Predicting how T cell receptors (TCRs) recognize peptide–MHC (pMHC) antigens is one of the central challenges in immunology today. It is also one of the hardest to solve. T cell recognition arises from complex molecular interactions, and even recent advances in protein structure prediction have not fully resolved which receptor-antigen pairs will lead to T cell activation. That question has broad relevance across immune-mediated diseases. In cancer, it could help researchers better understand antigen-specific immune responses relevant to target discovery and therapeutic design, while in autoimmune disease it may offer new ways to study the recognition events that drive immune dysregulation.

A recent Adaptive paper presented at Machine Learning for Health (ML4H) Symposium 2025 examines that challenge using ImmSET (Immune Synapse Encoding Transformer), a sequence-based transformer model trained on large-scale T-cell activation data. The research asks what becomes possible when models are trained directly on large-scale T-cell datasets rather than relying primarily on inferred structure.

What did the study examine?

Recent AI tools have expanded what is possible in structural biology. Deep learning systems such as AlphaFold have improved researchers’ ability to model proteins and protein complexes, opening new avenues for studying molecular function. But T cell recognition remains difficult to predict because the problem is not simply one of structure. It also requires understanding which receptor-antigen interactions are associated with T cell activation.

ImmSET was designed to test whether a sequence-based model could learn that relationship from data at scale. Trained from scratch using one of Adaptive’s large proprietary T-cell activation datasets, the model uses sequence information alone to predict T-cell receptor recognition of pMHC antigens.

Three findings from the study are especially notable.

Finding 1: Sequence-based prediction outperformed structure-based pipelines on one benchmark

When provided a sufficiently large training dataset, ImmSET outperformed AlphaFold2- and AlphaFold3-based pipelines on a benchmark assessing HLA-A*02:01 specificity prediction, while running orders of magnitude faster.

That result is notable because structure-based approaches have come to represent a major frontier in biological prediction. The study suggests that, for this task, sequence-based models trained on large T-cell activation datasets can provide a practical and scalable alternative. Rather than replacing structural approaches, the findings point to a complementary path—one that may be especially useful where speed, throughput, and dataset scale matter.

Finding 2: Performance improved as the dataset grew

A second finding was the consistency with which model performance improved as training data increased. The study reports performance gains as both the number of peptides increased and as the number of TCRs observed per peptide increased.

This suggests the model is not nearing an early ceiling. Instead, it points to a clear path for continued improvement as larger and more diverse T-cell activation datasets become available. In a field where data limitations have often constrained progress, that is an important signal.

Finding 3: Predictive signal began to extend across HLA alleles

The study also found evidence that increasing scale improved generalization beyond the allele on which the model was trained. Although ImmSET was trained only on A*02:01 data, the authors report measurable predictive signal on other HLA alleles as training data increased.

The cross-allele signal remains early, but it suggests the model may be learning features of T cell recognition that are generalizable across HLA alleles. HLA varies widely across the human population, so models need to do more than learn from the specific alleles in their training data. They need to generalize beyond them.

Why evaluation matters

The paper also addresses a methodological issue that has complicated prior work in this area: shortcut learning. According to the authors, some sequence-based models can appear to generalize even when peptide information is removed, effectively relying on memorized TCR motifs rather than learning meaningful receptor-antigen relationships.

To address that risk, the study emphasizes strict evaluation on truly unseen peptides. That is an important point. In problems as complex as TCR recognition, apparent performance gains can be misleading if evaluation is not designed carefully enough to test real generalization.

Why this matters across cancer and autoimmune disease

Taken together, the findings point to a practical direction for the field. Structural models have opened an important chapter in computational biology, but predicting immune recognition may also require approaches trained directly on large-scale T-cell activation data. The paper does not suggest that the mechanistic complexity of T cell recognition has been solved. It does suggest, however, that sequence-based models can provide fast, scalable, and increasingly generalizable predictions that complement structure-based methods as the field evolves.

That direction matters across immunology. In cancer, better prediction of T cell recognition could help inform antigen selection, including for personalized cancer vaccines, as well as T cell selection for cell therapies. In autoimmune disease, the same challenge applies in a different context: understanding which recognition events may contribute to pathogenic immune responses. Across both, improved prediction could help researchers study immune recognition more systematically.

Accepted and presented at ML4H 2025, the study adds to a growing body of work suggesting that progress in computational immunology may depend not only on better models, but on larger and more informative datasets.

Read the full paper on OpenReview.

The post Predicting T Cell Recognition: New Insights from Large-Scale Activation Data appeared first on Adaptive Biotech.

Size variants determination is a critical quality attribute for a therapeutic recombinant antibody, since they can impact the drug product safety, potency, and efficacy. Size exclusion chromatography is the most frequently employed technique for estimation of size variant and SE-HPLC principally separates out variants based on size, shape and molecular weight. A novel tri-specific antibody is recombinantly produced in Chinese hamster ovary (CHO) cells. Our studies exemplify the effectiveness of native SE-HPLC for separation of isoform variant (*P1) of Novel Tris-specific antibody. The study involves development of SE-HPLC method for Tri-specific antibody to differentiate the HMWP and the isoform variant of Tri-specific antibody. SEC-MALS and functional assay by ELISA results demonstrated that *P1 peak observed in SEHPLC contains structural isoform variant with molecular weight similar to main peak, and the enriched *P1 peak fraction had an equal potency to the main peak, confirming this isoform variant has similar access to the antigen-binding site.

Objective:

To identify and demonstrate the identity of *P1 peak observed in SE-HPLC. To demonstrate the potency of *P1 peak in comparison to main peak

Approach:

Size variants determination is a critical quality attribute of a therapeutic recombinant antibody, since the size variant can impact the drug product safety, potency, and efficacy. Size exclusion chromatography is most frequently employed for estimation of size variant and SEC principally separates out variants based on size, shape and molecular weight.

A Novel Tri specific antibody is recombinantly produced in Chinese hamster ovary (CHO) cells and based on SEC principle; our studies exemplify the effectiveness of native SEC for separation of hydrodynamic variant/isoform variant (Peak A) of Novel Trispecific antibody. The study involves development of SEC method for Trispecific antibody to differentiate the HMWP form of Tri specific antibody and the isoform form of Trispecific antibody. Electrospray ionization-mass spectrometry (ESI-MS), Capillary electrophoresis, Fluorescence and SEC-MALS results demonstrated that SE-HPLC Peak A contains structural isoform variant. Isolated Peak A fraction had an equal potency to the Main peak, confirming this isoform variant has similar access to the antigen-binding site

Conclusion:

1. The enriched Main peak and enriched *P1 + P1 + P2 were shown to have biological activity comparable to that of the control. On the other hand, there is no biological activity or potency in Enriched P3 and P4.
2. Confirmed by the SEC-MALS result, the peak left to Main peak peak (*P1) is not an HMW impurity peak and identified as main peak's conformational isoforms. The differential elution of *P1 isoforms could be attributed to differential size/shape in comparison to main peak
3. Further confirmation on isoforms will be carried out by HOS techniques like, NMR and HDX-MS

The post Kemwell’s Expertise in Size Exclusion Chromatography for Isoform Separation of a Novel Tri-specific Antibody appeared first on Kemwell Pharma.

Recent advances in biologics development are primarily focussed on development of high titre producing cell lines to substantiate the requirement of high dosage mAbs which may be as high as 600-700 mg drug per dose. This also brings in the requirement of subcutaneous injectables to alleviate the hassles of IV infusion. Therefore, to achieve both these objectives, substantial work has also been initiated for development of high concentration formulation along with high titre cell lines. One of the biggest challenges for the development of high concentration formulation is increase in viscosity which impact the injection force and patient comfort. As part of purification process flow, the concentration of the protein molecules is achieved by TFF unit operation which is also the unit operation where this challenge is encountered the most. This poster focuses on the strategy to mitigate these challenges and achieve a concentration of >200 mg/mL for mAbs as well as other mammalian recombinant proteins.

Barriers during TFF:

Key Contributors:

Conclusion:

During high-concentration TFF unit operations, pressure drop escalation and gel layer formation on the membrane surface are significant challenges to achieving concentrations >200 mg/mL at the drug substance stage. The strategies discussed in this work effectively mitigate these issues by reducing protein-protein interactions and minimizing gel layer formation on the membrane surface. This results in better displacement and higher recovery during the TFF process. By applying these approaches, consistent concentration >200 mg/mL was successfully achieved at the pilot scale and subsequently scaled up to 2000L at GMP.

The post Breaking Barriers: How Kemwell’s Mitigation Strategy Overcomes Viscosity-Induced High-Concentration TFF Challenges appeared first on Kemwell Pharma.

Milestone expands India’s role in global biologics manufacturing; collaboration with Cipla underscores commitment to quality and reliability

Bengaluru, India – October 28, 2025 – Kemwell Biopharma Pvt Ltd (“Kemwell”), a leading global biologics Contract Development and Manufacturing Organization (CDMO), announced today that its drug-product manufacturing facility in Bengaluru has successfully completed a U.S. Food and Drug Administration (FDA) pre-approval inspection (PAI) and is now cleared for the commercial manufacturing and testing of injectable products destined for the U.S. market.

This milestone marks the approval of the first product to get U.S. FDA clearance for commercial supply from Kemwell’s Bengaluru drug-product facility since the site became a dedicated biologics campus eight years ago. It demonstrates the company’s ability to meet stringent current Good Manufacturing Practice (cGMP) and aseptic drug-product manufacturing standards.

“Achieving FDA clearance for commercial manufacturing from our Bengaluru site marks a defining moment in Kemwell’s growth journey,” said Anurag Bagaria, Chairman and CEO, Kemwell Biopharma. “It reflects our team’s steadfast focus on quality, compliance, and scientific excellence. We are proud to support our partners in accelerating the delivery of important therapies to patients in the United States and around the world.”

The inspection was conducted as part of a Pre-Approval Inspection (PAI) following a product technology transfer from Cipla Limited, a leading global pharmaceutical company. The outcome underscores the confidence that major pharmaceutical innovators place in Kemwell’s capabilities and the reliability of its manufacturing network.

“Our partnership with Kemwell aligns with Cipla’s strategy of building a resilient and globally trusted supply chain,” said Pradeep Bhadauria, Chief Scientific Officer, Cipla Limited. “The successful completion of the FDA inspection at Kemwell’s Bengaluru site reinforces our commitment to delivering safe, high-quality medicines to patients in regulated markets.”

Kemwell’s FDA-approved site features advanced single-use and stainless-steel bioreactors, with over 5000L+ of installed upstream capacity, commercial fill-finish lines for liquid and lyophilized vials, and pre-filled syringe systems equipped with isolator technology for enhanced sterility assurance. In addition to drug-product (DP) manufacturing, the site supports process development, analytical testing, and drug-substance (DS) production—enabling fully integrated biologics programs from cell-line to commercial supply.

The company’s services span a wide spectrum of biologic modalities—including Monoclonal Antibodies, recombinant proteins, bispecific and trispecific antibodies, biosimilars and New Biological Entities—and extend to advanced therapies with cGMP cell-therapy development and manufacturing capabilities, positioning Kemwell as a trusted partner for global biopharma companies pursuing regulated-market approvals.

This achievement further strengthens Kemwell’s strategy to expand capacity, invest in advanced manufacturing technologies, and deliver compliant, scalable solutions that meet the evolving needs of the biologics industry in the United States and around the world.

About Kemwell Biopharma

Kemwell Biopharma Pvt Ltd is a leading biologics CDMO headquartered in Bengaluru, India, offering fully integrated services spanning cell-line development, process and analytical development, formulation, drug substance, and sterile drug product manufacturing for both novel biologics and biosimilars. Kemwell enables seamless technology transfer, validation, and scale-up across development, clinical, and commercial supply.

With more than 45 years of manufacturing excellence and regulatory accreditations including U.S. FDA, Indian FDA, CDSCO (Central Drugs Standard Control Organization), Malaysia NPRA (PIC/S), and recent Qualified Person (QP) audits, Kemwell partners with global pharmaceutical and biotech innovators to accelerate development and reduce time-to-market for biologics.

About Cipla Limited

Established in 1935, Cipla Limited is a global pharmaceutical company headquartered in Mumbai, India, with a strong presence across India, South Africa, North America, and other regulated and emerging markets. The company’s portfolio spans more than 1,500 products across key therapeutic areas including respiratory, urology, cardiology, and anti-infectives. Guided by its purpose of “Caring for Life,” Cipla continues to deliver affordable, high-quality medicines to patients worldwide.

