing country	United States
Document type	Case Reports
ZDB-ID	2747273-5
ISSN	2168-8184
ISSN	2168-8184
DOI	10.7759/cureus.34835
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Article ; Online: FRET-GP - A Local Measure of the Impact of Transmembrane Peptide on Lipids.

Thakur, Garima C N / Uday, Arunima / Jurkiewicz, Piotr

Langmuir : the ACS journal of surfaces and colloids

2023 Volume 39, Issue 50, Page(s) 18390–18402

Abstract: Reconstitution of a transmembrane protein in model lipid systems allows studying its structure and dynamics in isolation from the complexity of the natural environment. This approach also provides a well-defined environment for studying the interactions ... ...

Abstract	Reconstitution of a transmembrane protein in model lipid systems allows studying its structure and dynamics in isolation from the complexity of the natural environment. This approach also provides a well-defined environment for studying the interactions of proteins with lipids. In this work, we describe the FRET-GP method, which utilizes Förster resonance energy transfer (FRET) to specifically probe the nanoenvironment of a transmembrane domain. The tryptophan residues flanking this domain act as efficient FRET donors, while Laurdan acts as acceptor. The fluorescence of this solvatochromic probe is quantified using generalized polarization (GP) to report on lipid mobility in the vicinity of the transmembrane domain. We applied FRET-GP to study the transmembrane peptide WALP incorporated in liposomes. We found that the direct excitation of Laurdan to its second singlet state strongly contributes to GP values measured in FRET conditions. Removal of this parasitic contribution was essential for proper determination of GP
MeSH term(s)	Fluorescence Resonance Energy Transfer ; Laurates ; Liposomes/chemistry ; Peptides ; Fluorescent Dyes
Chemical Substances	laurdan (Y97FBL93VW) ; Laurates ; Liposomes ; Peptides ; Fluorescent Dyes
Language	English
Publishing date	2023-12-04
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2005937-1
ISSN	1520-5827 ; 0743-7463
ISSN (online)	1520-5827
ISSN	0743-7463
DOI	10.1021/acs.langmuir.3c02505
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Article ; Online: Continuous manufacturing of monoclonal antibodies: Dynamic control of multiple integrated polishing chromatography steps using BioSMB.

Nitika, Nitika / Thakur, Garima / Rathore, Anurag S

Journal of chromatography. A

2023 Volume 1690, Page(s) 463784

Abstract: We propose a strategy for automation and control of multi-step polishing chromatography in integrated continuous manufacturing of monoclonal antibodies. The strategy is demonstrated for a multi-step polishing process consisting of cation exchange ... ...

Abstract	We propose a strategy for automation and control of multi-step polishing chromatography in integrated continuous manufacturing of monoclonal antibodies. The strategy is demonstrated for a multi-step polishing process consisting of cation exchange chromatography in bind-and-elute mode followed by mixed-mode chromatography in flowthrough mode. A BioSMB system with a customized Python control layer is used for automation and scheduling of both the chromatography steps. Further, the BioSMB valve manifold is leveraged for in-line conditioning between the two steps, as tight control of pH and conductivity is essential when operating with multimodal resins because even slight fluctuations in load conditions adversely affect the chromatography performance. The pH and conductivity of the load to the multimodal chromatography columns is consistent, despite the elution gradient of the preceding cation exchange chromatography step. Inputs from the BioSMB pH and conductivity sensors are used for real-time control of the 7 pumps and 240 valves to achieve in-line conditioning inside the BioSMB manifold in a fully automated manner. This is confirmed by showcasing different elution strategies in cation exchange chromatography, including linear gradient, step gradient and process deviations like tubing leakage. In all the above cases, the model was able to maintain the pH and conductivity of multimodal chromatography load within the range of 6 ± 0.1 pH and 7 ± 0.3 mS/cm conductivity. The strategy eliminates the need for using multiple BioSMB units or integrating external pumps, valves, mixers, surge tanks, or sensors between the two steps as is currently the standard approach, thus offering a simple and robust structure for integrating multiple polishing chromatography steps in continuous downstream monoclonal antibody purification trains.
MeSH term(s)	Cricetinae ; Animals ; Antibodies, Monoclonal/chemistry ; Chromatography, Ion Exchange/methods ; CHO Cells ; Cations/chemistry
Chemical Substances	Antibodies, Monoclonal ; Cations
Language	English
Publishing date	2023-01-07
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	1171488-8
ISSN	1873-3778 ; 0021-9673
ISSN (online)	1873-3778
ISSN	0021-9673
DOI	10.1016/j.chroma.2023.463784
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Article ; Online: Continuous manufacturing of monoclonal antibodies: Automated downstream control strategy for dynamic handling of titer variations.

