Dr. Rahul Bhambure Demonstrates How PAT Tools Are Transforming India’s Continuous Biomanufacturing from Reactive to Predictive
The Principal Scientist and bioprocess engineering lead at CSIR-NCL Pune brings two landmark case studies to PharmaCore India 2026 — a continuous refolding platform for romiplostim and an in-process intact mass monitoring method for tenecteplase — showing that the integration of process analytical technology into biosimilar manufacturing is not theoretical aspiration but verified, scalable practice, Rashmi Kumariof Neo Science Hub reports from Jio Centre, Mumbai
The afternoon Expert Insights track at PharmaCore India 2026 had, by 4:00 pm, covered the strategic vision for India’s biologics future, the analytical instrumentation framework that underpins it, and the operational manufacturing challenges that test it at commercial scale. What remained — and what Dr. Rahul Bhambure, Principal Scientist at CSIR-National Chemical Laboratory (CSIR-NCL), Pune, provided in the session’s final full presentation — was proof of concept: actual experimental data from his laboratory showing, in granular molecular detail, how PAT tools can be integrated into continuous biopharmaceutical manufacturing to monitor critical quality attributes in real time, and what the yield, purity, and timeline consequences of that integration look like in practice.
His talk, “PAT Tools for Continuous Biomanufacturing,” was, by design, a departure from the macro-strategic register of the earlier sessions. It was a working scientist’s report — two case studies, specific molecules, specific instruments, specific numbers — and its value lay precisely in that specificity. Where the morning sessions had argued for what India must do, Dr. Bhambure showed what is already being done, in a publicly funded laboratory in Pune, with the kind of rigour and industry engagement that positions India’s academic science as a genuine partner in the country’s biopharma ambitions rather than merely a policy footnote.
NCL and the Industry Partnership Model
CSIR-NCL’s Chemical Engineering and Process Development Division, where Dr. Bhambure leads his bioprocess engineering group, focuses on high-throughput chromatography development, continuous purification platforms, protein refolding, and QbD-compliant process design for biosimilar therapeutics. The NCL model, as Dr. Bhambure described it, is explicitly industry-oriented — functioning as an IP-generating problem-solving partner for three main categories of client: biosimilar and recombinant peptide manufacturers (typically operating at scales below 6,000-litre fermentation); biocatalyst and recombinant enzyme producers; and fermentation-origin API and nutraceutical manufacturers.
The problems these clients bring to NCL are consistently of two types. First: IP clearance. Given that innovator companies patent not only their molecules but their cell lines, process conditions, and formulations, biosimilar developers need to validate IP-free development pathways before investing in manufacturing infrastructure. NCL provides freedom-to-operate (FTO)-anchored solutions for clone development, upstream process design, and downstream processing. Second, and equally pressing: cost of goods (COGS). “The problem definitions are focused towards reducing costs in order to match existing import prices,” Dr. Bhambure said with the pragmatic directness of someone who takes industry briefs rather than writes academic papers about them. The competitive reference point is Chinese import pricing — a benchmark that forces a discipline on Indian biosimilar developers that no regulatory guideline can replicate.
Dr. Bhambure’s team at CSIR-NCL has worked in formal research collaboration with Lupin Ltd., supported by DST funding, to develop a novel continuous purification process for a biosimilar monoclonal antibody therapeutic — a collaboration that represents exactly the kind of industry-academia axis that both Dr. Jain in the morning and Dr. Tiwari in the preceding session had identified as critical to India’s biologics capability development.
The PAT Framework
Before presenting his case studies, Dr. Bhambure situated PAT within the broader manufacturing philosophy that the afternoon’s talks had been building toward. The central problem of biologics manufacturing is that the product — a large, complex, post-translationally modified protein — cannot be fully characterised by final product testing alone. The quality is built in during the process, and that process must be monitored continuously if deviations are to be caught before they become defects.
Conventional batch manufacturing separates the process from its analysis: samples are taken, shipped to the analytical laboratory, and results returned hours or days later. By the time a glycan profile deviation is identified, the batch that generated it may already be in the downstream processing queue. PAT, as defined by the FDA’s 2004 initiative and the ICH Q8/Q9/Q11 quality-by-design framework, is the philosophy of integrating measurement into the process itself — placing sensors and analytical instruments at-line, on-line, or in-line, so that quality data is generated continuously and process decisions can be made in real time. The result, when implemented correctly, is a shift from reactive quality control — finding problems after they occur — to proactive quality assurance — preventing them through continuous feedback. Dr. Tiwari had introduced this concept in the preceding session; Dr. Bhambure was about to demonstrate it with data.