Media Contacts:

Kemwell Biopharma Pvt Ltd
Email: info.india@kemwellbiopharma.com
Website: www.kemwellbiopharma.com

Cipla Limited

Email: corpcomm@cipla.com
Website: www.cipla.com

The post Kemwell Biopharma Facility in Bengaluru Successfully Completes U.S. FDA Inspection for Commercial Drug-Product Manufacturing appeared first on Kemwell Pharma.

Karen is a blood cancer survivor with a zest for life — she is committed to patient advocacy and taking advantage of everything outdoors, including gardening, flyfishing and water sports. Prior to her diagnosis, she was a caretaker for her husband who is currently managing a rare blood disorder. Therefore, while familiar with doctor offices, she was not used to being the patient. That was until she experienced searing back pain in 2009 and the subsequent collapse of her spine. She was diagnosed with multiple myeloma (MM) and told she might never walk again.

Within a few months, Karen transitioned from an outdoor enthusiast to a debilitated “terminal cancer patient” with an estimated two years to live. With few options at the time, she decided to go through with chemotherapy, despite the toll it would take on her body. While successful, doctors told her she would have to remain on maintenance treatment indefinitely despite the slew of side effects; that is, until she met Dr. Hashmi about 10 years later.

After hearing Karen’s story, Dr. Hashmi recommended Adaptive’s Minimal Residual Disease (MRD) test (clonoSEQ®). With a negative result, they decided to alter her maintenance plan, which included eliminating chemotherapy. During our conversation with Karen, she told the Adaptive team, “You gave my life back to me.”

As for general advice to people who receive a cancer diagnosis, Karen has several suggestions to help improve the patient care experience:

• Conduct external research about doctors, and be intentional about choosing your provider
• Expand your external research into learning more about your cancer and potential diagnostic and treatment options, including MRD testing
• Ask questions to clearly understand all available options for care, and don’t be afraid to seek out a second opinion

From a physician perspective, Dr. Hashmi emphasizes the need for more clinicians to understand the latest technologies in the fight against cancer, such as MRD testing. These innovative tools are only beneficial if doctors understand how they work and what kind of information they provide. Armed with this knowledge, healthcare providers may be able to consider current medical advancements while tailoring their treatment recommendation.

Today, Karen shares her experience to comfort and inform people who are living with blood cancer, along with their caregivers and families. For example, when she accompanies her husband to blood transfusion centers, she walks the halls and speaks to patients who are hesitant to receive bone marrow transplants. She listens to them, helps guide them and is a true testament to us all that a full life beyond a cancer diagnosis is possible.

clonoSEQ® is available as an FDA-cleared in vitro diagnostic (IVD) test service provided by Adaptive Biotechnologies to detect minimal residual disease (MRD) in bone marrow from patients with multiple myeloma or B-cell acute lymphoblastic leukemia (B-ALL) and blood or bone marrow from patients with chronic lymphocytic leukemia (CLL). clonoSEQ is also available for use in other lymphoid cancers and specimen types as a CLIA-validated laboratory developed test (LDT). For important information about the FDA-cleared uses of clonoSEQ including test limitations, please visit clonoSEQ.com/technical-summary

The post A Reflection on Blood Cancer Awareness Month: Patient Spotlight appeared first on Adaptive Biotech.

Every March, people and organizations celebrate Multiple Myeloma Awareness Month – using their voices to raise awareness about efforts to diagnose, treat, and support patients with multiple myeloma as well as inspire others on their blood cancer journeys. Earlier this month, Adapters had the privilege of hearing from Tiffany Williams, a 55-year-old multiple myeloma patient. Tiffany is a retired pediatric nurse practitioner turned patient advocate. More than that – she’s a mother, wife, sister, daughter and a fiercely supportive friend.  

Nearly 10 years ago, Tiffany began experiencing back pain. The previous year she had been treated by a neurologist for a herniated disc, so she booked another visit. This time, things quickly turned serious when an MRI revealed lesions on her spine that had metastasized. Given her medical background, she knew the prognosis wasn’t good and started to give up hope for her future. Within two weeks from the onset of her back pain, Tiffany was diagnosed with multiple myeloma.  

After meeting with a specialist, however, hope was restored. She was told, “It’s incurable, but treatable.” She held onto those words and embarked on an eight-month journey of induction therapy, including chemotherapy infusion and then a stem cell transplant. During that period, in many ways, her life stopped. She was suffering from extreme fatigue and had to take a leave of absence from work. As a result, she struggled with her loss of independence and identity. Her family became her caregivers, and she leaned on those closest to her to make major decisions. Her circle of friends tightened.  

In 2021, after more than six years of maintenance therapy, Tiffany’s oncologist recommended minimal residual disease (MRD) testing – a test that measures the remaining number of cancer cells present in a patient’s body during and after treatment that can lead to disease recurrence. By using this tool to gain real-time insight into Tiffany’s disease status, her doctor was able to develop a personalized care plan for Tiffany that allowed her to feel informed in the face of so much uncertainty she had experienced throughout her cancer journey. Ultimately the results from the clonoSEQ® Assay confirmed she was MRD-negative, and her doctor told her she could safely discontinue maintenance therapy.  

Knowing that she was MRD-negative was truly freeing. For the first time in nearly a decade, she was able to take a deep breath, exhale, and enjoy the space she was in. Now, Tiffany can comfortably look forward to other monumental events in her life, such as her two children’s weddings (and hopefully grandkids!), knowing that she is free of cancer.  

Tiffany offers this advice to other multiple myeloma patients: “If there was one thing I could share, it’s to encourage them to not walk this journey alone. The path is so much easier when it’s walked alongside others. Allow yourselves to lean into being vulnerable enough to release the stress, the pain, the anger, all those emotions, and allow other people to help embrace you through it. I have a village of friends and family who I can count on and rely on, and who give me hope and inspire me to be healthy and live.” 

Adaptive is dedicated to recognizing patients like Tiffany and to contribute to the world’s understanding of the critical role that MRD can play in blood cancer patient care.

clonoSEQ® is available as an FDA-cleared in vitro diagnostic (IVD) test service provided by Adaptive Biotechnologies to detect minimal residual disease (MRD) in bone marrow from patients with multiple myeloma or B-cell acute lymphoblastic leukemia (B-ALL) and blood or bone marrow from patients with chronic lymphocytic leukemia (CLL). clonoSEQ is also available for use in other lymphoid cancers and specimen types as a CLIA-validated laboratory developed test (LDT). For important information about the FDA-cleared uses of clonoSEQ including test limitations, please visit clonoSEQ.com/technical-summary

The post Hope restored: The critical role that Minimal Residual Disease can play in blood cancer patient care appeared first on Adaptive Biotech.

This is why minimal residual disease (MRD) testing is so important. MRD testing can help to provide an accurate, ongoing picture of disease and risk status, offering a sense of control and comfort during a time when life may feel less manageable.

Adaptive recently supported a survey of multiple myeloma patients conducted by HealthTree Foundation (formerly Myeloma Crowd), to assess and learn from real-world patients about their current awareness, use, and perceptions of MRD testing. With this knowledge, Adaptive and HealthTree can ensure patients and clinicians are educated and supported to get the care they need.

The survey of 635 patients with multiple myeloma across the U.S. delivered a meaningful insight that cancer care providers nationwide need to know: The majority of myeloma patients experience both clinical and emotional benefits from MRD testing.

• 96% of patients who used MRD testing found the test useful.
• 81% of respondents said that MRD testing brought them comfort in their treatment journey by keeping them consistently informed.
• 78% of patients said that the results helped inform their provider’s treatment decisions.
• More than 90% of those who had MRD testing said they would recommend it to others.

These results affirmed the high value of MRD while underscoring the huge opportunity for more patients to benefit from MRD testing. Over half of all survey respondents were only somewhat or less familiar MRD testing. Furthermore, 99% of those surveyed who had not yet received MRD testing reported being interested in learning more about it.

Jenny Ahlstrom, a multiple myeloma survivor and founder of HealthTree Foundation, reflected on the survey results, stating, “Education is power. Patients need access to the most up-to-date information, including the most advanced diagnostics, to feel confident asking questions, making decisions, and speaking with their health care providers openly. The survey results show that more education is needed on this important type of useful testing.”

We look forward to working together with HealthTree Foundation to identify more opportunities to expand education around real-time MRD testing with clonoSEQ® to both patients and physicians.

For those interested in learning more about the uses and benefits of MRD testing, visit https://www.myelomacrowd.org/myeloma or clonoSEQ.com.

clonoSEQ® is available as an FDA-cleared in vitro diagnostic (IVD) test service provided by Adaptive Biotechnologies to detect minimal residual disease (MRD) in bone marrow from patients with multiple myeloma or B-cell acute lymphoblastic leukemia (B-ALL) and blood or bone marrow from patients with chronic lymphocytic leukemia (CLL). clonoSEQ is also available for use in other lymphoid cancers and specimen types as a CLIA-validated laboratory developed test (LDT). For important information about the FDA-cleared uses of clonoSEQ including test limitations, please visit clonoSEQ.com/technical-summary

The post Among Multiple Myeloma Patients Receiving MRD Testing, Majority Report Meaningful Benefits appeared first on Adaptive Biotech.

The findings represented a huge step forward for understanding how we should be personalizing treatment decisions based on MRD-negative status. Specifically, it examined the cessation of therapy in newly diagnosed multiple myeloma patients who showed deep MRD-negative responses to treatment. Data from the final analysis of MASTER with extended follow-up time were published earlier this fall, so we checked back in with Dr. Costa to discuss what else we’ve learned and hear his perspective on how the findings might be implemented into practice.

1. Can you speak to the additional findings from the MASTER final analysis vs. the initial publication? What else have we learned?

With the extended follow-up (median 42.2 months vs. 23.8 months in the initial publication) we learned a few important things. Perhaps the most important is that most patients with standard risk multiple myeloma (MM) will achieve and sustain MRD-negative response without ongoing therapy. A longer follow-up gives us more confidence in this approach. We could also demonstrate that the use of MRD-adapted treatment modulation appears to negate the prognostic impact of MRD after transplant. This is a very important step to demonstrate the utility of adjusting therapy to depth of response.

2. Can you share your insights on the subset of patients whose outcomes were better with sustained MRD negativity at 10^-6?

With longer follow-up we could also examine the impact of sustained (12 months) MRD negativity. We demonstrated that among patients with sustained MRD<10^-5, those with sustained MRD<10^-6 have better progression free survival (PFS) than those with sustained MRD between 10^-5 and 10^-6. This further supports the notion that the lower the threshold for “positivity,” the better the assay will be able to identify patients with exquisitely good prognosis.

3. How have you implemented the findings from this study into clinical practice and how do you feel others can implement findings?

That has been an important practical lesson from this study, that sequential MRD assessment can be incorporated in the management of patients with NDMM and can be employed to inform depth of response. In an era where most patients will achieve responses deep enough to render serum and urine paraprotein unquantifiable, MRD becomes the only way to quantitatively track disease burden. We have fully incorporated this tool in our patient management, and we routinely use this information to decide on post-transplant consolidation and to defer subsequent therapy in patients without ultra-high-risk MM who have sustained MRD negativity.

4. We at Adaptive have been so thrilled to see the first published results from a trial in MM where MRD is used not only as a correlative but as an actionable parameter. In what other ways do you think we should be studying MRD to continue to generate actionable insights?

Absolutely. Like others, we have an ongoing trial employing experimental, enhanced consolidation for patients with MRD>=10^-5 after ASCT.  We also have the ongoing MASTER-2 trial, asking different questions for patients who are MRD-positive and MRD-negative after completion of induction. If successful, this can solidify MRD assessment post induction as a parameter to decide on subsequent therapy. Other trials are underway on the same premise. MRD is not a perfect biomarker, and we don’t have a perfect biomarker in any cancer. But I have no doubt it is the best indicator of treatment success and the strongest prognostic factor. The notion of asking different treatment questions to populations with different prognoses and/or achieving different levels of response was always a natural element of developing therapies for malignancies we now consider curable. We should do the same for MM.

