This panel discussion was originally published in the eBook

“ Monoclonal Antibody Manufacturing Trends, Challenges, and Analytical Solutions to Eliminate Bioprocessing Bottlenecks”

You can download all the articles in the series, by downloading the eBook.

Panel discussion members:

Carrie Mason – Associate Director, R&D at Lonza Biologics
Laura Madia – Independent Industry Consultant
Alan Opper – Director of HaLCon Sales at RedShiftBio
David Sloan, PhD – Senior Vice President, Life Sciences at RedShiftBio
Brandy Sargent, Editor in-chief, Cell Culture Dish and Downstream Column (Moderator)

In this panel discussion, we talked with industry experts about antibody process development and manufacturing. Specifically focusing on current antibody titer expectations, analytical challenges and how real time titer measurement is a game changer for bioproduction moving forward.

Where is the industry at today with titer expectations and what are the best practices for measuring titer?

Laura Madia

With respect to expectations regarding titer over the years, what we’ve seen is a need for increased titer
within the upstream development of a drug. As an industry, we have moved from the 80s where titers were closer
to .2 to .5 grams per liter to the early 2000s where concentrations of titer production rose to 3 to 5 grams per
liter. What we see today is a continued increase in titer concentrations, which creates a challenge to make sure
that you have technologies that can accurately measure titer concentration without introducing any errors.

The other thing that we have seen within the industry is the need for more data to not only understand what is
happening in the tank, but also to be able to make decisions about the product as the process is running or
shortly after.

Lastly, it is important to consider people and resources. It has been exacerbated by COVID, but it is difficult
to find people to work within the industry and there are fewer people within a production suite. This has helped
to drive the need for online and remote monitoring and automation to make it easier to get the necessary
measurements.

David Sloan

To follow up on the lack of workers, one of the things that we constantly hear from the customers we are
working with is that training employees can be a real challenge and a very time-intensive process. Technologies
that are easier to use and require less expertise help get people up and running and minimize errors amongst new
users of a technology.

Laura Madia

As for the current best practices for measuring titer, HPLC is the gold standard. But HPLC presents some
challenges including training and HPLC requires a highly skilled person to get accurate results. There is a need
for something that is simple and easy to use when it comes to measuring titer. You will still need HPLC results
for approval and decisions at the end, but to be able to monitor titer throughout the process is important.

What are the challenges associated with the way that titer is measured today and what can we do as an industry
to improve?

Laura Madia

One of the challenges is that most of the assays available today are batch processes, so that lends itself to
providing a retrospective look and means that most people don’t run samples throughout the process. This is
because most people save these tests until the end when they can run a batch and make it more cost effective,
and it is typically a long time to result so running it during the process isn’t helpful. Systems today are more
for batch process and are not set up for at-line measurement, unless you are lucky enough to be able to have an
HPLC that’s dedicated to that tank.

Another challenge is speed and accuracy. Many of the techniques that are offline today are longer assays
because they’re running as a batch. You must wait for the entire batch, which is a long time to first result.
That is where having a system like the HaLCon, which you can move online and do a simple one or two injections
to get the concentration is a nice thing. To be able to move measurements online and closer to the tank with an
easy to use system is critical.

David Sloan

One of the challenges with some technologies is that they just don’t have the dynamic range that’s required to
cover from the low concentrations you see earlier in the tank to those high concentrations that you’re getting
later or at the end of the production run. With a technology like HaLCon, you can measure from the low
concentrations of the .1 grams per liter all the way up to the higher concentrations of 8-10 grams per liter
without needing to do any sort of dilutions or serial dilutions.

What it really comes down to is accuracy and how closely does it agree with HPLC and do you have the dynamic
range that’s required to be able to cover the whole concentration range without needing to do multiple
dilutions. As soon as you start bringing dilutions into the equation, you’re increasing the chance of error and
the variability sample to sample, run to run, and user to user. If you have a technique that is user friendly
and even better if it’s automated to some extent, you minimize the human aspect of it and the potential error
that the lab analyst brings to the assay, thus providing a more reliable and reproducible result.

Alan Opper

I typically see that the instruments out there, except for HPLC, are not fit for purpose. They are used for a
lot of different things. For example, there are some instruments that can only measure titer after it’s
purified, not prior to purification or not in the upstream lab. There are other instruments that analyze dozens
of metabolites, and they can measure titer, however, they’re using methods such as turbidity, which is not as
accurate throughout the whole entire production run. Results could be anywhere from 10 to 30% off the expected
value.