Case Study One: Continuous Refolding of Romiplostim — Monitoring Disulfide Bond Formation in Real Time
The first case study centred on romiplostim (sold commercially as Nplate® by Amgen), a peptibody therapeutic used in the treatment of chronic immune thrombocytopenia — a condition characterised by dangerously low platelet counts. The choice of molecule was deliberate and instructive. Peptibodies — hybrid molecules consisting of a therapeutic peptide fused to the Fc region of an antibody — represent a structurally novel class with 13 molecules currently approved globally, most of them expressed in E. coli. They offer the tissue penetration advantages of small peptides combined with the extended half-life conferred by the Fc portion, and the cost and speed advantages of bacterial expression systems. But they are molecularly complex in a way that creates a specific manufacturing challenge: romiplostim is a dimer of two identical 269-amino-acid subunits, with a total of six disulfide bonds — two inter-chain bonds linking the two subunits, and two intra-chain bonds within each subunit — all of which must form correctly to yield a functional therapeutic.
When expressed in E. coli, the protein accumulates as inclusion bodies (IBs) — insoluble, non-functional protein aggregates. The manufacturing process must solubilise the inclusion bodies, refold the protein under controlled conditions that allow the six disulfide bonds to form in the correct sequence and geometry, and then capture and purify the correctly refolded dimer. The challenge is that refolding is inherently difficult to control: the kinetics of disulfide bond formation are time-dependent, the correctly folded form is thermodynamically only marginally more stable than misfolded intermediates, and the refolding yield in conventional dilution-assisted batch processing does not typically exceed 50 per cent.
Dr. Bhambure’s laboratory addressed this by building a molecular kinetics map of the refolding process — characterising, at multiple time points during refolding, both the covalent interactions (disulfide bond formation, tracked by mass spectrometry under reduced and non-reduced conditions using trypsin and glucoamylase-based peptide digestion with both CID and ETD fragmentation modes) and the non-covalent structural changes (tracked by intrinsic and extrinsic fluorescence spectroscopy, which reveals the tertiary structure of the protein as it transitions from the denatured inclusion-body state to the native conformation). The fluorescence data showed a characteristic blue-shift in emission wavelength upon refolding — the spectroscopic signature of tryptophan residues moving from a solvent-exposed to a buried hydrophobic environment, confirming that the tertiary structure is reaching its native state.
The mass spectrometry data mapped the kinetics of each disulfide bond individually: inter-chain disulfides began appearing after two hours of refolding, but the area under the curve for each species continued increasing until 72 hours. Intra-chain disulfides were not detectable until four hours, with formation continuing across the same 72-hour window. The conclusion was precise: complete, correctly paired disulfide formation — and the maximum refolding yield — requires 72 hours under dilution-assisted batch conditions. The yield ceiling at this point is approximately 50 per cent.
Armed with this mechanistic understanding, the team then designed a continuous refolding platform — a system in which solubilised inclusion bodies in Buffer A and the refolding buffer meet in a coiled reactor maintained at controlled temperature, allowing in-situ dilution to initiate refolding, with the refolding mixture immediately captured onto a Protein A affinity column. This simultaneous refolding-and-purification configuration — supported by arginine and cysteine in the buffer to promote correct disulfide formation — converts what was a 72-hour batch process into a continuous flow process that achieves comparable results in 15 hours. The overall refolding yield rises from approximately 50 per cent (batch) to 55–60 per cent (continuous), and purity increases from 67 per cent to approximately 80 per cent, because the integrated chromatography step removes misfolded and aggregated species as they form, rather than allowing them to accumulate.
For process monitoring in continuous mode, Dr. Bhambure’s team implemented time-based sampling with half-hour digest windows and direct injection mass spectrometry — providing a near-real-time readout of disulfide bond status throughout the continuous run. This is PAT in its most rigorous implementation: analytical data closing the loop on process control, not as a post-hoc quality check but as an active guidance signal. Dr. Bhambure noted that this platform is consistent with industrial manufacturing benchmarks — romiplostim is currently manufactured by Intas Pharmaceuticals and by Emcure in India, using batch refolding approaches — and that the continuous platform offers a quantifiable productivity and quality advantage.
Case Study Two: Intact Mass Monitoring of Tenecteplase — PAT for Heavily Glycosylated Proteins
The second case study addressed a fundamentally different analytical challenge: not disulfide bond integrity, but glycan mass — the monitoring of glycosylation patterns in heavily glycosylated proteins during cell culture, before downstream processing begins.
Tenecteplase (TNKase®) is a genetically engineered variant of tissue plasminogen activator (t-PA) used as a thrombolytic — a clot-dissolving agent in the treatment of acute myocardial infarction. It is a complex glycoprotein carrying multiple N-linked glycan chains that are critical both to its pharmacological activity and to its immunogenic profile. Glycosylation patterns in cell culture are sensitive to culture conditions — nutrient availability, pH, dissolved oxygen, feeding strategy — and changes in glycan composition can affect the molecule’s PK/PD behaviour and regulatory comparability profile. But conventional glycan analysis is slow, sample-intensive, and typically performed offline after downstream processing, by which point the upstream culture conditions that generated the deviation can no longer be corrected.