5. What are you most looking forward to seeing at the upcoming ASH meeting?

For MM, I think this will be an ASH about better understanding MM and our treatments rather than an ASH about new treatments. There is an explosion of good work geared towards understanding mechanisms of resistance to immunotherapy, both intrinsic to the clonal cells and related to the immune environment. This will be crucial as we learn how to best choose and sequence the many available new therapies.

clonoSEQ® is available as an FDA-cleared in vitro diagnostic (IVD) test service provided by Adaptive Biotechnologies to detect minimal residual disease (MRD) in bone marrow from patients with multiple myeloma or B-cell acute lymphoblastic leukemia (B-ALL) and blood or bone marrow from patients with chronic lymphocytic leukemia (CLL). clonoSEQ is also available for use in other lymphoid cancers and specimen types as a CLIA-validated laboratory developed test (LDT). For important information about the FDA-cleared uses of clonoSEQ including test limitations, please visit clonoSEQ.com/technical-summary

The post Clinically Actionable Insights: A Q&A with Dr. Luciano Costa on the Final Analysis of MASTER appeared first on Adaptive Biotech.

In a 2017 publication¹, Dr. Vinod Balachandran, surgical oncologist at Memorial Sloan Kettering Cancer Center (MSKCC), worked with Adaptive to leverage our T-cell receptor beta (TCRB) sequencing assay on research that found long-term survivors of PDAC could mount spontaneous T-cell responses against tumor-specific neoantigens not shared among patients. From there, he hypothesized that personalized mRNA vaccines could provide clinical benefit.

Results from Balachandran’s Phase 1 study² of a personalized mRNA vaccine were recently published in Nature, and demonstrated a potential breakthrough for PDAC treatment. Again, Adaptive Immunosequencing provided critical insight into the T-cell response elicited by the vaccine, and its correlation with delayed disease recurrence.

T-Cell Clonal Expansion as a Potential Prognostic Biomarker

To investigate the diversity and specificity of T-cell clonal expansion generated by the mRNA vaccine, the study authors developed a mathematical method using Adaptive Immunosequencing that analyzed peripheral blood pre- and post-vaccination. They identified vaccine-induced expanded clones in 8 of 8 responders and 1 of 8 non-responders, serving as a potential prognostic biomarker of disease recurrence. Adaptive Immunosequencing provided a quantitative and molecular characterization of T-cell clones, allowing for identification of vaccine vs immune checkpoint inhibitor (ICI) expanded T cells and uncovering a T-cell response in a non-responder that was missed by ELISpot (a traditional functional assay).  

About the Study

Balachandran and his team of MSKCC researchers shipped samples of PDAC patient tumors to scientists at BioNTech and Genentech. The tumors were sequenced to identify individual patient neoantigens to create a patient-specific vaccine, autogene cevumeran.

The vaccine was given in combination with an ICI and chemotherapy after surgery. At 18-months median follow-up, half of those treated (eight patients) who had T-cell responses to autogene cevumeran were recurrence-free, with a significantly longer recurrence-free survival compared to 13.4 months median in eight non-responders. 

A Critical Tool in Drug Development

This study highlights how Adaptive Immunosequencing can assess response and provide quantitative data to inform the development of new vaccines that can have significant impact on patient lives. This is the power of Immune Medicine in action.

References:

1. Balachandran VP, Leach SD, et al. Identification of unique neoantigen qualities in long-term survivors of pancreatic cancer. Nature (2017)
2. Rojas LA, Balachandran VP, et al. Personalized RNA neoantigen vaccines stimulate T cells in pancreatic cancer. Nature (2023)

The post Immune Medicine in Action: How Adaptive Immunosequencing Helped Validate a Personalized Vaccine Trial in PDAC appeared first on Adaptive Biotech.

In honor of Multiple Myeloma Awareness Month, we sat down with Dr. Hamza Hashmi, a hematologist-oncologist from Memorial Sloan Kettering Cancer Center, to understand how his family’s personal experience with cancer has influenced his approach to treating and caring for patients living with myeloma and other plasma cell disorders.

As an oncology specialist, Dr. Hashmi is accustomed to his friends and family turning to him for guidance or additional context when a loved one is diagnosed with cancer, as they seek ways to cope with the situation. His siblings are also physicians, and they often rely on one another for consults across their respective specialties. So, when he received a message from his brother with images of a patient’s labs in May 2023, he proceeded to provide his expertise as usual.

“I went through the case and immediately recognized the unmistakable signs of advanced-stage, high-risk myeloma. The indications were all there – high calcium, high protein, and poor kidney function,” said Dr. Hashmi. “But when I looked at the patient’s name, a realization hit me, it was Samia, our mother.”

This came as a big surprise as even the week prior, Samia seemed to be in perfect health, enjoying time with her children and grandchildren, and as an active member of her local yoga group. The shift in her health was sudden, and it continued to deteriorate quickly.

After her diagnosis, the siblings became deeply involved in her care decisions. Yet for Dr. Hashmi, each choice weighed heavily.  Despite his expertise, his emotions threatened to cloud his judgment and he began to second-guess himself at every turn. Dr. Hashmi sought counsel from peers and mentors, ensuring that he continued to pursue the best possible care for his mother.

“Up until this moment, I had seen thousands of patients with myeloma, treating them with rounds of chemotherapy, transplants and novel, state-of-the-art therapies. But I’d never been in the role of making these decisions as a caregiver. Part of my role is to determine whether patients are well enough to go through the rigors of a transplant. Knowing my mother’s age and seeing her frailness, I had to make the very difficult decision that unfortunately, she was not fit for this. She may not even survive it.”

During Samia’s hospital stays, Dr. Hashmi gained a newfound respect for the various players who keep our healthcare system running: the nurses, physical therapists and support staff who selflessly contribute to all aspects of patient care. He also came to recognize the dedication and commitment often required of caregivers as they bring their loved ones home.

As his mother underwent chemotherapy, the same treatment he had provided to countless patients over the years, Dr. Hashmi came to understand the treatment’s impact on a patient’s quality of life like never before. The side effects plaguing his mom – weakness, fatigue, aches, and pains – became far more than just symptoms listed in a medical chart.

“I now saw these side effects through the lens of a caregiver, and the major impact they had on my mother’s quality of life. I realized there’s so much more that I wanted to do to help not only her, but all the patients I support.”

This journey has transformed Dr. Hashmi’s approach to patient care, balancing his previous aim to completely eradicate cancer with a more patient-centric approach that considers each individual patient’s quality of life. This experience informs his philosophy as he advocates for more personalized treatment plans and even the potential cessation of therapy as appropriate to improve his patients’ well-being.

For several years, Dr. Hashmi has used measurable (also known as minimal) residual disease (MRD) testing, specifically clonoSEQ®, with many patients in his practice. This tool helps to detect, quantify, and monitor MRD – the residual malignant cells that can be present at very low levels in a patient’s body after treatment, which are a strong predictor of outcomes. Results from the test can help physicians monitor subtle but important changes in disease burden over time, enabling personalized treatment decisions, including if their patient can safely de-escalate or discontinue therapy – which can be key to helping to restore a patient’s quality of life.

While Dr. Hashmi’s mother is not yet ready to go off treatment, he plans to use clonoSEQ once she is ready to consider next steps for her treatment, enabling his family to prioritize quality of life.

clonoSEQ® is available as an FDA-cleared in vitro diagnostic (IVD) test service provided by Adaptive Biotechnologies to detect measurable residual disease (MRD) in bone marrow from patients with multiple myeloma or B-cell acute lymphoblastic leukemia (B-ALL) and blood or bone marrow from patients with chronic lymphocytic leukemia (CLL). clonoSEQ is also available for use in other lymphoid cancers and specimen types as a CLIA-validated laboratory developed test (LDT). To review the FDA-cleared uses of clonoSEQ, visit clonoSEQ.com/technical-summary.

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Understanding these signatures is crucial as they offer a window into the body’s adaptive immune responses, revealing how T cells differentiate and mobilize in the face of diverse threats. This knowledge is instrumental in developing targeted therapies and vaccines. By mapping the intricate relationship between T-cell responses and exposures, researchers can identify immune system malfunctions and craft interventions that bolster our defense mechanisms against diseases.

At Adaptive, we have spent over a decade generating the largest collection of immune receptor data from tens of thousands of donors. Together with our partners at Microsoft Research, we have developed computational models to infer the HLA type of a donor from their T-cell receptor (TCR) repertoire. We’ve also associated millions of “public” TCRs (TCRs that occur in many people) with the HLAs that present the antigens that the TCRs respond to. The combination of these two capabilities – to infer a donor’s HLA type and to catalog the public TCRs associated with HLAs – enables new kinds of discoveries.

Adaptive and Microsoft have released three manuscripts on bioRxiv, shedding light on this complex relationship and offering new avenues for research and clinical applications.

Large-scale mapping of TCRs to HLAs

In the first manuscript, “Large-scale statistical mapping of T-cell receptor β sequences to Human Leukocyte Antigens,” we analyze Adaptive Immunosequencing data from >4,000 subjects with known HLA type to statistically associate a million public TCRs with hundreds of HLAs. This in turn allows us to build accurate models for imputing HLA genotypes from TCR repertoires. The reason that these TCRs associate with an HLA is that each TCR responds to some HLA-presented antigen that is part of a prevalent immune exposure, and so these HLA-associated TCRs collectively represent the public responses to many different exposures.

Constructing ECOclusters: the public responses to individual immune exposures

The second manuscript, “Identifying immune signatures of common exposures through co-occurrence of T-cell receptors in tens of thousands of donors,” leverages HLA-associated TCRs and immune repertoires from more than 30,000 donors to construct “ECOclusters” (Exposure-associated Co-Occurrence clusters), groups of HLA-associated TCRs that tend to co-occur in the same people. Each ECOcluster putatively represents the “public” T-cell response to some virus, bacterium, or other prevalent exposure. We use T-cell repertoires from donors of known serological status for 7 exposures (Cytomegalovirus, SARS-CoV-2, HSV-1, HSV-2, EBV, Parvovirus and T. gondii) to identify a single ECOcluster associated with each exposure.

Exploring the Cytomegalovirus (CMV) ECOcluster, a new public resource

A third manuscript, “A catalog of the public T-cell response to Cytomegalovirus,” makes the 26,106-TCR ECOcluster associated with Cytomegalovirus (CMV) publicly available and explores it in detail. We use TCR sequence similarity within ECOclusters to identify groups of TCRs that appear to respond to the same antigen, and we find suggestions of different subgroups of CMV-exposed donors responding to different antigens. CMV has clinical relevance in certain settings, like transplant rejection. More broadly, the CMV ECOcluster represents the first comprehensive catalog of the public T-cell response to a virus with a profound impact on the T-cell repertoire.

Combined, these three manuscripts demonstrate important new capabilities but also highlight a great enigma still to be solved. We’ve defined the public T-cell responses to many prevalent immune exposures, and we can use them to construct a ledger of an individual’s historical exposures. However, most of the entries in that ledger remain a mystery: currently, we only know the identity of seven of them. Through future work with collaborators, and by experimentally associating more TCRs with their HLA-presented antigens, we will learn the exposures associated with more and more ECOclusters, adding labels to this new catalog of public immune responses.

These capabilities have applications beyond infectious disease. Adaptive trains AI/ML models to help accelerate our target and drug discovery efforts. For example, in autoimmune disorders, we can infer the HLA types of affected individuals, to isolate the HLA presentation of specific autoimmune responses and help identify their target antigens. We can also use our catalog of TCR responses to public exposures to explore the intersection between pathogen response and autoimmunity, building our knowledge of the mechanism of disease.

These new capabilities extend Adaptive’s reach as a key player in an era of novel target discovery and development of immune-mediated therapeutics to help tackle devastating diseases. We look forward to decoding more and more of the public responses to prevalent immune exposures, so we can realize a future where we fully leverage diagnostic and therapeutic power of the adaptive immune system.  

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Pride Month is an opportunity to celebrate the history and progress made by the lesbian, gay, bisexual, transgender, queer (LGBTQ+) community, while also reflecting on the ongoing challenges faced today. As the lead of the Adaptive Pride employee resource group (ERG), we arranged exciting programming with onsite lunches, social events, and speaking engagements focused on how to support, serve, and increase Adapter awareness and understanding of the LGBTQ+ community.

We kicked off the month in the sweetest way possible—with a rainbow cookie baking demonstration. As we whipped up these colorful treats, we were reminded how diversity enriches our lives and our workplace. Each ingredient, like each person, adds its own unique flavor to the mix, creating something truly wonderful when we come together.

Later in the month, we hosted a Lunch & Learn in collaboration with the Rainbow Center, a resource hub and safe space for the LGBTQ+ community located in Tacoma, Washington. Each year, the Rainbow Center provides direct services to 1,000 individuals, welcomes almost 4,000 visits, and touches the lives of thousands more through events and partnerships. 