Second, retrospective analysis. Why are we doing retrospective analysis? Why are people pulling samples at the
end of the run to send it to an analytical lab? Well, it’s because the current methods besides HPLC, which takes
a long time, are not very accurate. End users don’t trust the results or making process controlled decisions
based on the results.

Lastly, HPLC is a wonderful test, but it is complex to run. It’s often in a separate analytical lab which can
take, depending on the company and whether it’s production or development, anywhere from hours to days to weeks.

HPLC also requires a very well trained and well versed operator. With the HaLCon, it is a small instrument that
sits directly in the lab, it takes 5 minutes to run using liquid chromatography Protein A. It is very accurate,
and you don’t need to do batch methods of the cells because you could take daily samples and rest assured,
you’re going to know that those are very accurate and reproducible.

What benefits can be gained from real time titer measurement?

Carrie Mason

In the development laboratory, a lot of time you’ll do a batch study for bioreactors. You’re running small
scale bioreactors to optimize the process and taking samples. The process may run 10-15 days and then you batch
the samples all together and send them to get results.

One of the exciting aspects of real time titer measurement with the HaLCon is seeing that it really simplifies
the end use, and it allows the bioreactor operator to run a test on the HaLCon, as easy as injecting any of the
other types of methodologies, they use for monitoring a bioreactor. It allows you to get titer results within
the day, so now your researcher has an actual snapshot of what’s happening. They can start charting that
information and see what their titer looks like in relation to all the other bioreactor parameters. That’s very
powerful, because now instead of having to wait until the end when you gather up all your results and see if
bioreactor one was better than bioreactor two conditions, you can now start understanding what’s happening and
speed up the time to decide on what conditions are the best.

In a perfect world you can use this information to start making changes in your bioreactors, to start tweaking
media feeds or other conditions to get optimal titer. In the past you were just extrapolating cell growth as you
know best cell growth gave you the best titer, but that’s not always what we see in a bioreactor. So, in the
development lab, this gives a lot of power to the end user and now allows them to see insights in their process
and make faster decisions.

Looking at continuous manufacturing, you are not going to have the liberty of waiting for an HPLC result
because your process is running continuously. We are going to have to have controls within that process to
ensure that we’re running within the set points that we want the process to execute in. With technology like the
HaLCon and it’s fast results, you could have this sitting next to your continuous process and be taking small
samples across a day. This would ensure that what you’re putting into your downstream process that is coming out
of your continuous upstream process is exactly what you think it is. This would ensure good process control when
it comes to continuous manufacturing.

Alan Opper

Real time monitoring and real time data can lead to much more accurate process adjustments or real time process
adjustments, which will also increase your process understanding and that will enable you in the development
scheme to save a tremendous amount of time, resources and money in bringing the product to market.

On the flip side, when you get into CGMP production, typically they’ll close down the bioreactor and then take
the sample and measure titer. Based on the titer, they will properly load their columns, because Protein A can
be quite expensive. With the HaLCon, you can take a sample in real time and load your column immediately, which
can save you hours or even a production shift. This can save a tremendous amount of time because every hour lost
in the production environment is quite costly.

David Sloan

One last thought, it is all about changing bioprocess from a black box or a big unknown until the retrospective
analysis occurs to a data rich process where you understand what’s occurring in the process while it’s still
going on. There’s just so much additional information you can add to your understanding when you are able to add
titer measurement to all the other measurements that you are taking during the bioreactor run.

What does real time titer measurement mean for bioproduction moving forward?

Carrie Mason

If you look at several of the directives that are coming out from regulatory agencies and the push for more
information, it’s real time release. In my mind this is one step towards that pathway. All of this is really
building momentum to be able to understand what your process is doing and have much better process control,
which is what every regulatory agency wants to see. If we can say that we are taking these steps, even if there
are small steps along the way that we are increasing our process vision or process knowledge, making much better
process decisions and being able to verify that we’re running within our control strategy from the beginning,
this is significant.

In my world of looking at PAT and automation, this feels like a great place to be because it gives us
information that we can make decisions with and can be used for automation purposes. If we are looking at feed
streams as being continuous manufacturing, you could potentially look at this as a way to collect this data and
then use this data to determine time to shift to the next column or time to do another operation. In the past we
would not have had the ability to make on demand changes to processing.