Thakur, Garima / Bansode, Vikrant / Rathore, Anurag S

Journal of chromatography. A

2022 Volume 1682, Page(s) 463496

Abstract: Handling long-term dynamic variability in harvest titer is a critical challenge in continuous downstream manufacturing. This challenge is becoming increasingly important with the advent of high-titer clones and modern upstream perfusion processes where ... ...

Abstract	Handling long-term dynamic variability in harvest titer is a critical challenge in continuous downstream manufacturing. This challenge is becoming increasingly important with the advent of high-titer clones and modern upstream perfusion processes where the titer can vary significantly across the course of a campaign. In this paper, we present a strategy for real-time, dynamic adjustment of the entire downstream train, including capture chromatography, viral inactivation, depth filtration, polishing chromatography, and single-pass formulation, to accommodate variations in titer from 1-7 g/L. The strategy was tested in real time in a continuous downstream purification process of 36 h duration with induced titer variations. The dynamic control strategy leverages real-time NIR-based concentration sensors in the harvest material to continuously track the titer, integrated with an in-house Python-based control system that operates a BioSMB for carrying out capture and polishing chromatography, as well as a series of pumps and solenoid valves for carrying out viral inactivation and formulation. A set of 9 different methods, corresponding to the different harvest titers have been coded onto the Python controller. The methods have a varying number of chromatography columns (3-6 for Protein A and 2-10 for CEX), designed to ensure proper scheduling and optimize productivity across the entire titer variation space. The approach allows for a wide range of titers to be processed on a single integrated setup without having to change equipment or to re-design each time. The strategy also overcomes a key unexplored challenge in continuous processing, namely hand-shaking the downstream train to upstream conditions with long-term titer variability while maintaining automated operation with high productivity and robustness.
MeSH term(s)	Antibodies, Monoclonal ; Chromatography/methods ; Filtration ; Staphylococcal Protein A/chemistry ; Virus Inactivation
Chemical Substances	Antibodies, Monoclonal ; Staphylococcal Protein A
Language	English
Publishing date	2022-09-17
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	1171488-8
ISSN	1873-3778 ; 0021-9673
ISSN (online)	1873-3778
ISSN	0021-9673
DOI	10.1016/j.chroma.2022.463496
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Article ; Online: Continuous integrated manufacturing for biopharmaceuticals: A new paradigm or an empty promise?

Rathore, Anurag S / Thakur, Garima / Kateja, Nikhil

Biotechnology and bioengineering

2022 Volume 120, Issue 2, Page(s) 333–351

Abstract: Continuous integrated bioprocessing has elicited considerable interest from the biopharma industry for the many purported benefits it promises. Today many major biopharma manufacturers around the world are engaged in the development of continuous process ...

Abstract	Continuous integrated bioprocessing has elicited considerable interest from the biopharma industry for the many purported benefits it promises. Today many major biopharma manufacturers around the world are engaged in the development of continuous process platforms for their products. In spite of great potential, the path toward continuous integrated bioprocessing remains unclear for the biologics industry due to legacy infrastructure, process integration challenges, vague regulatory guidelines, and a diverging focus toward novel therapies. In this article, we present a review and perspective on this topic. We explore the status of the implementation of continuous integrated bioprocessing among biopharmaceutical manufacturers. We also present some of the key hurdles that manufacturers are likely to face during this implementation. Finally, we hypothesize that the real impact of continuous manufacturing is likely to come when the cost of manufacturing is a substantial portion of the cost of product development, such as in the case of biosimilar manufacturing and emerging economies.
MeSH term(s)	Technology, Pharmaceutical ; Biological Products/chemistry ; Drug Industry
Chemical Substances	Biological Products
Language	English
Publishing date	2022-09-28
Publishing country	United States
Document type	Journal Article ; Review ; Research Support, Non-U.S. Gov't
ZDB-ID	280318-5
ISSN	1097-0290 ; 0006-3592
ISSN (online)	1097-0290
ISSN	0006-3592
DOI	10.1002/bit.28235
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Article: Continuous manufacturing of monoclonal antibodies: Automated downstream control strategy for dynamic handling of titer variations

Thakur, Garima / Bansode, Vikrant / Rathore, Anurag S.