Mary Woodard, education manager of the Rainbow Center, joined us virtually to teach us about the history of the LGBTQ+ Rights Movement in the U.S.—from the founding of the earliest LGBT rights advocacy organization in Chicago (1924), to the Stonewall Riot (1969), the first Pride March (1970), through the codification of the 14th Amendment, and legal right to marry (2015). This was followed by a discussion on the current state of LGBTQ+ advocacy, including a deep dive into the ACLU anti-LGBTQ bills tracker, with an explanation of the various types of attacks on protections and rights. For allies looking to deepen their involvement in advocacy, Mary emphasized the importance of expanding their understanding of the issues or challenges the community may be facing in their region or state.

Pride Month is an opportunity to look back on LGBTQ+ history, recount how we arrived at this moment in time, and contemplate how we move into the future and practice making the world a more inclusive and respectful place. It’s a time when allies can learn more about the diverse experiences, challenges, expressions, and personalities in the community. These learnings will serve as an inspiration well beyond the month of June.

The post Pride Month at Adaptive: Celebrating Progress and Community appeared first on Adaptive Biotech.

At Adaptive, we focus on the development and commercialization of clinical products that improve people’s lives. We also measure success by recognizing the value of our people, who make it all happen. This is why we are on a mission to perpetually enhance our culture and create an environment that embraces and celebrates individuality. This is because we know that fundamentally, you can’t be your best if you can’t be yourself.

Given our steadfast mission and inclusive culture, we have made meaningful enhancements to our programs and benefits – including gender affirming care benefits, HIV prevention, services and pharmacy benefits, parental leave benefits, and our employee assistance program — and organized them into one LGBTQ+ Benefits Guide for easier accessibility. Additionally, throughout 2024, our PRIDE employee resource group has continued to host a number of social events and speaking engagements focused on how all of us can support, serve, and increase Adapter awareness and understanding of the LGBTQ+ community.

We want to personally thank every Team Member, Manager, and Senior Leader who took steps small and large to thoughtfully create an inclusive culture that allowed this community to be seen, to be celebrated, and to thrive.  And we are particularly grateful to the many members of the Adaptive PRIDE employee resource group who, over the last 5 years, have encouraged and challenged us as a company to continuously raise our bar ever higher.  

As we look ahead, we know that we must always be evolving to meet the ever-changing needs of our Adapters in the years to come.  Adaptive’s mission is to create the best conditions for everyone to bring their whole self to work, optimize their Adapter Experience, and ultimately maximize our collective individual and professional success.

The post Adaptive Earns Cornerstone Recognition for LGBTQ+ Workplace Inclusion in HRC’s 2025 Corporate Equality Index appeared first on Adaptive Biotech.

Facing cancer takes courage. It also requires collaboration: patients and their physicians working together to gain the information they need throughout the treatment journey. At Adaptive, we are proud to develop advanced testing methods that can provide these insights as patients and their care teams manage blood cancer care.

In honor of the important relationships between patients and their providers, this Blood Cancer Awareness Month, we sat down with Jim B., who lives with diffuse large B-cell lymphoma (DLBCL), and his hematologist, Dr. Jie Wang from Duke Blood Cancer Center, to discuss how clonoSEQ® testing supported his care.

Jim has always thrived on building connections with others. Married to his wife, Anne, for 47 years, Jim earned his degrees in cognitive psychology and information science before pursuing a successful career in user interface, software and network engineering at Bell Labs, and later running his own landscaping business. He is an avid cyclist, who together with his cycling club, has completed multiple long rides including a ride across North Carolina.

However, Jim’s life took an unexpected turn in 2023 as he prepared for the Ride Around Mt. Rainier in One Day (RAMROD) cycling event. After a 200-mile week of training, he noticed he wasn’t recovering as expected. “I know my body and knew what it took to recover, but another week went by, and I wasn’t feeling right,” Jim recalls. A visit to his doctor revealed low white and red blood cell counts. He was referred to Dr. Wang and was ultimately diagnosed with DLBCL, the most common form of non-Hodgkin lymphoma.

Jim’s relationship with Dr. Wang became central to his treatment journey. Dr. Wang has dedicated her life to treating cancer and prides herself on understanding and supporting her patients as individuals, not just by their disease. With this philosophy, Dr. Wang’s approach was not only to treat Jim but to consider how treatment might impact his overall well-being and active lifestyle. She made a concerted effort to balance the intensity of chemotherapy with Jim’s physical capabilities.

Dr. Wang and Jim made the decision to monitor for evidence of remaining cancer not only by PET/CT but also by clonoSEQ, which can detect minimal residual disease (MRD).  Clear PET CTs and consecutively negative clonoSEQ test results contributed to the decision to forego the 6th and final chemotherapy treatment. This timely adjustment in his treatment plan reduced unnecessary exposure to chemotherapy and its side effects.

Knowing that his MRD status was negative gave Jim his confidence back. He began reconnecting with friends and family. He and Anne embarked on a dream trip to Antarctica, followed by travels to Patagonia, Argentina and Chile. Ultimately, his IV port was removed, marking another milestone in his recovery. He now relies on clonoSEQ as an “early warning system” to monitor for potential recurrence of his cancer, giving him the reassurance he needs to continue doing what he loves, and getting back on his bike.

Jim’s journey underscores the importance of empowering patients to participate actively in their care and how collaboration can lead to successful outcomes in cancer treatment. As Dr. Wang says, “Jim and I are copilots in his story. We make decisions together.”

clonoSEQ® is available as an FDA-cleared in vitro diagnostic (IVD) test service provided by Adaptive Biotechnologies to detect measurable residual disease (MRD) in bone marrow from patients with multiple myeloma or B-cell acute lymphoblastic leukemia (B-ALL) and blood or bone marrow from patients with chronic lymphocytic leukemia (CLL). clonoSEQ is also available for use in other lymphoid cancers and specimen types as a CLIA-validated laboratory developed test (LDT). To review the FDA-cleared uses of clonoSEQ, visit clonoSEQ.com/technical-summary.

The post Jim’s Journey: The Power of Collaboration in Blood Cancer Care appeared first on Adaptive Biotech.

ARVYS Proteins Inc. provides a full spectrum of protein biochemistry services – recombinant protein expression in bacterial, insect and mammalian cells, protein purification, refolding, assays and assay development, protein characterization, fermentation and endotoxin removal.

Outsource your protein biochemistry projects to ARVYS and enjoy superior results, team expertise and customer support after project completion.

(This article belongs to the Special Issue Monoclonal Antibodies and Their Functional Fragments in Research, Diagnosis and Therapy)

Abstract

Antibodies and antibody-derived molecules are continuously developed as both therapeutic agents and key reagents for advanced diagnostic investigations. Their application in these fields has indeed greatly expanded the demand of these molecules and the need for their production in high yield and purity. While full-length antibodies require mammalian expression systems due to the occurrence of functionally and structurally important glycosylations, most antibody fragments and antibody-like molecules are non-glycosylated and can be more conveniently prepared in E. coli-based expression platforms. We propose here an updated survey of the most effective and appropriate methods of preparation of antibody fragments that exploit E. coli as an expression background and review the pros and cons of the different platforms available today. Around 250 references accompany and complete the review together with some lists of the most important new antibody-like molecules that are on the market or are being developed as new biotherapeutics or diagnostic agents.

1. coli as Microbial Expression Stem for the Production of Antibody Fragments
2. coli is one of the most well-established cell factories for the production of recombinant proteins (RPP) [43]. Currently, many molecular tools and protocols are available for the high-level production of heterologous proteins, including a vast catalog of expression plasmids and of engineered strains and many cultivation strategies. From a theoretical point of view, the steps needed for obtaining a recombinant protein are pretty straightforward. The gene of interest (GOI) is cloned in whatever available expression vector, is transformed into the host of choice, expression is induced and the protein is then ready for purification and biochemical, structural and functional characterization. Practically, however, many things can go wrong such as poor growth of the host, inclusion body (IB) formation, protein inactivity, and even lack of protein expression. Choosing the perfect combination is not possible a priori, thereby multiple conditions should be empirically tested to obtain a soluble and active recombinant protein.

Conclusions

The ever-increasing applications of antibody-based molecules as both therapeutic agents and key reagents for advanced diagnostic investigations have greatly expanded the demand of these crucial classes of molecules and the need for their production in high yield and purity. While the preparation of whole antibody molecules requires eukaryotic expression systems, antibody fragments like Fabs, scFvs and other similar surrogates that lack the glycosylation, can be conveniently prepared in E. coli backgrounds. The recent evolution of E. coli expression systems reinforces the use of this easy and cheap host-microorganism for an advisable production of antibody fragments in recombinant form. In Table 2, we report an updated list of antibody fragments described so far and the expression conditions utilized for their production, including expression localization (periplasmic, cytoplasmic), the type and format of antibody fragment, the vectors used, the inductor, the temperature and time of expression, the strain, the medium, the use of chaperones and the overall recovery. As explained in this review and reported in Table 2, during the last years, huge efforts have been done to adapt at best the E. coli machinery to the production of these “magic bullets” molecules, and a contribution has been also provided by structural biology and bioinformatics in addition to advanced molecular genetics, basic biology and chemical biology. Notable progress gas also emerged from the ever-increasing understanding of the unique structural features of the Ig-like domains as obtained by X-ray crystallography and accurate homology modeling studies. The structural knowledge of such basic units indeed provides a relevant contribution in the construct design for tuning the physicochemical and affinity properties and to improve the stability, the efficacy and the clinical potential of antibody-like molecules. Despite the high degree of similarity between the different components of this class of proteins and the growing availability of innovative strategies and robust tools, such as oxidizing mutant strains or plasmids for the overexpression of chaperones and foldases, and new growth media, it is evident that there is not a universal E. coli-based methodology for their efficient production, therefore, a trial and error optimization process is necessary for the determination of the successful experimental settings and to achieve scalability of antibody fragment expression processes at an industrial level.

To read the full journal, click here.

ARVYS Proteins Inc. provides a full spectrum of protein services to the life science, pharmaceutical and biotechnology communities.  Our work experience encompasses almost every aspect of protein biochemistry allowing us to contribute to projects regardless of whether they are at early research or late development stages.  We can be your partner in:

– generation and expression of recombinant proteins in bacterial, baculovirus and mammalian  expression systems,

– large-scale fermentation,

– cell culture,

– purification of recombinant proteins, antibodies or naturally occurring proteins,

– refolding from inclusion bodies,

– improvement of protein stability,

– protein labeling with fluorescent, biotin and enzyme probes,

– endotoxin removal and testing for in vivo studies,

– protein characterization to monitor its integrity and functionality

ARVYS Proteins Inc. is a Contract Research Organization (CRO) that Specializes in Custom Protein Services for Drug Discovery and Life Science Research.

The post A look at: “Evolution of Escherichia coli Expression” appeared first on Arvys Proteins.

Outsource your protein biochemistry projects to ARVYS and enjoy superior results, team expertise and customer support after project completion.

Department of Bioengineering, University of Illinois at Chicago, Chicago, IL 60607, USA and 2Institut National de Recherche en Informatique et en Automatique, Paris 75012, France

Abstract

Geometric and topological properties of protein structures, including surface pockets, interior cavities and cross channels, are of fundamental importance for proteins to carry out their functions. Computed Atlas of Surface Topography of proteins (CASTp) is a web server that provides online services for locating, delineating and measuring these geometric and topological properties of protein structures. It has been widely used since its inception in 2003. In this article, we present the latest version of the web server, CASTp 3.0. CASTp 3.0 continues to provide reliable and comprehensive identifications and quantifications of protein topography. In addition, it now provides: (i) imprints of the negative volumes of pockets, cavities and channels, (ii) topographic features of biological assemblies in the Protein Data Bank, (iii) improved visualization of protein structures and pockets, and (iv) more intuitive structural and annotated information, including information of secondary structure, functional sites, variant sites and other annotations of protein residues. The CASTp 3.0 web server is freely accessible at http://sts.bioe.uic.edu/castp/.

Introduction

Protein structures are complex and are sculpted with numerous surface pockets, internal cavities and cross channels. These topographic features provide structural basis

and micro-environments for proteins to carry out their functions such as ligand binding, DNA interaction and enzymatic activity. Identification and quantification of these topographic features of proteins are therefore of fundamental

importance for understanding the structure–function relationship of proteins (1), in engineering proteins for desired

properties (2) and in developing therapeutics against protein targets (3).