Dr. Bhambure’s team developed an MALDI-TOF mass spectrometry-based method for intact mass analysis of tenecteplase directly from cell culture samples — a method that completes the full analysis in three minutes, compared to the hours or days required by conventional ESI-MS approaches. The method enables real-time identification of the intact glycoprotein mass across multiple cell culture time points, providing immediate visibility into whether the glycan profile is drifting from target. Verification was performed by comparing MALDI-TOF intact mass results with ESI-MS data from de-glycosylated samples, confirming accuracy. The same analytical strategy was validated for recombinant erythropoietin and etanercept — two additional glycosylated biosimilar targets in the NCL development portfolio.
The practical consequence, as Dr. Bhambure framed it, is a transformation of the upstream optimisation cycle. When intact mass data is available in real time during cell culture, the feeding strategy — the nutrient supplementation regime that drives glycosylation towards the desired profile — can be adjusted before the batch is committed to downstream processing. “Minimal downstream process optimisation was needed when we have intact masses matching the innovator at the upstream level.” The analytical feedback loop compresses what would otherwise be a multi-batch, multi-week iteration cycle into a single, data-guided process run.
The NCL Portfolio and the Academic-Industry Bridge
In his closing remarks, Dr. Bhambure briefly catalogued the breadth of molecules his laboratory addresses using these PAT platforms: novel monoclonal antibodies including anti-rabies antibodies and anti-LIPA antibodies, recombinant enzymes, and various other categories of biosimilar and novel therapeutic proteins. The range is deliberate — NCL’s role is not to be a single-molecule specialist but to develop platform analytical and process approaches that can be transferred across the full spectrum of India’s biopharmaceutical development pipeline.
Dr. Bhambure’s postdoctoral training was in Chemical and Biomolecular Engineering at the University of Delaware — one of the world’s premier centres for bioseparation science — and his career at NCL has been defined by the application of that rigorous process engineering foundation to India’s specific manufacturing challenges: the cost pressure from Chinese competition, the IP navigation required around innovator patents, and the need to generate globally compliant analytical data with the resources available in a publicly funded research institute. The session moderator’s closing remark — “the process is actually the product” — was the afternoon’s running theme stated one final time, but in Dr. Bhambure’s presentation it acquired a precision it had not previously had. The process is the product only if the process is fully understood, continuously monitored, and analytically guided. Without the PAT infrastructure to close the feedback loop between what is happening in the bioreactor and what the scientist decides to do next, the principle remains aspiration. With it, it becomes engineering.
SPEAKER PROFILE
Dr. Rahul Bhambure, PhD Principal Scientist & Group Leader, Bioprocess Engineering, Chemical Engineering and Process Development Division, CSIR-National Chemical Laboratory (CSIR-NCL), Pune Postdoctoral training: Chemical and Biomolecular Engineering, University of Delaware Awards: DST Early Career Research Award Research focus: Biosimilar process development; continuous biomanufacturing; PAT integration; downstream process chromatography; protein refolding; high-throughput process development Led CSIR-NCL’s process development collaboration with Lupin Ltd. and DST for continuous purification of biosimilar monoclonal antibody therapeutics Consulting services: Bioprocess optimisation, analytics, FTO-based solutions for biosimilar and recombinant protein manufacturers
KEY TECHNICAL CONCEPTS FROM THE SESSION
PAT (Process Analytical Technology) — FDA-endorsed framework for integrating real-time measurement and control into biopharmaceutical manufacturing; enabling shift from reactive quality control to proactive quality assurance | Peptibody — hybrid therapeutic molecule consisting of a bioactive peptide fused to an antibody Fc region; 13 currently approved globally, majority E. coli-expressed | Inclusion bodies (IBs) — insoluble protein aggregates formed during E. coli recombinant protein expression; require solubilisation and refolding before downstream purification | Continuous refolding platform — in-situ dilution in a coiled reactor coupled with simultaneous Protein A affinity capture; demonstrated 15-hour cycle time vs. 72-hour batch, yield 55–60%, purity ~80% | Disulfide bond kinetics mapping — mass spectrometry-based characterisation of disulfide bond formation at multiple refolding time points using CID and ETD fragmentation; combined with fluorescence spectroscopy for non-covalent structural monitoring | MALDI-TOF intact mass analysis — 3-minute cell culture sample analysis for glycoprotein intact mass; enables real-time upstream feeding strategy correction | Romiplostim (Nplate®) — peptibody therapeutic for chronic immune thrombocytopenia; 6 disulfide bonds; case study molecule for continuous refolding platform | Tenecteplase (TNKase®) — heavily glycosylated thrombolytic; case study molecule for MALDI-TOF intact mass monitoring
