SMART Chromatography: purifying biomolecules direct from the fermenter without clarification

 

This article is for anyone working on downstream processing. If that describes you, I’d like you to consider this: how much you could save in time and costs if you were to carry out downstream processing without clarification? Here, I will describe a field-tested method of column chromatography purification called SMART Chromatography™, where SMART is an acronym of Simplified Method of Applied Radial Technology.

SMART Chromatography is used as a first step in downstream processing (DSP), where we apply a standard definition of DSP to mean that part of bioprocessing where the cell mass (from the upstream) is processed to meet purity and quality requirements. An overview of the typical bioprocessing workflow is illustrated below.

SMART Chromatography™ - Downstream Processing without Clarification

Figure 1: A typical bioprocessing workflow

The SMART Chromatography™ method has wide and general application to bioprocessing. It is used to purify products from mammalian, bacterial, insect and yeast cell cultures. Products that can be purified by the method include peptides, antibodies, enzymes and larger biomolecules such as DNA or vira.

So, what is it about the technique that makes it notable?

The two most notable feature of SMART Chromatography™ are:

  1. Feedstreams containing cells (or crude cell extract) can be loaded directly from the fermenter onto the purification column, i.e, no clarification of fermentation broth is required.
  2. SMART Chromatography column design is linearly scalable. This gives users the opportunity to rapidly prototype new processes and/or optimize and troubleshoot existing processes, with a smoother transition from R&D lab to production plant.

 

With the application of SMART Chromatography™, the typical bioprocessing workflow is radically altered:

SMART Chromatography™ - Downstream Processing without Clarification

Figure 2: Bioprocessing re-created. The bioprocessing workflow when SMART Chromatography™ is used. The clarification steps that are considered a regular part of the workflow are eliminated.

 

SMART Chromatography™ Eliminates the Need for Clarification in Downstream Processing

Preparation of the feed stream for product purification is a time-consuming and costly step. The problems associated with this step only increase as the bioreactor volume increases in scale. Removal of insolubles, or clarification, is normally seen as a required step in downstream processing. Typical clarification steps include, but are not restricted to, filtration, centrifugation, sedimentation, precipitation, flocculation, electro-precipitation and gravity settling. 

Cell removal also results in lower recovery of the desired product, whether it is intra- or extracellular. Product loss is inevitable during clarification steps. By eliminating filtration and centrifugation, SMART Chromatography™ makes the entire product titre within the fermenter available for capture on the purification column.

Are we claiming too much? We don’t think so. For those working in downstream processing, the elimination of clarification steps has clear positive consequences:

1.Saving Time

The amount of time spent on clarification in traditional bioprocessing can vary from hours to days, depending upon the volume of fermenter broth that needs to be processed. By removing the need for filtration and centrifugation, SMART Chromatography™ offers time savings for virtually all bioprocessing workflows. Sometimes these time savings can be considerable. For example, one of the early adapters of SMART Chromatography™ was able to reduce purification times by a factor of 5 (existing 16 hour DSP process reduced to 3 hours).

2. Reducing Costs

Our conversations with customers have revealed that clarification can often require more than a single step. In the case of filtration, for example, there might be several rounds of filtration, with new filters being required at each step. In such case, filtration costs end up being a significant proportion of the total costs for the downstream process.

SMART Chromatography™ removes other associated costs attached to clarifications, e.g., costs associated with consumables, buffer etc. These costs can form a significant part of the overall cost of downstream processing, especially in cGMP manufacturing environments.

Finally, the most obvious cost saving is a consequence of the time savings mentioned previously, in terms of personnel, equipment and other overheads that have a time-dependent cost factor.

3. Increasing Product Recovery

Estimates vary as to the amount of product that is lost from the original titre (present in the fermentation broth) to that which eventually gets added to the purification column. Anecdotally, we hear values of between 5% to 60% product loss due to clarification steps. Just like clarification has been an assumed step in downstream processing, this product loss has simply been considered a normal state of affairs, and is factored into product cost, plant design, timelines etc.