THE CASTp SERVER

The CASTp server aims to provide comprehensive and detailed quantitative characterization of topographic features of proteins (14,15). Since its release 15 years ago, the CASTp server has ∼45 000 visits and fulfills ∼33 000 calculation requests annually. It has been proven to be a useful tool for a wide range of studies, including investigations of signaling receptors (1), discoveries of cancer therapeutics (16), understanding of mechanism of drug actions (17), studies of immune disorder diseases (18), analysis of protein–nanoparticle interactions (19), inference of protein functions (20) and development of high-throughput computational tools (21,22).

To provide additional useful information and to deliver improved user experience, we introduce here an updated server called CASTp 3.0. All important features of the previous versions of the server are retained, including detecting and characterizing cavities, pockets and channels of protein structures (Figure 1). In addition, we have substantially extended its functions by providing pre-computed topographic features of biological assemblies in the PDB database, as well as imprints of negative volumes of these

Read more, click here.

ARVYS Proteins Inc. provides a full spectrum of protein services to the life science, pharmaceutical and biotechnology communities.  Our work experience encompasses almost every aspect of protein biochemistry allowing us to contribute to projects regardless of whether they are at early research or late development stages.

The post A look at: “CASTp 3.0: computed atlas of surface topography of proteins” appeared first on Arvys Proteins.

Outsource your protein biochemistry projects to ARVYS and enjoy superior results, team expertise and customer support after project completion.

Affiliations

1. Oxford Expression Technologies Ltd, Bioinnovation Hub, Oxford, UK.
2. Department of Biological Medical Sciences, Oxford Brookes University, Oxford, UK.

Abstract

Over 35 years since it was established to make recombinant proteins, the baculovirus expression vector system continues to develop and improve. Early systems for recombinant virus selection were laborious, but better methods were rapidly devised that enabled non-virologists to use baculovirus vectors successfully in a wide range of applications. These applications include multiple gene expression for complex molecules, production of adeno-associated virus-like particles for gene therapy, the use of baculovirus budded virus for the same purpose, numerous potential human and animal vaccines, and for other therapeutic proteins. A number of products for human and veterinary use are now on the market, which attests to the utility of the systems. Despite these successes, baculovirus vectors essentially remain in a relatively primitive state of development. Many proteins, particularly membrane-bound or secreted products, continue to be difficult to produce. Various research groups are working to identify potential areas of improvement, which if combined into an ideal vector might offer considerable advances to the system. This chapter will review some of the most recent reports and highlight those that might have generic application for recombinant protein synthesis in insect cells. We also summarize parallel developments in host cells used for baculovirus expression and how culture conditions can influence protein production.

Future Developments

3.0 Future developments

The two original publications on the use of baculoviruses as expression vectors were significant milestones in demonstrating that both secreted eukaryotic proteins as well as bacterial products could be synthesised in insect cells (Smith et al., 1983; Pennock et al., 1984). These were later followed by the use of the silkworm baculovirus (BmNPV) as a convenient vehicle for protein expression in whole insect larvae (Maeda et al., 1985). The complete genome sequences of both AcMNPV and BmNPV were published subsequently and have furnished valuable information on virus gene function that has aided the further development of baculovirus expression vectors (Ayres et al., 1994; Gomi et al., 1999, respectively). However, to date such modifications made to baculovirus expression have been relatively minor in their scope. The deletion or silencing (2.5) of a few virus genes have helped improve protein expression is some cases. Conversely, the addition of various genes that encode “helper” functions such as chaperones (2.4) have also been beneficial. Another very significant recent milestone was the production of a synthetic baculovirus genome based on AcMNPV (Shang et al., 2017). This virus (AcMNPV-WIV-Syn1) was assembled using a combination of PCR and transformation-associated recombination in yeast. It was fully infectious for both cell cultures and insect larvae. This technically very difficult feat opens the way to the design of minimal baculovirus replicons that might offer improved features as expression vectors. Baculovirus genes could be omitted or other genes added to optimize the synthesis of different recombinant proteins. We have shown in this brief review, how many groups have shown advantages to various strategies for improving baculovirus expression vectors. What is apparent is how difficult it is to extrapolate results from one system to another. The challenge now is to draw these modifications together in a rational way as well as paying attention to how these might be integrated with improvements in the host cells currently used for protein expression.

Read more, click here.

ARVYS Proteins Inc. provides a full spectrum of protein services to the life science, pharmaceutical and biotechnology communities.  Our work experience encompasses almost every aspect of protein biochemistry allowing us to contribute to projects regardless of whether they are at early research or late development stages.

The post A look at: “Recent Developments in the Use of Baculovirus Expression Vectors” appeared first on Arvys Proteins.

Outsource your protein biochemistry projects to ARVYS and enjoy superior results, team expertise and customer support after project completion.

Affiliation – Stockholm Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, The Arrhenius Laboratories for Natural Sciences, Stockholm, SE-10691, Sweden.

Abstract

Membrane proteins control a large number of vital biological processes and are often medically important-not least as drug targets. However, membrane proteins are generally more difficult to work with than their globular counterparts, and as a consequence comparatively few high-resolution structures are available. In any membrane protein structure project, a lot of effort is usually spent on obtaining a pure and stable protein preparation. The process commonly involves the expression of several constructs and homologs, followed by extraction in various detergents. This is normally a time-consuming and highly iterative process since only one or a few conditions can be tested at a time. In this article, we describe a rapid screening protocol in a 96-well format that largely mimics standard membrane protein purification procedures, but eliminates the ultracentrifugation and membrane preparation steps. Moreover, we show that the results are robustly translatable to large-scale production of detergent-solubilized protein for structural studies. We have applied this protocol to 60 proteins from an E. coli membrane protein library, in order to find the optimal expression, solubilization and purification conditions for each protein. With guidance from the obtained screening data, we have also performed successful large-scale purifications of several of the proteins. The protocol provides a rapid, low cost solution to one of the major bottlenecks in structural biology, making membrane protein structures attainable even for the small laboratory. 

Results:

We set out to design a protocol for rapid microscale solubilization and affinity purification in a 96‐well format, in order to be able to quickly evaluate whether a particular membrane protein is amenable to production for structural studies or not. For comparative purposes, we selected a battery of 16 commonly used detergents of varying size and head group chemistry, divided into five detergent families (Supporting Information Table SI). The screening protocol was engineered to be as fast and small‐scale as possible, while still simulating large‐scale preparation procedures closely; the latter to be able to identify conditions that allow for upscaling.

For the screening trials, 60 proteins were selected from a folding reporter GFP fusion membrane protein library where the membrane protein is followed by a TEV protease cleavage site, a C‐terminal folding reporter GFP molecule and a C‐terminal His‐tag [Fig. ​[Fig.11(A)].21 The proteins were chosen to cover a wide range of molecular weights and globular/transmembrane domain ratios (Table 1).

To buy the full journal, click here.

ARVYS Proteins Inc. provides a full spectrum of protein services to the life science, pharmaceutical and biotechnology communities.  Our work experience encompasses almost every aspect of protein biochemistry allowing us to contribute to projects regardless of whether they are at early research or late development stages.  We can be your partner in:

– generation and expression of recombinant proteins in bacterial, baculovirus and mammalian  expression systems,

– large-scale fermentation,

– cell culture,

– purification of recombinant proteins, antibodies or naturally occurring proteins,

– refolding from inclusion bodies,

– improvement of protein stability,

– protein labeling with fluorescent, biotin and enzyme probes,

– endotoxin removal and testing for in vivo studies,

– protein characterization to monitor its integrity and functionality

The post A Look At: “A rapid expression and purification condition screening protocol for membrane protein structural biology” appeared first on Arvys Proteins.

Outsource your protein biochemistry projects to ARVYS and enjoy superior results, team expertise and customer support after project completion.

Affiliation – Caroline Goussen, Laëtitia Goldstein, Corinne Brèque, Bruno You, Stéphane Boyer,Damien Bataille, Ludovic Burlot

LFB Biotechnologies, Direction Générale du Développement, 3 avenue des Tropiques, 91940 Les Ulis, France

Abstract

In response to the strong demand of biological protein therapeutics, such as monoclonal antibodies (MAbs), continuous downstream process was developed to deliver these molecules while maintaining desired product consistency and quality attributes, and providing manufacturing e ciency and  exibility. Viral safety is a cri- tical quality attribute for biopharmaceuticals, such as MAbs. Evaluation of the viral clearance by the downstream process is a key component of risk mitigation. Protein A chromatography is typically used as an initial capture step for MAbs and e cient for the removal of process-related impurities like Host Cell Proteins (HCP). This step can also contribute to the clearance of potential viral contaminants.

Murine Minute Virus (MMV)-spiking experiments were performed at small scale to investigate the impact on the viral clearance e ciency of the way the Protein A chromatography step is carried out, whether in batch or multicolumn mode. Protein A chromatography step using Novasep Sequential MultiColumn Chromatography (SMCC) technology demonstrated no statistical di erence in the viral reduction with reduction factor (RF) of 3.7 log10 (vs. RF of 3.8 log10 for batch). The experiments showed also similar viral distribution over the puri cation cycles and columns.

Data con rmed that the viral clearance capacity by the continuous Protein A chromatography step using SMCC technology is maintained and e cient.

Results:

MAb yield and HCP reduction in Protein A eluate fraction from continuous bio-chromatography and batch studies design are presented in Table 3. For continuous bio-chromatography, a MAb yield of 99% was reached with an HCP reduction of 3.3 log10. Evolution of MAb yield and HCP reduction through the 7 puri cation cycles is presented for one column (column 3) in Table 4. A small and limited variability of MAb yield and HCP reduction was observed from cycle 1 to 7. Variation in yield after cycle 3 was due to the use of a second starting material from cycle 4 to cycle 7. The starting material (HCCF) was stored frozen. To limit aggregation of the thawed starting material during the ex- periment, two preparations were performed. The rst was used until cycle 3 and the second from cycle 4 to the end. The di erences between the two feed stocks are considered without impact on the study results. The starting material was ltrated. For continuous bio-chromatography experiment, the yield was improved compared to puri cation in batch but HCP reduction was 1 log10 lower.

To read the full journal, click here.

ARVYS Proteins Inc. provides a full spectrum of protein services to the life science, pharmaceutical and biotechnology communities.  Our work experience encompasses almost every aspect of protein biochemistry allowing us to contribute to projects regardless of whether they are at early research or late development stages.  We can be your partner in:

– generation and expression of recombinant proteins in bacterial, baculovirus and mammalian  expression systems,

– large-scale fermentation,

– cell culture,

– purification of recombinant proteins, antibodies or naturally occurring proteins,

– refolding from inclusion bodies,

– improvement of protein stability,

– protein labeling with fluorescent, biotin and enzyme probes,

– endotoxin removal and testing for in vivo studies,

– protein characterization to monitor its integrity and functionality

The post A Look At – Viral clearance capacity by continuous Protein A chromatography step using Sequential MultiColumn Chromatography appeared first on Arvys Proteins.

Without any doubt, one of the greatest advances in molecular biology is the development of simple and easy to carry out protocols for the expression of proteins from Escherichia coli cells transformed by a plasmid. These plasmids are synthesized in the lab and typically contain three major regions of interest: an antibiotic resistance marker (for selection of successfully transformed cells), a lacZ promoter (for activation of a gene of interest upon addition of lactose), and a gene corresponding to the protein of interest. While other cell lines (and even cell-free systems) are capable of taking up plasmids to express a protein of interest, E. coli is most widely used because of its fast growth and high transformation efficiency. Even though recombinant protein expression is a relatively recent development, its rapid expansion into all facets of molecular biology and biochemistry has led to a remarkable standardization into the optimal expression conditions for E. coli. However, one should be aware of the purposes of these conditions; and the potential benefits of deviating from them.

One such condition that researchers seldom give any thought to is the temperature at which to express E. coli after induction. The vast majority of protocols call for expression to be carried out at 37℃ for anywhere from 2-4 hours. Since the optimal temperature for growing E. coli is 37℃, it makes sense that this is considered the default. While expressing at 37℃ is ideal for many proteins, it can introduce issues for others. For this reason, there are a myriad of benefits to expressing at lower temperatures between 10℃ – 15℃. The most significant benefit to a low-temperature expression is an increase in the solubility of the expressed protein. This can be due to many factors. For example, lower temperatures greatly increase the time proteins have to fold, which reduces overall protein aggregation. This factor in particular aids the expression of larger proteins, or those with highly complex folding processes. Another reason to express at lower temperatures is the increase of properly folded proteins. One major drawback to E. coli is that the rates of transcription and translation are not optimized for all proteins, which reduces the overall time that newly expressed proteins spend in bacterial chaperones, increasing the fraction of misfolded protein present in the sample. Lastly, expressing at lower temperatures decreases the amount of protein degradation that occurs during expression. E. coli contain endogenous proteases that remain active during a standard 37℃ expression, which can lead to a loss of yield. While rare, autoproteolysis can also occur during 37℃ expression, an issue that is also abated by expressing at low temperatures. The addition of most commercially available protease inhibitors can be detrimental to cell growth, so lowering temperature is the best bet for preventing degradation events from occurring.