Journal of chromatography. 2022 Oct. 25, v. 1682

2022

Abstract	Handling long-term dynamic variability in harvest titer is a critical challenge in continuous downstream manufacturing. This challenge is becoming increasingly important with the advent of high-titer clones and modern upstream perfusion processes where the titer can vary significantly across the course of a campaign. In this paper, we present a strategy for real-time, dynamic adjustment of the entire downstream train, including capture chromatography, viral inactivation, depth filtration, polishing chromatography, and single-pass formulation, to accommodate variations in titer from 1–7 g/L. The strategy was tested in real time in a continuous downstream purification process of 36 h duration with induced titer variations. The dynamic control strategy leverages real-time NIR-based concentration sensors in the harvest material to continuously track the titer, integrated with an in-house Python-based control system that operates a BioSMB for carrying out capture and polishing chromatography, as well as a series of pumps and solenoid valves for carrying out viral inactivation and formulation. A set of 9 different methods, corresponding to the different harvest titers have been coded onto the Python controller. The methods have a varying number of chromatography columns (3–6 for Protein A and 2–10 for CEX), designed to ensure proper scheduling and optimize productivity across the entire titer variation space. The approach allows for a wide range of titers to be processed on a single integrated setup without having to change equipment or to re-design each time. The strategy also overcomes a key unexplored challenge in continuous processing, namely hand-shaking the downstream train to upstream conditions with long-term titer variability while maintaining automated operation with high productivity and robustness.
Keywords	Python ; automation ; chromatography ; equipment ; filtration ; purification methods
Language	English
Dates of publication	2022-1025
Publishing place	Elsevier B.V.
Document type	Article
ZDB-ID	218139-3
ISSN	0021-9673 ; 0378-4355 ; 0376-737X
ISSN	0021-9673 ; 0378-4355 ; 0376-737X
DOI	10.1016/j.chroma.2022.463496
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Charge of a transmembrane peptide alters its interaction with lipid membranes.

Thakur, Garima C N / Uday, Arunima / Cebecauer, Marek / Roos, Wouter H / Cwiklik, Lukasz / Hof, Martin / Jurkiewicz, Piotr / Melcrová, Adéla

Colloids and surfaces. B, Biointerfaces

2024 Volume 235, Page(s) 113765

Abstract: Transmembrane (TM) proteins interact closely with the surrounding membrane lipids. Lipids in the vicinity of TM proteins were reported to have hindered mobility, which has been associated with lipids being caught up in the rough surface of the TM domains. ...

Abstract	Transmembrane (TM) proteins interact closely with the surrounding membrane lipids. Lipids in the vicinity of TM proteins were reported to have hindered mobility, which has been associated with lipids being caught up in the rough surface of the TM domains. These reports, however, neglect one important factor that largely influences the membrane behavior - electrostatics of the TM peptides that are usually positively charged at their cytosolic end. Here, we study on the example of a neutral and a positively charged WALP peptide, how the charge of a TM peptide influences the membrane. We investigate both its dynamics and mechanics by: (i) time dependent fluorescent shift in combination with classical and FRET generalized polarization to evaluate the mobility of lipids at short and long-range distance from the peptide, (ii) atomic force microscopy to observe the mechanical stability of the peptide-containing membranes, and (iii) molecular dynamics simulations to analyze the peptide-lipid interactions. We show that both TM peptides lower lipid mobility in their closest surroundings. The peptides cause lateral heterogeneity in lipid mobility, which in turn prevents free lipid rearrangement and lowers the membrane ability to seal ruptures after mechanical indentations. Introduction of a positive charge to the peptide largely enhances these effects, affecting the whole membrane. We thus highlight that unspecific peptide-lipid interactions, especially the electrostatics, should not be overlooked as they have a great impact on the mechanics and dynamics of the whole membrane.
MeSH term(s)	Lipid Bilayers/chemistry ; Peptides/chemistry ; Membrane Lipids/chemistry ; Membrane Proteins/chemistry ; Molecular Dynamics Simulation
Chemical Substances	Lipid Bilayers ; Peptides ; Membrane Lipids ; Membrane Proteins
Language	English
Publishing date	2024-01-23
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	1500523-9
ISSN	1873-4367 ; 0927-7765
ISSN (online)	1873-4367
ISSN	0927-7765
DOI	10.1016/j.colsurfb.2024.113765
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