With SMART Chromatography™, one of the major causes of product loss within bioprocessing (namely, clarification) is removed. Instead, the entire amount of product from the fermenter is now made available for purification. In test cases with customers who have tried SMART™ Chromatography we have seen up to 98% recovery of the product titre that was in the fermenter.

 

Summary of the Main Parameters Affected by Using SMART Chromatography™

ParameterResult
Process timeReduced
Buffer consumptionReduced
ProductivityIncreased
Recovery of ProductIncreased
Retention of biological activityIncreased
Overall EffectImproved process at lower cost

 

What technology lies behind SMART Chromatography™?

SMART Chromatography results from combining two technologies:

1. ZetaCell™ resin

2. Radial flow column chromatography

The properties of these components of SMART Chromatography™ are examined in greater detail below.

1. ZetaCell™ Resin

SMART Chromatography™ - Downstream Processing without Clarification

Figure 3: Representation of a ZetaCell™ bead. Each bead is a large particle, based on highly cross-linked agarose. The beads contain channels that allow the molecule of interest to enter and be captured (blue sphere). Large objects, such as cells or other debris (green sphere) are unable to enter the bead and pass around it.

SMART Chromatography™ uses ZetaCell solid phases. Each ZetaCell resin is composed of beads that are are based on large particle, highly cross-linked agarose. Modifications are made on all exposed surfaces of each bead (both external and internal surfaces) that provide the desired binding functionality. The target of interest is captured using whatever strategy is preferred, i.e., IEX or affinity.

See the table in the Appendix for an overview of the main capture functionalities available for ZetaCell resins.

The way that ZetaCell beads work is easy to imagine. Large molecules, such as cells, cell debris and other solid matter in the feedstream are too large to enter the bead. Thus, they pass around each bead. However the target molecule of interest gets bound to  the bead by whichever binding functionality is being used.

So, why doesn’t the column get blocked, which would happen with a regular chromatography column? This is where the characteristics of radial flow chromatography come into play.

SMART Chromatography™ - Downstream Processing without Clarification

Figure 4: Inside a SMART Chromatography™ column, cells and large debris from the fermenter feedstream pass around the ZetaCell™ beads and out of the column.

2. Radial Flow Chromatography

SMART Chromatography™ - Downstream Processing without Clarification

Figure 5: 2-D representation of Radial Flow Chromatography (RFC). Unike conventional chromatography columns that typically uses vertical (top-bottom) flow, RFC columns use horizontal flow. Bed height and chromatographic properties remain, while footprint and weight reduce dramatically.

Radial Flow Chromatography (RFC) is an efficient, low pressure technology for bio-molecule fractionation. Thanks to the column geometry, RFC is very suitable for high throughput adsorptive separations in process, pilot or lab scale.

SMART Chromatography™ results when ZetaCell Resin is combined with RFC. RFC columns have a larger surface area for column loading than regular (i.e. axial flow) chromatography columns. For example, an RFC column will typically have twenty times the frit surface area compared to an equivalent standard column with the same bed length and bed volume. This increased column loading surface area means that feedstreams containing cell and other debris are spread over a much larger surface area when entering the column. Inside the column, cells pass around the ZetaCell™ Resin, where the target of interest is captured. Cells pass through and out of the column, without blocking. After washing, to remove residual cells, the product can be eluted from the column by the preferred methodology.

There are many advantages to using RFC, including dramatic reductions in column weight/ footprint and easy packing properties. However, one of the most useful features within a bioprocessing setting is that RFC is linearly scalable. The advantages of this linear scalability are advantageous in a production setting and are expanded upon in the next section.

 

SMART Chromatography™ Allows for Rapid R&D, Troubleshooting and Optimization of Bioprocessing

SMART Chromatography™ - Downstream Processing without Clarification

Figure 6: The linear scalability of SMART Chromatography™ allows for more rapid process development cycles. An R&D column of about 5 mL can be further developed at 200 mL scale-up and validation scale, before going to final production scale. Alternatively, if changes need to be made to an existing SMART Chromatography™ process, process engineers can try out the modified process at R&D scale again, relying on the linear scalability of RFC to enable faster implementation in production.