While there are many advantages to expressing at low temperatures, there are a number of downsides as well. First, low-temperature expressions decrease all metabolic processes associated with cell growth, including protein production. This can lead to a reduced yield, especially without compensating for this loss of activity by increasing the overall expression time. Second, for best results, specialized E. coli cell lines should be used. These cell lines typically have recombinantly altered chaperones or polymerases that are most active at lower temperatures. Lastly, low-temperature expressions greatly increase the time for sufficient quantities of protein to be produced, which will reduce experimental throughput. These downsides can be mitigated by expressing at room temperature, which will also serve to lessen the downsides of standard 37℃ expressions. Room temperature expressions require less time than cold expressions (though still not as fast as 37℃), have greater yield, and can be done using typical E. coli cell lines.  The major takeaway from all of the factors should be that expression is not always ideal at 37℃, and the optimal temperature for expression should be optimized for each protein.

Which Temperature Should I Use?

Ideally, each protein should be fully characterized and its optimal expression temperature determined through repeated trials at each major temperature point (hot, cold, room temperature). Unfortunately, this process is laborious and incredibly time-consuming, so it is better to predict which temperature is best depending on the protein’s properties and working from there. A good place to start with a relatively unknown protein is the standard 37℃ protocol. If solubility is not an issue and the yield remains high, 37℃ should be used for the ideal temperature. If the protein is large, aggregation-prone, and/or is put into inclusion bodies, a low-temperature expression between 10℃ – 15℃ would be more suitable. For moderately insoluble proteins, expression conditions can be changed to room temperature to improve the overall yield and reduce time. While no substitute for a rigorous empirical comparison, these guidelines are a great place to start optimizing the temperature conditions for protein expression.

References:

https://aem.asm.org/content/73/15/4849

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC167914/

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The popularization of methods to obtain large quantities of proteins, peptides, and other large biomolecules has led to the advent of fast protein liquid chromatography (also known as fast-performance liquid chromatography or medium pressure liquid chromatography). FPLC utilizes a peristaltic pump to accurately control the speed at which the protein sample (mobile phase) passes through the chromatography column of choice (solid phase). Unlike other chromatography techniques like high-pressure liquid chromatography (HPLC), FPLC operates at a much lower pressure, ensuring that protein structure is not disrupted during purification. While many FPLC systems have been introduced since the technique’s introduction in the early 1980s, many research labs today utilize protein purification systems. ӒKTA models combine all components of a typical FPLC workflow (pump, pressure/UV sensors, fraction collector, etc.) into an automated, modular system compatible with most types of chromatography columns. The versatility and user-friendliness of these systems make them the workhorses of protein research in academic and industrial labs alike. For those seeking to switch from manual protein purification to an automated system like an ӒKTA, attempting to determine which of the many models currently available, and those that have come and gone, are right for you can be a daunting task. In this article, the differences between the four state-of-the-art ӒKTA systems will be compared to allow consumers to make the most informed decision as to which system is ideal for their lab. These systems are the ӒKTA start, ӒKTA go, ӒKTA pure, and ӒKTA avant. While other ӒKTA models have existed (notably the ӒKTA explorer and the ӒKTA purifier), these have been discontinued and are not readily available anymore. Thus, this article will focus on the four models that are still supported.

ӒKTA Start

ӒKTA Start

While at the lowest end of the available systems, the ӒKTA Start remains a desirable choice for those still on the fence about switching to an FPLC vs. traditional manual gravity-flow columns. The ӒKTA start may lack some of the bells and whistles of the higher-end systems, but affinity chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, and multimodal (mixed mode) chromatography can still be fully automated with this system. Size exclusion chromatography is doable as well, but due to the flow rate and pressure limitations (0.5 to 5.0 mL/min and 0.5 MPa, respectively), not all size exclusion columns are compatible with the ӒKTA start. Thus, for a lab that relies heavily on size exclusion chromatography, a higher-end ӒKTA with a wider range of available operating conditions would be more suitable. Regardless, the wide variety of applications makes the ӒKTA start ideal for most low-scale purifications.

ӒKTA Go

ӒKTA Go

Those who rely heavily on techniques like size exclusion chromatography or who would like more options in their setup than the ӒKTA start can offer should consider the ӒKTA Go. The ӒKTA go features a greatly enhanced flow rate (0.01 to 25 mL/min) and pressure tolerance (up to 5 MPa) which removes the limitations on size exclusion imposed by ӒKTA start. The ӒKTA go also features a modular fraction collection attachment, which in addition to normal fraction tubes can also accommodate deep-well plates, microplates, or small fraction tubes. This fraction collection attachment can also be set up underneath the system itself, which reduces the overall footprint of the system and saves on valuable bench space. An optional pH meter can be installed onto this system to track the pH of eluted fractions in addition to UV and conductivity readouts.

ӒKTA Pure

ӒKTA Pure

The ӒKTA Pure may be very similar to the ӒKTA go in terms of technical specifications and capabilities but offers much more in terms of customizability. The ӒKTA pure is offered in a variety of models, the two main categories of which are the ӒKTA pure 25 and the ӒKTA pure 150, the latter of which boasts a staggering 150 mL/min maximum flow rate for immensely scaled up protein purifications. Multiple columns can be hooked up to the ӒKTA pure simultaneously and can be automatically switched between using more advanced purification programs that this system is capable of. Much like the ӒKTA go, this automation is extended to the optional fraction collection system, which can accommodate deep-well plates, microplates, or small fraction tubes in addition to normal fraction tubes. More advanced features like resin scouting, pH monitoring, air sensors, etc. come in the form of optional upgrades that can augment the ӒKTA pure’s modular design with ease. These upgrades, in addition to the two base models, make the ӒKTA pure the most versatile among the available ӒKTA systems as it can be tailor-made to suit each individual’s needs.

ӒKTA Avant

ӒKTA Avant

The Avant is the highest end of all available ӒKTA systems, integrating many of the optional upgrades of the ӒKTA pure as standard features. These include pH monitoring, multi-wavelength detection, air sensors, and extended buffer inlets (18 inlets standard, option to upgrade further to 46). The ӒKTA Avant also has a couple of features that are exclusive to this higher-end system. The first is a fully integrated fraction collection system, which is enclosed and refrigerated, keeping protein fractions stable for long periods. The second is a buffer mixing system that utilizes the Avant’s numerous inlets to create column buffers automatically. This system is ideal for testing a variety of conditions for optimal protein purification, as opposed to making each buffer and testing them one at a time. The myriad of convenience and analytical tools that the ӒKTA Avant offers make it an ideal choice for those seeking to massively increase their throughput of protein purification or transition to higher scale preps.

Which One is Right For Me?

There are many different factors one should consider before purchasing an FPLC system like an ӒKTA. The first is how heavily your lab relies on protein purification services. For labs that seldom require producing their protein and have been running columns manually, a lower-end system like the ӒKTA start will likely suit their needs with its automation of many different types of chromatography. For labs that do purify proteins regularly and want to automate more advanced chromatography techniques like size exclusion, the ӒKTA go would be a good system to start with. If more advanced purification protocols with complex readouts are a common occurrence in your lab, a highly customizable system like the ӒKTA pure will satisfy all of those conditions while providing opportunities for future upgrades. For labs that purify proteins around the clock and need to scale up their workflow while increasing throughput, a high-end system like the ӒKTA Avant would be most ideal. While many intricate details of all ӒKTA systems have been omitted from this article for the sake of brevity, the general descriptions of the capabilities these systems offer should be sufficient to get prospective buyers looking in the right direction.

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The ability to synthesize proteins using recombinant DNA techniques has revolutionized molecular and cell biology research by enabling the study of target proteins in a laboratory setting. Most commonly, this is done by transforming live cells with a plasmid that contains the gene for the protein of interest and transcriptional activators. The affordability and high yields this method produces make it a desirable first choice for those seeking to study a particular protein of interest. However, in vivo protein expression is very laborious and time-consuming, often taking days to weeks to complete a single preparation. In recent years, several methods concerning in vitro protein expression have been developed that reduce this multi-day approach to a single day, sometimes even a few hours with the right amount of preparation. Cell-free protein synthesis (CFPS), also commonly referred to as in-vitro translation (IVT), seeks to enable researchers to produce proteins in the lab without spending multiple days or weeks culturing cells. While many CFPS systems have been characterized (E. coli cell lysates, wheat germ extracts, etc.) the process is generally the same: cells are cultured and lysed to produce a whole-cell extract that contains all of the biological machinery necessary to synthesize a protein from a gene sequence (DNA/RNA polymerases, ribosomes, chaperones, etc.). This extract can then be added to a master mix that contains all of the other necessary components, namely those necessary for the synthesis of all proteins (all essential amino acids, ATP/GTP energy sources for powering transcriptional/translational enzymes). Ancillary components can also be added to this master mix that is specific for certain proteins of interest, such as cofactors for metal-binding proteins or isotopic amino acids for synthesizing radiolabeled proteins. Once the final reaction mix is assembled, genetic information in the form of an mRNA sequence or even a recombinantly produced plasmid/linear expression template can be added to generate the protein of interest. CFPS is fast, easy, and has several advantages/disadvantages over traditional in vivo protein synthesis.

ScienceDirect.com

Advantages

As mentioned previously, the main advantage of CFPS is its speed. All components necessary for this reaction can be prepared in advance (cell extract, reaction master mix, genetic information) and stored for long periods. Rather than scheduling days of work to culture cells for expression, running a CFPS experiment is as trivial as thawing all of these components, setting up the reactions, and letting them run. Despite enabling this highly convenient workflow, the open environment of CFPS provides additional benefits. First, the lack of an enclosed environment means that reactions can be monitored in real-time using spectrophotometry. Samples for gel electrophoresis can be taken at various time points throughout the synthesis reaction to test for the presence of properly produced protein. CFPS is also commonly used for synthesizing proteins that are toxic to cells, and thus cannot be made in large quantities from the traditional in vivo method. These toxic proteins rarely affect the transcription/translation machinery necessary for producing them, thus CFPS is not negatively impacted by their presence. Lastly, CFPS allows for the easy incorporation of unnatural or modified amino acids into protein structure. These amino acids can simply be added to the master mix to ensure their eventual incorporation into the finished protein. This technique is ideal for producing radiolabeled protein, wherein amino acids that contain radioactive isotopes are added to the master mix so that the final protein can be quantified using a scintillation counter. 

Limitations

Even though there are many merits to CFPS, it has a few major flaws that prevent it from being the de-facto technique for laboratory protein synthesis, the first being the overall yield. The typical protocols for CFPS are simply incapable of producing large quantities of protein that are required for structural techniques or proteomics. While it is possible to scale up CFPS, the materials required to satisfy the high concentrations required for those techniques are simply not feasible under typical laboratory conditions, especially when compared to in vivo protein expression. Due to this limitation, CFPS is most commonly used for applications where only small quantities of a protein of interest are required. The second major limitation is sensitivity to nucleases or proteases. CFPS relies on the uninterrupted pairing of transcription and translation, meaning that mRNA messengers and final protein products are highly susceptible to degradation by nucleases/proteases, respectively, that are a part of the cell extract. While this is also an issue with in vivo protein synthesis, it is especially a problem for CFPS because of the lack of functional compartmentalization between individual cellular components. In other words, the cell extract contains the nucleases/proteases of many different cells whereas in vivo synthesis would contain discrete populations of protein products and enzymes. 

Should I Use Cell-Free Protein Synthesis?

The main consideration in whether a researcher should use CFPS is the desired application for the protein that is being produced. Is it one where a large concentration per sample is necessary? Does this application require multiple trials with the same protein? If the answer to either of these questions is “yes”, then CFPS is not the best expression choice. However, if low concentration samples will suffice, and many different proteins will need to be tested as part of this experiment, then CFPS will save countless hours of lab time due to its speed and compatibility with high-throughput experimentation. If none of these criteria apply and the experiment simply requires a small amount of protein that is toxic to cells or contains unnatural amino acids, then CFPS may also be the best choice for you.

