Publications by authors named "Anurag Khetan"

11 Publications

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Metabolomic and quality data for early and late passages of an antibody-producing industrial CHO cell line.

Data Brief 2020 Dec 26;33:106591. Epub 2020 Nov 26.

Biologics Development, Global Product Development and Supply, Bristol-Myers Squibb Company, Devens, MA 01434, USA.

In this article, we provide four data sets for an industrial Chinese Hamster Ovary (CHO) cell line producing antibodies during a 14-day bioreactor run. This cell line was selected for further evaluation because of its significant titer loss as the cells were passaged over time. Four conditions that differed in cell bank ages were run for this dataset. Specifically, cells were passaged to passage 12, 21, 25, and 37 and then used in this experiment. Once the run commenced the following datasets were gathered: 1). Glycosylation data for each reactor 2). Size Exclusion Chromatography (SEC) data for the antibodies produced which allowed for the identification of high and low molecular weight species in the samples (N-Glycan and SEC data was taken on day 14 only). 3/4). Metabolites levels measured using Nuclear Magnetic Resonance (NMR) and liquid chromatography-mass spectroscopy (LC-MS) for all reactors over the time course of days 1, 4, 6, 8, 12, and 14. We also provide a graph of the glutamine levels for cells of different ages as an example of the utility of the data. These metabolomics data provide relative amounts for 36 metabolites (NMR) and 109 metabolites (LC-MS) over the 14-day time course. These data were collected in connection with a co-submitted paper [1].
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http://dx.doi.org/10.1016/j.dib.2020.106591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724160PMC
December 2020

Osmolality as a lever to modulate the N-glycolylneuraminicacid (Neu5Gc) level of a recombinant glycoprotein produced in Chinese hamster ovary cells.

Biotechnol Prog 2020 11 10;36(6):e3038. Epub 2020 Jul 10.

Manufacturing Science and Technology, Bristol Myers Squibb Co, Devens, Massachusetts, USA.

Glycoproteins could be highly sialylated, and controlling the sialic acid levels for some therapeutic proteins is critical to ensure product consistency and efficacy. N-acetylneuraminic acid (Neu5Ac, or NANA) and N-glycolylneuraminic acid (Neu5Gc, or NGNA) are the two most common forms of sialic acids produced in mammalian cells. As Neu5Gc is not produced in humans and can elicit immune responses, minimizing Neu5Gc formation is important in controlling this quality attribute for complex glycoproteins. In this study, a sialylated glycoprotein was used as the model molecule to study the effect of culture osmolality on Neu5Gc. A 14-day fed-batch process with osmolality maintained at physiological levels produced high levels of Neu5Gc. Increase of culture osmolality reduced the Neu5Gc level up to 70-80%, and the effect was proportional to the osmolality level. Through evaluating different osmolality conditions (300-450 mOsm/kg) under low or high pCO , we demonstrated that osmolality could be an effective process lever to modulate the Neu5Gc level. Potential mechanism of osmolality impact on Neu5Gc is discussed and is hypothesized to be cytosol NADH availability related. Compared with cell line engineering efforts, this simple process lever provides the opportunity to readily modulate the Neu5Gc level in a cell culture environment.
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http://dx.doi.org/10.1002/btpr.3038DOI Listing
November 2020

Applications of small molecules in modulating productivity and product quality of recombinant proteins produced using cell cultures.

Biotechnol Adv 2020 11 12;43:107577. Epub 2020 Jun 12.

Biologics Development, Global Product Development & Supply, Bristol Myers Squibb Company, New Brunswick, NJ 08903, United States of America.

Mammalian cell cultures have been used extensively for production of recombinant protein therapeutics such as monoclonal antibodies, fusion proteins and enzymes for decades. Small molecules have been investigated as media supplements to improve process productivity and reduce cost of goods. Those chemicals can lead to significant yield improvement through different mechanisms such as cell cycle modulation, cellular redox regulation, etc. In addition to productivity, small molecules have also been routinely used to regulate post-translational modifications of recombinant proteins. This review summarizes key applications of small molecules in protein productivity improvement and product quality control.
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http://dx.doi.org/10.1016/j.biotechadv.2020.107577DOI Listing
November 2020

Enabling speed to clinic for monoclonal antibody programs using a pool of clones for IND-enabling toxicity studies.