One of the critical challenges of any bio-manufacturing process is moving from R&D to production, a process commonly referred to as “scale-up.” SMART Chromatography™ uses a major feature of RFC to make the process of scale-up more straightforward – linear scalability.

Small- and medium-scale RFC columns are representative of larger, process-scale columns. Put another way, a smaller-scale RFC column is representative of the larger whole (see figure 7, below). For any given bed length, properties such as flow rates, cell density, resin binding, column washing parameters etc. remain the same over all column scales.

Linear scalability allows for accelerated process development, because many fundamental properties of the purification process that are set at the R&D stage can be implemented at larger scales.

Conversely, the linear scalability of RFC works in scale-down. Scale-down is more straightforward with SMART Chromatography™ than regular column chromatography approaches.

SMART Chromatography™ - Downstream Processing without Clarification

Figure 7: Due to the linear scalability of radial flow chromatography (RFC), purification parameter for a process can be determined on R&D and development-scale RFC columns. For a given column bed length, the properties of these smaller columns will represent what will occur in the large-scale final production column.

 

SMART Chromatography™ Works on a Wide Range of Cell Types and Target Molecules

SMART Chromatography™ has been implemented at, or is currently undergoing evaluation at 20+ different sites globally. EMP Biotech has accumulated experience in testing and roll-out of the use of SMART Chromatography™ for:

  • Yeast (Saccharomyces cerevisiae)
    • Purification of recombinant protein by IEX, using ZetaCell SP resin.
    • Currently at 90% recovery.
SMART Chromatography™ - Downstream Processing without Clarification


Figure 8: Scale Up SMART Chromatography™ Study on Yeast. Using a 200 mL column, 6 cm bed length (centre of picture). Yeast cells loaded directly from fermentation broth (left hand side) and passing through column to waste (beaker, right hand side).

SMART Chromatography™ - Downstream Processing without Clarification

Figure 9: Scale Up SMART Chromatography™ Validation on Yeast. Analytical RP-HPLC shows excellent purification (blue trace) compared with untreated and adjusted fermentation broth (black and pink traces)

  • Mammalian cells (CHO).
    • Purification of recombinant monoclonal IgG using ZetaCell Protein A resin.
    • Recovery of IgG: 98%
SMART Chromatography™ - Downstream Processing without Clarification

Figure 10: R&D scale SMART Chromatography™ column being loaded with CHO cells. The red colour is due to the presence of phenol red in the cell culture medium.

At the BioProcessing Network (BPN) conference (Adelaide, Australia, 2017), CSIRO (the premier Australian research centre) presented data for the use of SMART Chromatography™ to purify monoclonal antibodies from mouse hybridoma cell lines (Martin, J et. al, 2017). The SMART Chromatography™ column showed about 93% antibody capture, without the need for removal of cells from the media applied to the purification column.

SMART Chromatography™ has also been used with other cell systems such as E. coli and HEK-293, as well as cell systems which cannot be disclosed in this article due to confidentiality agreements between emp Biotech and their clients.

 

Using SMART Chromatography™ in Production

SMART Chromatography™ - Downstream Processing without Clarification

Figure 11: Production scale SMART Chromatography™ set-up

SMART Chromatography™ columns have the property (a function of RFC) of dramatically reduced footprint and weight, compared to a vertical column of equivalent bed height. This means that SMART Chromatography™ columns require less physical space, which is often an issue in a bioprocessing environment. The picture above shows a 250 litre column. With a diamter of 70 cm, the  column will fit through a standard doorway. As a “rule of thumb” the columns have around 25% of the footprint of the equivalent axial flow column. 

Interested?

We’d like to hear from you. Please submit your enquiries using the form at the foot of this page, or send a mail to info@youdobio.com. Alternatively, just reach for your  phone and call or text using our number at the top of this page.

An expert in SMART Chromatography™ will visit you at your convenience.

Frequently Asked Questions (FAQs)

Below is a list of common questions that arise in discussions about SMART Chromatography™. If you have your own questions and cannot find an answer below, or would like more specifics, please submit your question using the form at the foot of the page.

Q1. How quickly can I get started with SMART Chromatography™?