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History of Spike Protein


The first infections caused by coronaviruses affected livestock and were observed in the early 20th century. Coronaviruses would not get their name, however, until the 1960s when viral particles were isolated and visualized using transmission electron microscopy (TEM) [1]. These particles possessed a round capsid and crown-shaped protrusions (“corona” being Latin for crown), which would later become known as spike protein. The spike protein first came to prominence in the early 2000s, when a new disease-causing virus began rapidly spreading through regions of mainland China. This disease would later become known as Severe Acute Respiratory Syndrome (SARS) and would be the first instance of widespread human-infecting coronaviruses. Aside from the aforementioned spike protein, other key coronavirus proteins were subsequently identified, these being the envelope (E), membrane (M), and nucleocapsid (N) proteins [2]. While SARS spread rapidly after its initial emergence, proper quarantine procedures prevented it from becoming a global pandemic. The next coronavirus to emerge caused the Middle Eastern Respiratory Virus (MERS) outbreak in 2012. Spike proteins were subsequently discovered to be heavily glycosylated, which spawned several hypotheses into how this facet of spike protein structure plays a role in its function [3,4]. Structural studies suggest that glycosylation is important not only for the recognition of potential host cells but also for proper folding and stabilization of the spike protein fusion trimers. Much like SARS-CoV, MERS-CoV consists of a protein capsid enclosed by a lipid membrane, with spike protein trimers decorating the surface. The conservation of spike protein structure across many different types of coronaviruses would further cement its importance. Spike protein research would explode in popularity after the outbreak of the COVID-19 pandemic, which spurred interest in all facets of coronavirus structure to develop better therapeutics for these types of viruses [4]. While many other types of coronaviruses have been discovered, both with human and non-human hosts, the main three of interest are SARS-CoV, MERS-CoV, and SARS-CoV2. While slight differences in spike protein structure between these three viruses do exist, the key domains of protein structure are mostly consistent.

Ways to Characterize It


Like all proteins, the function of spike protein is dictated primarily by its structure. Thus, spike protein is characterized by methods that allow researchers to obtain high-resolution structural data about its subunits in isolation and a fully assembled trimer. The predominant technique for studying spike protein structure is cryo-electron microscopy (Cryo-EM). This technique is a modification of traditional transmission electron microscopy (TEM) wherein aqueous samples are embedded onto a carbon-film grid then rapidly cooled. Samples prepared in this manner are protected from degradation by the high-energy electron beam and the vacuums that are associated with TEM, furthermore, the crystalline arrangement of biomolecules in the sample provides a myriad of perspectives that are used to generate a 3D image of the sample [5]. Cryo-EM has been used successfully to obtain structures of individual proteins, however, this becomes increasingly difficult with smaller proteins [5]. To circumvent this size issue, Cryo-EM is primarily used for large proteins or protein complexes such as spike protein trimers. Cryo-EM can also be used for the structure of two different proteins in association, such as a spike protein bound to its receptor. All of this structural data is vital to characterizing spike protein, whether it be the changes in structure between variants of coronaviruses or the key residues that are associated with spike protein binding to human receptors. Another commonly used structural technique is Nuclear Magnetic Resonance (NMR) spectroscopy. In NMR, a sample is exposed to a powerful magnetic field which causes molecular dipoles (the sources of which are typically carbon and hydrogen atoms for protein NMR) to align with this magnetic field. Once the magnetic field dissipates, the dipoles then shift back to their original positions. This shift is then detected by the instrument and then deconvoluted by a series of Fourier transforms to yield a final 2D spectrum [6]. This 2D spectrum is sufficient to determine the structure of small molecules, for proteins, a 3D spectrum is required. To obtain a 3D spectrum, all carbons and nitrogens in the protein must be isotopically labeled so that the final plot has individual peaks that each correspond to a single amino acid in the protein. After conducting a series of experiments to assign each peak to its corresponding amino acid, the protein’s structure can finally be solved. Much like Cryo-EM, there is a size limitation to the samples that can be studied with NMR. Traditional solution-based NMR is ideal for studying smaller molecules, so unlike Cryo-EM, it is primarily used for obtaining structures of the individual subunits of spike protein rather than the fully assembled complex [6].

What Systems are Used?


By a wide margin, the most commonly used expression system for proteins of interest is the prokaryotic organism E. coli. This system is commonly used because of its fast growth, high yield, and reliability. However, for applications that rely on specific immune-complex interactions, such as ELISA assays or vaccines, prokaryotic systems are not ideal [7].

Furthermore, it is very difficult to express the receptor-binding domain in prokaryotic systems because these systems cannot carry out glycosylation processes that are important for the proper folding of that domain. For the produced spike protein to be used in clinical applications such as antigen testing or vaccine development, it must be expressed using a mammalian system. Of these mammalian expression systems, there are a myriad of choices, but the most commonly used cell line for this purpose is the HEK293 cell line, with Expi293 performing the best [7]. Apart from the specificity that is required from spike protein produced from mammalian cells, it is also important that these expression systems produce a protein that can form stable trimers to better represent the spike proteins found on the mature virus. While the recent COVID-19 pandemic has shifted most of the focus towards clinical applications, and mammalian expression systems along with them, traditional prokaryotic expression systems are still in use. E. coli expression systems are ideal for producing large amounts of stable spike protein subunits, save for the aforementioned receptor-binding domain [8]. These subunits can be used for structural characterization and can even be used to form complete trimers (provided all three subunits are expressed) that can be used for antigen-detection assays such as ELISAs (though not at the specificity required for the aforementioned clinical applications) [8]. For example, E. coli expression systems are used for the structural characterization of the receptor-binding domain of spike protein. Yeast expression systems can also be used to purify spike protein, though not in as large quantities as from E. coli. Yeast expression systems are still superior to mammalian systems in terms of yield and scalability, and their eukaryotic nature overcomes the glycosylation issues that prevent the receptor-binding domain from being synthesized in large quantities from prokaryotic cells [9]. This makes them a candidate choice for an alternate expression system to mammalian cell lines, though the difference in final protein structure from yeast expression compared to mammalian expression is still under investigation.

What Constructs?


While not as important as the type of organism in which to express spike protein, plasmid constructs can still be vital to the final yield and the functionality of the final product. For prokaryotic expression, standard subcloning approaches may be utilized to insert a PCR-amplified spike protein subunit gene into plasmid vectors. While the type of plasmid to use varies depending on the desired application and expression workflow, some examples from the literature include pET32a, pETGEX-4t-2, and pET-MBP [10]. Keep in mind, these expression vectors should not be used to amplify the gene sequence before subcloning. For that application, a vector such as pET-S would be more appropriate. The process for making constructs for mammalian systems is very similar, though different plasmid vectors must be used. For example, the mammalian plasmid vector pCDNA3.1 was used to express the receptor-binding domain of spike protein along with a poly-Histidine tag and two extraneous sequences tailor-made for that specific experiment [11]. This trend also holds true for expression in yeast, the subcloning process and desired constructs are more or less identical with the only major difference being the template vector that the gene of interest is cloned into, an example for yeast would be pPICZalpha. This flexibility associated with construct design has certainly aided research into spike protein, though challenges still exist in the purification process, which will be discussed in a later section. Regardless, it is important to consider the characteristics of each expression system before choosing a template vector to make the final construct.

What is the Current Interest?


Like any viral protein, most of the current interest centered on spike protein relates to clinical applications such as vaccines and antibody screenings. This is especially true after the COVID-19 pandemic, wherein spike protein became the target of vaccine-based research. Since coronavirus infections are always initiated by the binding of spike protein to the ACE2 receptor, targeting antibodies to this protein will prevent viral particles from starting new infections and help to target cells that are currently undergoing the process of infection. Since spike protein is subject to degradation by the innate immune system if injected directly, key vaccine strategies involve the production of spike protein mRNA to induce human cells to produce the protein on their own [10]. The purpose of this is to utilize the acquired immune system to produce antibodies that will recognize virus particles should they enter the body. While most previously discussed interest in spike protein has related to its structure, this key application also relies heavily on sequencing. While one of the biggest hurdles to any vaccine development is the delivery aspect, mRNA vaccines simply would not be possible without the fully sequenced spike protein gene from which to make mRNA. Given the immense amount of diversity between different types of coronaviruses, and the fact that the spike protein, in particular, is subject to rapid mutation, getting a useful gene sequence is not as simple as sequencing the already well-characterized SARS and MERS viruses. Fortunately, after the identification of SARS-CoV2 in late 2019, the virus was isolated and had its genome sequenced to provide a consensus sequence for the spike protein in early 2020. This spurred a further investigation into the comparison between the gene sequences between SARS-CoV2 and other human infecting coronaviruses to better ascertain the type of receptor that it binds to. Antibodies that are produced in this manner, as well as antibodies that are produced as part of a natural immune response to the virus, can be detected using a diagnostic test to assess whether a person has been previously infected with SARS-CoV2 or has produced antibodies against spike protein. Further interest in spike protein is centered on the aforementioned structural studies to better understand viral infection, subunit assembly, and receptor binding [11]. All of these factors are critical to understanding how coronaviruses are capable of infecting hosts and how they assemble in living cells. Additionally, the high rate of mutation among these viruses means that structures are subject to slight variations over time. While the structures of the membrane and nucleocapsid proteins are highly conserved, spike protein tends to accumulate slight changes that correspond with host-virus interactions. For example, SARS-CoV2 has already produced variants (namely, the Delta variant) that have shown to be more lethal and/or infectious than the original strain that originated from Wuhan, China in 2019. Since spike protein is responsible for starting viral infections, most of the differences between these variants likely lie in the structure of spike protein, as more efficient receptor binding will lead to a greater infection rate. Initial data on spike protein structure of the delta variant showed changes in the furin cleavage site (a proteolysis sequence essential to viral infection), the deletion of residues 156-157 of the N-terminal domain, and the point mutation of amino acid 158 to a glycine (N terminal mutations of this nature typically correspond to reduced detection from the immune system). While mutations in the other viral proteins can certainly lead to a more deadly virus, spike protein will remain the center of attention due to its clinical importance.

What are the Challenges to Purifying Spike Protein?


Despite the vast amount of attention that has been centered around spike protein over the past decade, there remain purification issues that hinder further research. The first is that pre-fusion spike protein requires post-translational modification to adopt its mature form [12]. Traditional prokaryotic expression systems are not capable of delivering the post-translational modification required to study the entire protein, so truncated forms that do not require this modification are used instead. This approach is sufficient when focusing on individual parts of the protein, such as the receptor-binding domain, but is inadequate for use in diagnostic assays or other applications that require a fully-assembled spike protein trimer. When spike protein is expressed in prokaryotic systems, the resulting protein forms inclusion bodies [13]. Obtaining soluble protein from these inclusion bodies involves aggressively denaturing the inclusion bodies with chaotropic chemicals before dialyzing these chemicals out to refold soluble spike protein monomers. This approach is not ideal as its yield is very low due to a subpopulation of protein that is irreversibly damaged by the aggressive denaturation step [13]. To produce full-length subunits, production must be conducted in a eukaryotic expression system that is capable of carrying out the post-translational modification required. The main drawback to this approach is that the yields from most well-characterized yeast and mammalian cell lines are very low when compared to prokaryotic expression. While it is possible to get high yields from eukaryotic expression systems, a standardized protocol is yet to be seen in the literature. Even when full-length subunits are produced, some challenges remain. Spike protein is found in two major conformations: “up” and “down”. Spike protein can only bind to a host receptor when found in the “up” conformation, thus making it the more physiologically relevant form. Unfortunately, spike proteins found in the “up” conformation are very unstable in solution, even when kept at four degrees Celsius [12]. While the “down” conformation is much more stable, it cannot be used for applications such as receptor binding assays. Further research into spike protein could be aided significantly by developing a method to stabilize the “up” conformation in solution. Spike protein trimers are also extremely difficult to purify due to their size (~670 kDa), leading many groups to purify the monomers and allow them to oligomerize after purification. While there are certainly many challenges associated with expressing and purifying spike protein, many of them have already been addressed with promising directions for future groups to take them.

What is Known About Its Structure?