MAbs 2020 Jan-Dec;12(1):1763727

Biologics Development, Bristol Myers Squibb Co , New Brunswick, NJ, USA.

The importance of speed to clinic for medicines that may address unmet medical needs puts pressure on product development timelines. Historically, both toxicology and first-in-human clinical materials are generated using the same clonal-derived cells to ensure safety and minimize any development risks. However, cell line development with single cell cloning is time consuming, and aggravated by the time needed to screen for a lead clone based on cell line stability and manufacturability. In order to achieve faster timelines, we have used pools of up to six clones for earlier production of drug substance for regulatory filing-enabling toxicology studies, and then the final single clone was selected for production of clinical materials. This approach was enabled by using platform processes across all stages of early development, including expression vectors, host cell lines, media, and production processes. Through comprehensive cell culture and product quality analysis, we demonstrated that the toxicology material was representative of the clinical material for all six monoclonal antibody programs evaluated. Our extensive development experience further confirmed that using a pool of clones for toxicology material generation is a reliable approach to shorten the early development timeline.
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http://dx.doi.org/10.1080/19420862.2020.1763727DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7531531PMC
May 2020

Understanding the effect of high gas entrance velocity on Chinese hamster ovary (CHO) cell culture performance and its implications on bioreactor scale-up and sparger design.

Biotechnol Bioeng 2020 06 28;117(6):1684-1695. Epub 2020 Feb 28.

Cell Culture, Process Science, Boehringer Ingelheim Fremont, Inc., 6701 Kaiser Drive, Fremont, California.

There are three main potential sources for cell shear damage existing in stirred tank bioreactors. One is the potential high energy dissipation in the immediate impeller zones; another from small gas bubble burst; and third is from high gas entrance velocity (GEV) emitting from the sparger. While the first two have been thoroughly addressed for the scale-up of Chinese hamster ovary (CHO) cell culture knowing that a wide tolerable agitation range with non-damaging energy dissipation exists and the use of shear protectants like Pluronic F68 guard against cell damage caused by bubble burst, GEV remains a potential scale-up problem across scales for the drilled hole or open pipe sparger designs. GEV as high as 170 m/s due to high gas flow rates and relatively small sparger hole diameters was observed to be significantly detrimental to cell culture performance in a 12,000 L bioreactor when compared to a satellite 2 L bioreactor run with GEV of <1 m/s. Small scale study of GEV as high as 265 m/s confirmed this. Based on the results of this study, a critical GEV of >60 m/s for CHO cells is proposed, whereas previously 30 m/s has been reported for NS0 cells by Zhu, Cuenca, Zhou, and Varma (2008. Biotechnol. Bioeng., 101, 751-760). Implementation of new large scale spargers with larger diameter and more holes lowered GEV and helped improve the cell culture performance, closing the scale-up gap. Design of such new spargers was even more critical when hole plugging was discovered during large scale cultivation hence exacerbating the GEV impact. Furthermore, development of a scale down model based on mimicry of the large scale GEV profile as a function of time was proven to be beneficial for reproducing large scale results.
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http://dx.doi.org/10.1002/bit.27314DOI Listing
June 2020

Suitability of a generic virus safety evaluation for monoclonal antibody investigational new drug applications.

Biotechnol Prog 2019 09 9;35(5):e2850. Epub 2019 Jun 9.

Product Development, Bristol-Myers Squibb, Princeton, New Jersey.