For a new process, we expect it to take about 6 months to go from R&D to production. We have experience of re-developing an existing process, for purification of a registered pharmaceutical – this took 12 months. These figures represent a rough estimate – it will depend on the process.

Q2. Is SMART Chromatography™ more expensive to implement than what I’m currently using?

Small volume columns, e.g. 5 mL – 200 mL, are slightly more expensive than traditional axial flow columns. At large scale (> 5L) the price of columns and resin is similar to similar-scale axial  flow columns. In any case, SMART Chromatography™ will save you money in the long run.

Q3. How easy is SMART Chromatography™ to implement?

Not difficult. A typical process work-up will involve using R&D columns at 5 mL scale, validation columns at 200 mL scale, before going to process scale. EMP Biotech has developed the SMART Chromatography™ method and will be on-hand to ensure that implementation goes smoothly.

EMP Biotech can provide small-scale columns and resin for proof-of-principle studies. Such columns can be fitted to standard chromatography equipment, e.g. ÄKTA purification systems. SMART Chromatography columns at small-scale can be loaded with a peristaltic pump.

Q4. Can we pack and repack SMART™ Chromatography columns ourselves?

SMART Chromatography™ columns are remarkably straightforward to pack. Columns can be packed using a packing station. Alternatively, the nature of the column design means that columns can be packed in a “low tech” way with resin slurry using a pump and a pressure.

Q5. How are SMART Chromatography™ columns cleaned?

SMART Chromatography™ columns are cleaned in the same way as traditional axial flow columns.  Cell and cellular debris are washed through the column and a short back flush will remove any solids trapped in the inlet frit.  The resins used for SMART Chromatography™ are generally tolerant of harsh CIP reagents such as 0.5 – 1 M NaOH, although obviously this is ligand dependent.

Q6. Who are EMP Biotech?

EMP Biotech is a 25-year old privately-held company, based in Berlin, Germany. The company has three active manufacturing sites, with a fourth one the way. EMP is ISO certified (ISO 9001:2015). The company has passed dozens of audits by major organizations using a wide range of manufacturing products.

The EMP team consists of accomplished chemists and engineers who professionally deliver a wide range of products within purification, chemistry and biochemistry.

You can learn more about EMP Biotech from their website.

EMP Biotech is represented in Denmark, Sweden and Norway by You Do Bio.

 

Appendix: main purification functionalities available for ZetaCell™ resins

Resin name Functionality Ligand Target
ZetaCell Protein AAffinityProtein AAntibodies
ZetaCell Protein A EndureAffinityBase-stable protein AAntibodies
ZetaCell Protein GAffinityProtein GAntibodies
ZetaCell IDAAffinityIminodiacetic acidHis-tagged proteins
ZetaCell NTA (uncharged)AffinityNitrilotriacetic acidHis-tagged proteins
ZetaCell Cu-NTAAffinityNitrilotriacetic acid Cu2+His-tagged proteins
ZetaCell Co-NTAAffinityNitrilotriacetic acid Co2+His-tagged proteins
ZetaCell Ni-NTAAffinityNitrilotriacetic acid Ni2+His-tagged proteins
ZetaCell Zn-NTAAffinityNitrilotriacetic acid Zn2+ His-tagged proteins
ZetaCell PhenylHICPhenyl (C6H5)Protein purification
ZetaCell QIEX (Strong Anion)Quaternary ammoniumNeg. charged molecules
ZetaCell Q BoostIEX (Strong Anion)Quaternary ammoniumNeg. charged molecules
ZetaCell SPIEX (Strong Cation)SulphopropylPos. charged molecules
ZetaCell SP BoostIEX (Strong Cation)SulphopropylPos. charged molecules
ZetaCell DEAEIEX (Weak Anion)Diethyl-aminoethylNeg. charged molecules
ZetaCell DEAE BoostIEX (Weak Anion)Diethyl-aminoethylNeg. charged molecules
ZetaCell CMIEX (Weak Cation)Carboxy-methylPos. charged molecules
ZetaCell CM BoostIEX (Weak Cation)Carboxy-methylPos. charged molecules
ZetaCell Aldehyde activatedPrimary amine bindingClient providedBinding to specific ligand

 

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