Individual spike protein monomers are composed of three major domains. These domains, in order of N to C terminal, are the N-terminal glycan binding, the ACE2 receptor binding, fusion peptide, and a heptad repeat coiled-coil [14]. Spike proteins can adopt two different conformations: one with the receptor-binding domain pointing up, and another with the receptor-binding domain pointing down. Coronaviruses can only bind a host cell with the spike protein in the up conformation, but this is less stable and makes it more vulnerable to immune system responses, so it spends most of its time in the down conformation [14]. The human enzyme angiotensin-converting enzyme 2 (ACE2) was identified as the receptor for the spike proteins of both SARS-CoV and SARS-CoV2, though MERS-CoV binds to dipeptidyl peptidase 4 (DPP4) [15]. Once fully assembled into the mature trimer, there are three distinct regions of interest: S1, S2, and IC. IC anchors the protein to the transmembrane region of the viral envelope (coronaviruses cloak themselves in lipid membranes that surround the protein-composed capsid). S1 is the crown-shaped region that contains the receptor-binding domain. Once a host cell is bound, S1 is proteolyzed which allows S2 to present a fusion peptide that further buries the virus into the host cell. Once this is achieved, the viral envelope merges with the cell membrane to complete the infection [15]. Spike protein is also decorated in glycosylation sites throughout the S1 and S2 regions. Attachment of sugars to the protein is theorized to help evade immune system responses.

Figure 1, from Wrapp et al. (2020) Science 367:1260. A) Sequence map of spike protein, with highlighted monomers. B) Cryo-EM structure of prefusion spike protein monomer with one RBD in the receptor-accessible “up” conformation.

Conclusion


Spike protein is a highly conserved trimeric protein that is essential for the survival of all coronaviruses. Despite knowledge of coronavirus infections dating back to the early 20th century, the many mysteries associated with this class of viruses are only beginning to be unraveled. Coronavirus research has taken off dramatically after the COVID-19 pandemic, leading to many developments in the structural characterization of spike protein as well as developing new ways to produce it in the lab. Structural characterization techniques such as NMR and Cryo-EM have revealed that spike protein has two primary conformations: a receptor-accessible “up” and receptor-inaccessible “down”. It is theorized that this sheathing of the receptor-binding domain helps to stabilize the protein for traveling long distances and shielding it from recognition by the host’s immune system. While many facets of spike protein structure and function have been elucidated, many challenges remain. An optimal expression system has yet to be determined, while there are benefits and downsides to all of the popular choices an ideal system has not been identified that can produce viable spike protein for all applications. Two applications of recent importance are serological assays and vaccine development, both of which are critical to clinical efforts against SARS-CoV2. Bacterial expression systems do not have the post-translational machinery necessary to produce full-length monomers, and mammalian expression systems are not high-throughput enough for the astronomically high demand imposed by this pandemic. While an impressive amount of progress has been achieved in just under two short years, there are still many challenges left to overcome.

References

1. Kahn, J. S., & McIntosh, K. (2005). History and Recent Advances in Coronavirus Discovery. Pediatric Infectious Disease Journal, 24(11). https://doi.org/10.1097/01.inf.0000188166.17324.60
2. Belouzard, S., Millet, J. K., Licitra, B. N., & Whittaker, G. R. (2012). Mechanisms of Coronavirus Cell Entry Mediated by the Viral Spike Protein. Viruses 2012, Vol. 4, Pages 1011-1033, 4(6), 1011–1033. https://doi.org/10.3390/V4061011
3. Du, L., He, Y., Zhou, Y., Liu, S., Zheng, B.-J., & Jiang, S. (2009). The spike protein of SARS-CoV — a target for vaccine and therapeutic development. Nature Reviews Microbiology 2009 7:3, 7(3), 226–236. https://doi.org/10.1038/nrmicro2090
4. Kirchdoerfer, R. N., Cottrell, C. A., Wang, N., Pallesen, J., Yassine, H. M., Turner, H. L., Corbett, K. S., Graham, B. S., McLellan, J. S., & Ward, A. B. (2016). Pre-fusion structure of a human coronavirus spike protein. Nature, 531(7592). https://doi.org/10.1038/nature17200
5. Bai, X. chen, McMullan, G., & Scheres, S. H. W. (2015). How cryo-EM is revolutionizing structural biology. In Trends in Biochemical Sciences (Vol. 40, Issue 1). https://doi.org/10.1016/j.tibs.2014.10.005
6. Mahajan, M., Chatterjee, D., Bhuvaneswari, K., Pillay, S., & Bhattacharjya, S. (2018). NMR structure and localization of a large fragment of the SARS-CoV fusion protein: Implications in viral cell fusion. Biochimica et Biophysica Acta – Biomembranes, 1860(2). https://doi.org/10.1016/j.bbamem.2017.10.002
7. Esposito, D., Mehalko, J., Drew, M., Snead, K., Wall, V., Taylor, T., Frank, P., Denson, J.-P., Hong, M., Gulten, G., Sadtler, K., Messing, S., & Gillette, W. (2020). Optimizing high-yield production of SARS-CoV-2 soluble spike trimers for serology assays. Protein Expression and Purification, 174. https://doi.org/10.1016/j.pep.2020.105686
8. Chen, J., Miao, L., Li, J.-M., Li, Y.-Y., Zhu, Q.-Y., Zhou, C.-L., Fang, H.-Q., & Chen, H.-P. (2005). Receptor-binding domain of SARS-Cov spike protein: Soluble expression in E.coli, purification and functional characterization. World Journal of Gastroenterology : WJG, 11(39), 6159. https://doi.org/10.3748/WJG.V11.I39.6159
9. Consortium, A. A. (2020). Structural and functional comparison of SARS-CoV-2-spike receptor binding domain produced in Pichia pastoris and mammalian cells. Scientific Reports 2020 10:1, 10(1), 1–18. https://doi.org/10.1038/s41598-020-78711-6
10. Malik, J. A., Mulla, A. H., Farooqi, T., Pottoo, F. H., Anwar, S., & Rengasamy, K. R. R. (2021). Targets and strategies for vaccine development against SARS-CoV-2. Biomedicine & Pharmacotherapy, 137, 111254. https://doi.org/10.1016/J.BIOPHA.2021.111254
11. Zhu, N., Zhang, D., Wang, W., Li, X., Yang, B., Song, J., Zhao, X., Huang, B., Shi, W., Lu, R., Niu, P., Zhan, F., Ma, X., Wang, D., Xu, W., Wu, G., Gao, G. F., & Tan, W. (2020). A Novel Coronavirus from Patients with Pneumonia in China, 2019. Https://Doi.Org/10.1056/NEJMoa2001017, 382(8), 727–733. https://doi.org/10.1056/NEJMOA2001017
12. Edwards, R. J., Mansouri, K., Stalls, V., Manne, K., Watts, B., Parks, R., Janowska, K., Gobeil, S. M. C., Kopp, M., Li, D., Lu, X., Mu, Z., Deyton, M., Oguin, T. H., Sprenz, J., Williams, W., Saunders, K. O., Montefiori, D., Sempowski, G. D., … Acharya, P. (2021). Cold sensitivity of the SARS-CoV-2 spike ectodomain. Nature Structural and Molecular Biology, 28(2), 128–131. https://doi.org/10.1038/s41594-020-00547-5
13. Piao, D. C., Lee, Y. S., Bok, J. D., Cho, C. S., Hong, Z. S., Kang, S. K., & Choi, Y. J. (2016). Production of soluble truncated spike protein of porcine epidemic diarrhea virus from inclusion bodies of Escherichia coli through refolding. Protein Expression and Purification, 126, 77–83. https://doi.org/10.1016/j.pep.2016.05.018
14. Wrapp, D., Wang, N., Corbett, K. S., Goldsmith, J. A., Hsieh, C.-L., Abiona, O., Graham, B. S., & McLellan, J. S. (2020). Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science, 367(6483). https://doi.org/10.1126/science.abb2507
15. Walls, A. C., Park, Y.-J., Tortorici, M. A., Wall, A., McGuire, A. T., & Veesler, D. (2020). Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell, 181(2). https://doi.org/10.1016/j.cell.2020.02.058

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Beer’s Law

The simplest and perhaps most common method of quantifying proteins is by using the innate fluorescence of the amino acid tryptophan. The absorbance of tryptophan can then be converted into a concentration measurement by using Beer’s formula A = ε b c, where ε is the molar extinction coefficient of the protein (in M-1cm-1), b is the path length (typically 1 cm), and c is the concentration (in M). Since tryptophan absorbs at 280 nm, the procedure for a Beer’s law quantitation is as simple as reading the absorbance of the protein-containing solution at 280 nm, correction for the dilution factor (if any), and using Beer’s formula to calculate the concentration. While this method is simple and requires no added reagents, it has several major limitations. First, Beer’s law calculations are fairly inaccurate and imprecise compared to some of the other methods in this article. Because Beer’s law directly relates the absorbance of the sample to the final concentration, slight fluctuations in the absorbance will lead to an inaccurate concentration. Second, Beer’s law requires the protein to be spectroscopically active. If a protein does not have any tryptophans, or has no other spectroscopic properties, it cannot be quantified using this method. While other spectroscopically active amino acids do exist (phenylalanine, tyrosine), the strength of their absorbance pales in comparison to tryptophan, and solutions need to be highly concentrated to get an accurate signal. Third, the molar extinction coefficient of the protein must be known. While it is possible to approximate the molar extinction coefficient of an unknown protein by using the number of tryptophans and tyrosines in its amino acid sequence, this is not ideal because it will yield a less accurate measurement compared to other techniques. 

Pierce 660 Assay

The pierce 660 assay is a fast-binding colorimetric assay that relies on the binding of a proprietary dye compound to proteins in solution before quantifying the dye by reading its absorbance at 660 nm. Since the amount of free dye is corrected for by a non-protein containing blank, the absorbance of the dye can then be correlated to the absorbance of the protein. In order to convert this absorbance value to the concentration of the protein sample, a standard curve using standards of a known amount of protein (typically BSA) must first be constructed. Since the pierce 660 assay is most accurate between the concentrations of 25 ug/mL and 2000 ug/mL, the standards all fall along this range. After the standard curve is constructed, a linear trend can be fit to the data and used to calculate the concentration of the protein-containing sample. As with all standard curves, absorbance readings that fall outside of the range set by the standards cannot be used as they are not covered by the linear range of the trend. The endpoint of the dye-protein complex is stable, so samples can sit for long periods of time before being read. One major drawback of the pierce 660 assay is that it is not compatible with ionic detergents, so membrane proteins cannot be quantified in their native state. 

Bradford Assay

The Bradford assay (also known as the coomassie assay) is very similar in principle to the Pierce 660 assay, except that its dye is known. This assay utilizes the spectroscopic shift of coomassie brilliant blue G-250 under different pH conditions to quantify the amount of protein that it is bound to. At neutral pH, the coomassie dye absorbs at 465 nm, shifting once it binds to a protein. The high amount of localized amine groups on a protein’s surface causes the coomassie dye to respond as if it was a more acidic environment, inducing its spectral shift to 595 nm. The blank absorbance at 465 nm is then subtracted from the absorbance at 595 nm to yield a corrected absorbance that is compatible with the typical standard curve quantitative seen in the pierce 660 assay. Much like the pierce 660 assay, the effective range of the Bradford assay is between 25 and 2000 ug/mL. Due to the pH sensitive nature of the dye, the Bradford assay should be avoided with protein samples at highly acidic or basic conditions. Additionally, the dye-protein complex is disrupted in the presence of any detergents, so these should be avoided as well.

Bicinchoninic Acid Assay

The bichinoic acid (BCA) assay, a modification of the widely-used Lowry assay, utilizes the color shift of the bicinchoninic acid-copper complex with protein in solution to relate the absorbance of this final complex with the amount of protein in solution. The BCA working reagent contains bicinchoninic acid, sodium carbonate, sodium bicarbonate, sodium tartrate, and copper(II) sulfate pentahydrate. When this solution is first mixed, the bicinchoninic acid and copper(II)sulfate pentahydrate form a complex that can then interact with proteins when the working reagent is added. The absorbance of the BCA-Cu complex is shifted to a wavelength of 562 nm by the amino acid side chains tyrosine, tryptophan, and cysteine. BCA assay samples are incubated at 40℃ in order to assist in the formation of peptide bonds to the BCA-Cu complex, which increases the sensitivity of the assay while reducing protein to protein variation. Much like the pierce 660 and Bradford assays, a standard curve must first be constructed before the protein concentration can be determined. The BCA assay should be avoided with any samples that contain reducing agents, as these can disrupt the BCA-Cu complex. Strong acids and bases should also be avoided, as these can also damage the BCA-Cu complex. Additionally, the BCA assay has no true endpoint, so samples must be read within an hour after incubation for accurate results. 

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