Biologics produced from CHO cell lines with endogenous virus DNA can produce retrovirus-like particles in cell culture at high titers, and other adventitious viruses can find their way through raw materials into the process to make a product. Therefore, it is the industry standard to have controls to avoid introduction of viruses into the production process, to test for the presence of viral particles in unclarified cell culture, and to develop purification procedures to ensure that manufacturing processes are robust for viral clearance. Data have been accumulated over the past four decades on unit operations that can inactivate and clear adventitious virus and provide a high degree of assurance for patient safety. During clinical development, biological products are traditionally tested at process set points for viral clearance. However, the widespread implementation of platform production processes to produce highly similar IgG antibodies for many indications makes it possible to leverage historical data and knowledge from representative molecules to allow for better understanding and control of virus safety. More recently, individualized viral clearance studies are becoming the rate-limiting step in getting new antibody molecules to clinic, particularly in Phase 0 and eIND situations. Here, we explore considerations for application of a generic platform virus clearance strategy that can be applied for relevant investigational antibodies within defined operational parameters in order to increase speed to the clinic and reduce validation costs while providing a better understanding and assurance of process virus safety.
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http://dx.doi.org/10.1002/btpr.2850DOI Listing
September 2019

Cell culture media for recombinant protein expression in Chinese hamster ovary (CHO) cells: History, key components, and optimization strategies.

Biotechnol Prog 2018 11 5;34(6):1407-1426. Epub 2018 Oct 5.

Biologics Process Development, Bristol-Myers Squibb, Pennington, New Jersey, United States.

The culture of Chinese Hamster Ovary (CHO) cells for modern industrial applications, such as expression of recombinant proteins, requires media that support growth and production. Such media must support high viable cell densities while also stimulating the synthesis and extracellular transport of biologic products. Early media development efforts in this area yielded basic formulations to sustain growth, viability, and cellular function, albeit comprising animal sourced components, and complex constituents used in batch culture mode. Subsequent improvements included the development of serum-free and chemically defined (CD) media, the identification of critical nutrients, growth factors, and potentially inhibitory or toxic cellular metabolites, and the use of fed-batch and perfusion culture techniques to optimize nutrient delivery while minimizing accumulation of unwanted waste products. This review is comprised of sections covering milestones in the evolution of mammalian cell culture media, nutrient composition and formulation requirements, optimization strategies, consistency and scalability of powder and liquid media preparation for industrial applications, and key recent advances driving progress in CHO cell culture medium design and development. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1407-1426, 2018.
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http://dx.doi.org/10.1002/btpr.2706DOI Listing
November 2018

Gradient elution behavior of proteins in hydrophobic interaction chromatography with U-shaped retention factor curves.

J Chromatogr A 2018 Apr 9;1547:53-61. Epub 2018 Mar 9.

Department of Chemical Engineering, University of Virginia, Charlottesville, VA, USA. Electronic address:

Protein retention in hydrophobic interaction chromatography is described by the solvophobic theory as a function of the kosmostropic salt concentration. In general, an increase in salt concentration drives protein partitioning to the hydrophobic surface while a decrease reduces it. In some cases, however, protein retention also increases at low salt concentrations resulting in a U-shaped retention factor curve. During gradient elution the salt concentration is gradually decreased from a high value thereby reducing the retention factor and increasing the protein chromatographic velocity. For these conditions, a steep gradient can overtake the protein in the column, causing it to rebind. Two dynamic models, one based on the local equilibrium theory and the other based on the linear driving force approximation, are presented. We show that the normalized gradient slope determines whether the protein elutes in the gradient, partially elutes, or is trapped in the column. Experimental results are presented for two different monoclonal antibodies and for lysozyme on Capto Phenyl (High Sub) resin. One of the mAbs and lysozyme exhibit U-shaped retention factor curves and for each, we determine the critical gradient slope beyond which 100% recovery is no longer possible. Elution with a reverse gradient is also demonstrated at low salt concentrations for these proteins. Understanding this behavior has implications in the design of gradient elution since the gradient slope impacts protein recovery.
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http://dx.doi.org/10.1016/j.chroma.2018.03.012DOI Listing
April 2018

Advancement in bioprocess technology: parallels between microbial natural products and cell culture biologics.

J Ind Microbiol Biotechnol 2017 05 9;44(4-5):785-797. Epub 2017 Feb 9.

Department of Chemical Engineering and Materials Science, University of Minnesota, 421 Washington Avenue SE, Minneapolis, MN, 55455-0132, USA.