I think that this is a key tool when we talk about what’s going to enable manufacture in the future. I see this
as being a really strong partner for driving a lot of that forward and giving us the tools we need to make our
products for our patients in a much more controlled manner.

Laura Madia

Historically the HaLCon is unique because now you can put real time titer measurement inside the manufacturing
suite, and you can put the titer measurement in the hands of the people that are manufacturing the product, not
sending it out to QC. That’s a huge paradigm shift in how the process works and where the equipment goes within
the traditional workflow.

Another thing to add is that with the HaLCon, titer can now be a data point that you monitor just like pH and
DO. This gives you a better picture of where everything is happening because you’re measuring the titer and the
cell concentration at the time it’s being made, not after it’s been frozen for a few days and then analyzed so
that’s really a much better snapshot of what’s happening in the tank.

How does HaLCon compare to HPLC in both conducting the testing and results?

David Sloan

We get this question a lot from customers that we’re working with and we also get customers who present us with
samples that they’d like to test on the HaLCon. Frequently they already have HPLC data for those samples from
their bioanalytical core, so we get to do a head-to-head analysis quite regularly, and the HaLCon compares
favorably with HPLC. We presented some of the data back to the bioanalytical core itself and they are always
overwhelmed and happy that the HaLCon agrees so well with their HPLC results because in many cases the
bioanalytical core is also extraordinarily happy not to have to run titer samples, not to have to be presented
with stat titers that need to be done.

We see very similar absolute concentration and agreement between the concentration numbers, the gram per liter
numbers that HaLCon is reporting. HaLCon agrees well from a data perspective and from a repeatability and
reliability perspective when compared to HPLC.

As far as conducting the test is concerned, it’s a different story. HPLC is complicated and requires expertise,
a lot of setup including buffers to be prepared. HaLCon couldn’t be farther from that scenario. It is super
straightforward to run and the system comes with a set of buffers, so there’s nothing for the end user to
prepare. The buffers are ready and optimized for the single purpose of measuring antibody titers. The Protein A
column is loaded on the system and it is always ready to run. When you get a sample, it is as simple as
injecting that sample and getting your concentration about 5 minutes later.

Carrie Mason

Looking at PAT and automation, one of the biggest challenges that I face is trying to use a different
methodology for a test. If people are very comfortable with doing one methodology for their test, you know
that’s what they file. That’s what they understand and that’s what their process is characterized on. So, if I
was to say, I would like to introduce a spectroscopic method for modeling your titer in a process, we may get a
little bit more pushback, because now there’s a lot more work that must be put into validating that system.

What really attracted me to this technology is the fact that it’s like for like. I can look at this and say,
it’s a miniaturized Protein A, HPLC assay. The modality is the same, so I don’t have to prove that this is a new
novel way of measuring, because in essence really this isn’t a new novel way of measuring. It’s measuring the
same way with affinity chromatography and detection very similar to what we do on our HPLC. But the fact that
it’s been so focused and purpose built for this one process and that it takes all the complexity out of it is
what makes it great. It allows me to share that with colleagues and with customers, that it looks different, but
fundamentally the science is the same. That is a great advantage of this technology.

When talking about ease of use, if you look at beyond just the batching, but if you go into having to do sample
handling and sample analysis, you’re really relying on humans to look at that information to do those
manipulations. So really what I want to do with PAT and automation is look at ways to remove human error and
human deviations from our processes and control as much as we can in a manner that is more reproducible and more
robust. The fact that the dynamic range of the HaLCon is such that you don’t have to do dilutions is extremely
powerful. To be able to just take a sample and inject it instead of having to do dilution not only saves time,
but also saves potential error.

How does the HaLCon fit into a traditional bioprocess workflow?

Laura Madia

The HaLCon is based on chromatography, so it will correlate directly to HPLC data that’s already been generated
and we know that it aligns with results within the QC lab. The way it fits within the bioprocess workflow is
because it is small and compact. It can fit on any bench top very easily. It comes self-contained with the
consumables, the reagents and columns that are needed. It has a very small footprint, simple interface, and can
connect right beside your bioreactors or on a bench space. You can put it beside the tank and anytime you want
to check what the titer is within the tank; you just load a small sample and later you’ll have the results. The
other benefit is that it’s so simple and easy to use. You don’t need the skills required that you would for an
HPLC, anybody can get a result quickly and easily.