The emergence of natural products and industrial microbiology nearly eight decades ago propelled an era of bioprocess innovation. Half a century later, recombinant protein technology spurred the tremendous growth of biologics and added mammalian cells to the forefront of industrial producing cells in terms of the value of products generated. This review highlights the process technology of natural products and protein biologics. Despite the separation in time, there is a remarkable similarity in their progression. As the new generation of therapeutics for gene and cell therapy emerges, its process technology development can take inspiration from that of natural products and biologics.
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http://dx.doi.org/10.1007/s10295-017-1913-4DOI Listing
May 2017

Control of misincorporation of serine for asparagine during antibody production using CHO cells.

Biotechnol Bioeng 2010 Sep;107(1):116-23

Biogen Idec, Inc., Cambridge, Massachusetts 02142, USA.

A recombinant monoclonal antibody produced by Chinese hamster ovary (CHO) cell fed-batch culture was found to have amino acid sequence misincorporation upon analysis by intact mass and peptide mapping mass spectrometry. A detailed analysis revealed multiple sites for asparagine were being randomly substituted by serine, pointing to mistranslation as the likely source. Results from time-course analysis of cell culture suggest that misincorporation was occurring midway through the fed-batch process and was correlated to asparagine reduction to below detectable levels in the culture. Separate shake flask experiments were carried out that confirmed starvation of asparagine and not excess of serine in the medium as the root cause of the phenomenon. Reduction in serine concentration under asparagine starvation conditions helped reduce extent of misincorporation. Supplementation with glutamine also helped reduce extent of misincorporation. Maintenance of asparagine at low levels in 2 L bench-scale culture via controlled supplementation of asparagine-containing feed eliminated the occurrence of misincorporation. This strategy was implemented in a clinical manufacturing process and scaled up successfully to the 200 and 2,000 L bioreactor scales.
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http://dx.doi.org/10.1002/bit.22771DOI Listing
September 2010

Heterogeneous distribution of lysine 6-aminotransferase during cephamycin C biosynthesis in Streptomyces clavuligerus demonstrated using green fluorescent protein as a reporter.

Microbiology (Reading) 2000 Aug;146 ( Pt 8):1869-1880

Department of Chemical Engineering and Materials Science1 and Department of Microbiology and Biological Process Technology Institute2, Box 196 1460 Mayo Memorial Building, 420 Delaware Street SE, University of Minnesota, Minneapolis, MN 55455-0312, USA.

The cellular distribution of the cephamycin biosynthetic enzyme lysine 6-aminotransferase (LAT) has been studied in Streptomyces clavuligerus hyphae by confocal microscopy using the S65T mutant of green fluorescent protein (GFP) as a reporter. LAT mediates the first committed step in the biosynthesis of the secondary metabolite cephamycin C by S. clavuligerus. The enzymic activity of LAT varies with time during the growth of S. clavuligerus in liquid medium. To investigate if this temporal variation occurs uniformly amongst all hyphae, S. clavuligerus was transformed with a plasmid containing the LAT-encoding gene translationally fused to the GFP-encoding gene. The LAT-GFP fusion product displayed fluorescence spectral characteristics of GFP, and showed similar temporal characteristics of LAT activity compared to the wild-type strain of S. clavuligerus. The transformed strain exhibited a heterogeneous distribution of fluorescence in mycelia grown in liquid cultures. This distribution varied significantly as the batch progressed: only a fraction of the mycelia fluoresced in the early growth phase, whereas nearly all hyphae fluoresced by the late growth phase. Thereafter, a non-uniform distribution of fluorescence was again observed in the declining growth phase. A large fraction of the non-fluorescent cells in the declining growth phase were found to be non-viable. Observations of S. clavuligerus colonies grown on solid agar also showed variation of LAT-GFP expression at different stages of growth. These observations in the solid phase can be explained in terms of nutrient deprivation and signalling molecules. The results suggest that physiological differentiation of S. clavuligerus mycelia leading to cephamycin C biosynthesis is both temporally and spatially distributed. The findings also revealed that the observed heterogeneity was independent of the position of individual cell compartments within the hypha. The potential of GFP as a reporter for the quantitative study of cephamycin biosynthesis at the cellular level has also been demonstrated.
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http://dx.doi.org/10.1099/00221287-146-8-1869DOI Listing
August 2000