Alan Opper

Typically, the current workflow of the laboratory, involves taking daily samples and measuring them, but it’s
more of a trend because the inaccuracies can be quite great. Therefore, samples are usually pulled at the end of
the run, sent to an analytical lab for measurement on HPLC and it takes hours to days or even weeks in some
cases to obtain results.

With the HaLCon, the proposed workflow is to take daily samples with accurate, real time results. As a result,
process control decisions can be made based on titer and cell productivity and there’s no need to pull all the
samples at the end of the run and wait days for the analytical lab to get back with results.

In the CGMP environment, the typical workflow is to pull samples at the end of the bioproduction run, obtain
measurements and then load the chromatography column. With the HaLCon, results are delivered right away, so the
column can be loaded immediately saving valuable time and effort.

Is the HaLCon compatible with automated sampling systems and how do you see it fitting into PAT initiatives?

David Sloan

Yes, HaLCon is compatible with automated sampling systems. It works with online systems, such as the MAST®
system as well as the Seg-Flow® system from Flownamics. For groups that hook it up to an online sampling system,
they can, in an automated fashion, grab samples from the bioreactor. Those samples are pushed directly to HaLCon
and this triggers HaLCon to take the measurement and then record that measurement and make it available within
the designated Laboratory Information Management System (LIMS), online notebooks, or automated data collection
system. This permits collecting, collating, and making all the data available in real time to the development
and manufacturing scientists.

For me, PAT is all about data and utilizing the analytical tools that are available to generate the data that’s
required to create a high quality, high yield product as quickly and easily as possible. PAT is about putting
analytical power into the hands of the scientists so they can ensure processes are going smoothly and will
result in the product and ultimately the drug that the patients need.

Carrie Mason

Interconnectivity of all these systems is critical. Looking at PAT tools, giving another lab bench tool to an
operator has its value, but where I really see the most value is if this can be integrated into the entire
ecosystem. So, not only are you looking at integrating and liquid handling for samples coming in, but then also
the integration of the data coming out. We don’t want to have another source of paper. With devices like the
HaLCon it is so critical for us to be moving forward and connecting the system to put the data into our
distributed control systems, that’s critical for new PAT tools. From what I’ve seen, the technology is moving in
that direction and has shown what it can do when it comes to being connected with liquid handling systems for
automated sampling. If I was to just say, here’s one more tool, I don’t think that it would be as well received
as if I say, this fits into our ecosystem.

What do you find or what do your customers find most exciting after using the Halycon?

Alan Opper

What customers really like about the HaLCon is that it’s fast, it’s accurate, and it’s very easy to use
compared to other methods. They also like the fact that it uses the same Protein A liquid chromatography method
that is used with HPLC. They don’t have to reinvent the wheel trying to validate another type of technology.
However, the difference between this and typical HPLC is that the HaLCon doesn’t require the method development,
the preparatory work or the specialized training required with HPLC.

Also, with the HaLCon, as we’ve discussed, there’s no sample dilution needed or purified samples needed with
this instrument. Because it runs through a range of .1 to 10 grams per liter, that’s quite a wide range. We
don’t need to have any human intervention. The system doesn’t need frequent recalibration either.

Lastly, it eliminates reliance on other labs, but also allows you to make critical decisions sooner which will
ultimately save a lot of time and money for the customer or the company.

Carrie Mason

What I like about it is the fact that it is equivalent to an HPLC method. In a company where you have various
groups all over the world that are all trying to measure titer, it is important to be able to recognize that
this is a slightly different system, but I get a comparable result. It is critical, especially if you look at
facilities that are going from small scale R&D through the development process, scale up, clinical and
commercial, we want to know that we’re basing our decisions and making our process control agreement strategies
based on data that is equivalent. We don’t want to say that there’s something different between one facility and
the other. So, by being able to have that equivalency across between HPLC and this system is very critical
because it allows us to then implement it in a controlled manner and to have confidence that this system will
provide the results that we need.

For customers and even development scientists to be able to have titer information rapidly available and to be
able to have the ability to intervene or to make a process decision there’s a lot of value in that.

If we can do real time analysis within the day, it allows us to speed up our development iterations and be able
to go through that process much quicker, which in the end ultimately gets the product to the market quicker.

This panel discussion was originally published in the eBook

“ Monoclonal Antibody Manufacturing Trends, Challenges, and Analytical Solutions to Eliminate Bioprocessing Bottlenecks”

You can download all the articles in the series, by downloading the eBook.